Analyse Des Wirtszellproteoms Und Der Expression Viraler Proteine In

Analyse des Wirtszellproteoms und der Expression viraler Proteine in virusinfizierten Gewebekulturen durch quantitative Massenspektrometrie mit metabolisch eingeführten stabilen Isotopen

I n a u g u r a l d i s s e r t a t i o n

zur

Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) an der Mathematisch-Naturwissenschaftlichen Fakultät der Ernst-Moritz-Arndt-Universität Greifswald

vorgelegt von

Martin Skiba

geboren am 11.12.1980

in Bochum

Greifswald, 18.11.2010

Dekan: Prof. Dr. Klaus Fesser

1. Gutachter : Prof. Dr. Dr. h.c. Thomas C. Mettenleiter

2. Gutachter: Prof. Dr. Beate Sodeik

Tag der Promotion: 04.03.2011

Inhaltsverzeichnis

I. Einleitung ...... 1

I.1 Vorwort ...... 1

I.2 Das Pseudorabiesvirus ...... 1

I.2.1 Die Taxonomie der Herpesviren ...... 1

I.2.2 Die Pathogenese der Pseudorabiesvirus-Infektion ...... 2

I.2.3 Der morphologische Aufbau des Herpesvirus Partikels ...... 3

I.2.4 Der Genomaufbau ...... 4

I.2.5 Der Replikationszyklus des Pseudorabiesvirus ...... 4

I.2.6 Die Expression herpesviraler Gene ...... 6

I.2.7 Einfluss der Infektion mit Herpesviren auf die Expression und posttranslationale Modifikation zelleigener Proteine ...... 8

I.3 Das Newcastle Disease Virus ...... 12

I.3.1 Taxonomische Einordnung und Pathogenese ...... 12

I.3.2 Morphologie und Genomorganisation des NDV ...... 12

I.3.3 Die Transkription der NDV-Gene ...... 14

I.3.4 NDV als viraler Vektor für rekombinante Impfstoffe ...... 15

I.4 Proteomanalyse ...... 16

I.4.1 Quantitative Proteomics ...... 17

I.4.2 Proteomanalysen von Viruspartikeln ...... 19

I.4.3 Proteomanalysen und Expressionsstudien mit PrV-infizierten Zellen ...... 20

II. Zielstellung ...... 27

II.1 Erstellung einer Proteomkarte PrV-infizierter MDBK Zellen ...... 27

II.2 Proteomanalysen an Zellfraktionen nach Infektion mit PrV-Wildtyp und einer Kinase-negativen Deletionsmutante ...... 28 II.3 Quantitative Analyse der Proteinexpression von Fremdgenen in rekombinanten Newcastle Disease Viren ...... 28

III. Zusammenfassende Darstellung und Diskussion der Ergebnisse ...... 29

III.1 “Quantitative whole-cell proteome analysis of pseudorabies virus-infected cells.” .29

III.2 “Gene expression profiling of Pseudorabies virus (PrV) infected bovine cells by combination of transcript analysis and quantitative proteomic techniques.” ...... 31

III.3 “Influence of Insertion Site of Avian Influenza Virus Hemagglutinin (HA) Gene Within the Newcastle Disease Virus Genome on HA.” ...... 32

IV. Literaturverzeichnis ...... 34

V. Quantitative whole-cell proteome analysis of pseudorabies virus-infected cells...... 63

VI. Gene expression profiling of Pseudorabies virus (PrV) infected bovine cells by combination of transcript analysis and quantitative proteomic techniques...... 75

VII. Influence of Insertion Site of Avian Influenza Virus Hemagglutinin (HA) Gene Within the Newcastle Disease Virus Genome on HA Expression...... 83

VIII. Zusammenfassung der Dissertation ...... 100

IX. Summary ...... 103

X. Anhang ...... 106

X.1 Abkürzungsverzeichnis ...... 106

X.2 Publikationen ...... 109

X.2.1 Veröffentlichungen ...... 109

X.2.2 Tagungsbeiträge: ...... 109

X.3 Eidesstattliche Erklärung ...... 110

X.4 Lebenslauf ...... 111

X.5 Danksagung ...... 112

I. Einleitung

I.1 Vorwort In der vorliegenden Arbeit wurden Proteinexpressionsanalysen an Gewebekulturen durchgeführt, die mit dem Pseudorabiesvirus (PrV) oder mit rekombinanten Newcastle Disease Viren (NDV) infiziert worden waren. Die quantitative Analyse erfolgte nach zweidimensionaler Proteinelektrophorese (2DE) massenspektrometrisch unter Verwendung metabolisch eingeführter stabiler Isotope. Um in die Thematik einzuführen, sollen zunächst die verwendeten Krankheitserreger und die Grundlagen der verwendeten Protein- und Massenanalyse vorgestellt werden.

I.2 Das Pseudorabiesvirus I.2.1 Die Taxonomie der Herpesviren

Die Herpesviren werden in drei Familien innerhalb der Ordnung der Herpesvirales unterteilt. Zu der Familie der Herpesviridae werden die Herpesviren der Säuger, Vögel und Reptilien gezählt. Die Familie der Alloherpesviridae umfasst die Herpesviren der Fische und Amphibien, und die Familie Malacoherpesviridae enthält das Herpesvirus der Auster (Davison et al., 2009). Aufgrund ihres unterschiedlichen Wirts- und Zelltropismus werden die Vertreter der Familie der Herpesviridae weiter in die drei Unterfamilien Alpha-, Beta- und Gammaherpesvirinae klassifiziert (Roizmann et al., 1992). Die Alphaherpesviren weisen im Allgemeinen ein breites experimentelles Wirtsspektrum, einen kurzen Replikationszyklus und eine schnelle lytische Ausbreitung in der Zellkultur auf. Häufig wird eine reaktivierbare Latenz in sensorischen Ganglien des Wirts etabliert. Die Unterfamilie wird von vier Genera gebildet. Dem Genus Simplexvirus werden die humanpathogenen Herpes Simplex Viren Typ 1 und 2 (HSV-1 und -2; HHV1 und 2), sowie das Herpes B Virus (McHV1) zugeordnet. Das humanpathogene Varizella-Zoster Virus (VZV; HHV3) und die tierpathogenen Bovine Herpesviren 1 und 5 (BoHV1 und 5), Equinen Herpesviren 1 und 4 (EHV1 und 4) und das Pseudorabiesvirus (PrV; SuHV1) gehören zum Genus Varicellovirus (Roizmann et al., 1992). Die beiden weiteren Genera Mardivirus, sowie Iltovirus beinhalten u.a. die Erreger der Marek´schen Krankheit (MDV; GaHV2 und 3) bzw. der Infektiösen Laryngotracheitis (ILTV; GaHV1) der Hühner (Minson et al., 2000). Die Vertreter der Betaherpesviren zeichnen sich durch ein engeres Wirtsspektrum und einen längeren Replikationszyklus aus. Typische Merkmale sind eine langsamere Ausbreitung in der Zellkultur, häufig verbunden mit einer abnormen Vergrößerung infizierter Zellen (Zytomegalie). Latenz etabliert sich in sekretorischen Drüsen, lymphoretikulären Zellen, der Niere und in anderen Geweben.

Gebildet wird die Unterfamilie von den vier Genera Cytomegalovirus mit dem humanpathogenen Humanen Zytomegalievirus (HCMV; HHV5), Muromegalovirus, mit dem Murinen Zytomegalievirus (MCMV; MuHV1), Roseolovirus mit den Humanen Herpesviren Typ 6 und 7 (HHV6 und 7) und Proboscivirus mit dem Endotheliotropen Elefantenherpesvirus (EIHV1) als einzigem Vertreter. Die Gammaherpesviren besitzen ein sehr enges Wirtsspektrum und infizieren produktiv und latent B- und T-Lymphozyten. Unterteilt wird diese Unterfamilie in das Genus Lymphocryptovirus mit dem Epstein-Barr- Virus (EBV; HHV4), dem Genus Rhadinovirus mit dem Kaposi´s sarcoma associated herpesvirus (KSHV; HHV8), dem Genus Macavirus u.a. mit dem bösartigen Katarrhalfieber (CpHV2) und dem Genus Percavirus. In den Familien der Alloherpesviridae und der Malacoherpesviridae finden sich Vertreter, die zwar nur geringe Sequenzhomologien zu anderen Herpesviren aufweisen, aufgrund morphologischer und biologischer Eigenschaften aber dennoch diesen zugeordnet werden. So finden sich bei den Alloherpesviridae Herpesviren der Fische und der Amphibien mit den bekanntesten Vertretern Channel Catfish Virus (IcHV1), Koi-Herpesvirus (CyHV3) und den Froschherpesviren RaHV1 und 2. Die Familie der Malacoherpesviridae umfasst die Herpesviren der Weichtiere, mit dem bislang einzigen bekannten Vertreter bei den Invertebraten, dem Herpesvirus der pazifischen Auster (OsHV1) (Davison et al., 2005; Davison et al., 2009; Farley et al., 1972).

I.2.2 Die Pathogenese der Pseudorabiesvirus-Infektion

Das PrV ist der Erreger der Aujeszky‘schen Krankheit (AK, auch Pseudowut oder Aujeszky’s disease [AD]), die erstmals 1902 von dem ungarischen Tierarzt Aladár Aujeszky beschrieben wurde (Aujeszky, 1902). Es handelt sich um eine fieberhafte Allgemeinerkrankung, die von neurologischen Symptomen begleitet wird. Der Erreger kann aufgrund seines breiten Wirtsspektrums zahlreiche Säugetiere infizieren. Von wirtschaftlicher Bedeutung ist die Erkrankung aber vor allem beim Hauptwirt, dem Schwein. Die Erkrankung verläuft bei den meisten Spezies tödlich, Schweine können jedoch eine Infektion überleben (Pensaert & Kluge, 1989; Wittmann, 1984) und werden nach Etablierung einer latenten Infektion zu dauerhaften Virusträgern. Einhufer und höhere Primaten, einschließlich des Menschen, bilden eine Ausnahme und sind gegen die PrV-Infektion resistent (Mettenleiter, 1994b). Die Inkubationszeit und das klinische Erscheinungsbild beim Schwein sind stark vom Alter der erkrankten Tiere, von der Virulenz des Virusstammes und vom Infektionsweg abhängig. Bei erwachsenen Tieren verläuft die Infektion nahezu inapparent, wohingegen Saugferkel meist nach 2-3 Tagen an den Folgen einer Gehirn- und Rückenmarksentzündung verenden (Sabo et al., 1969). Klinische Symptome reichen von Apathie und Erbrechen bis zu charakteristischen zentralnervösen Symptomen, die von Erregungszuständen und 2

Koordinationsstörungen bis zu Ataxien, Lähmungen der Hintergliedmaßen, sowie Krämpfen der Rückenmuskulatur und der Extremitäten mit Ruderbewegungen geprägt sind (Pensaert & Kluge, 1989; Wittmann, 1984; Wittmann, 1991). Die Virusaufnahme erfolgt hauptsächlich oronasal über den Respirationstrakt durch das Nasensekret. Die primäre Virusreplikation findet in den Epithelzellen des Nasen- und Rachenraums statt. Danach gelangen die Viren in die Ganglien und breiten sich schnell durch axonal-retrograden Transport im zentralen Nervensystem aus. In den Ganglien, aber auch im Bulbus olfactorius und in den Tonsillen, etabliert sich eine latente Infektion, wodurch die Schweine zu lebenslangen Virusträgern und damit potentiellen Überträgern der Krankheit werden (Wittmann, 1991; Wittmann & Rziha, 1989).

Der Einsatz einer Glykoprotein (g)E-negativen Markervakzine, die eine serologische Unterscheidung zwischen geimpften und Feldvirus-infizierten Tieren erlaubt, führte in Eradikationsprogrammen dazu, dass die Krankheit heute in vielen westeuropäischen Ländern als ausgerottet gilt (Mettenleiter, 1994a; Mettenleiter, 1994b; Van Oirschot et al., 1996). Neben der Bedeutung als Tierseuchenerreger gilt PrV als gut untersuchtes Modellvirus zur Erforschung der Herpesvirusinfektion auf molekularbiologischer, immunologischer und neuroanatomischer Ebene (Mettenleiter, 2008). Günstig für Untersuchungen in Zellkultur und in Tierversuchen sind die biologischen Eigenschaften des PrV, wie fehlende Humanpathogenität, schnelles Wachstum in Zellkultur, leichte Manipulierbarkeit des Genoms und die Möglichkeit, Tierexperimente im natürlichen Wirt durchzuführen.

I.2.3 Der morphologische Aufbau des Herpesvirus Partikels

Alle reifen Herpes-Virionen besitzen einen typischen morphologischen Aufbau, der durch vier elektronenmikroskopisch unterscheidbare Komponenten charakterisiert ist (Abb. 1). Das Innere wird von einem elektronendichten Kern (Core) gebildet, in dem das mit Proteinen assoziierte lineare doppelsträngige virale DNA-Genom vorliegt (Furlong et al., 1972; Minson et al., 2000). Umschlossen wird dieser Kern von einem ca. 100 nm großen Kapsid ikosaedrischer Symmetrie, das aus 162 Kapsomeren (150 Hexone und 12 Pentone) besteht (Schrag et al., 1989). Das Nukleokapsid ist seinerseits von einem elektronenmikroskopisch amorphen Tegument umgeben. Das Kapsid und die Tegumentschicht werden von einer Hüllmembran umgeben, die zellulären Ursprungs ist und virale (Glyko-) Proteine enthält (Gingsberg, 1988).

Abb. 1 Morphologie eines Herpesviruspartikels

Der Aufbau eines Pseudorabiesvirus Partikels ist anhand der elektronenmikroskopischen Aufnahmen (links negativ kontrastiert und rechts im Ultradünnschnitt) und einer schematischen Übersicht (Mitte) dargestellt (modifiziert nach (Granzow et al., 1997)).

I.2.4 Der Genomaufbau

Das Genom des Pseudorabiesvirus umfasst ca. 143 Kilobasenpaare (kbp), codiert für 70

Proteine und wird durch Regionen mit inversen Sequenzwiederholungen (IRS und TRS) in eine „unique long“ (UL) und eine „unique short“ (US) Region gegliedert (Ben-Porat et al.,

1983; Klupp et al., 2004). Aufgrund der beiden repetitiven Regionen kann die US-Region in

Relation zur UL-Region in zwei antiparallelen Orientierungen auftreten, wodurch zwei isomere Genomformen entstehen (Ben-Porat et al., 1983; Davison & Wilkie, 1983). Diese Anordnung charakterisiert das Genus Varicellovirus und wird als Gruppe D-Genom bezeichnet. PrV trägt im Unterschied zu anderen Alphaherpesviren eine ca. 40 kbp große

Inversion in der UL-Region (Ben-Porat et al., 1983; Klupp et al., 2004). Das PrV-Genom besitzt drei Replikationsursprünge (origins of replication, ori) (Fuchs et al., 2000; Wu et al., 1986).

I.2.5 Der Replikationszyklus des Pseudorabiesvirus

Der Replikationszyklus der Alphaherpesviren ist durch elektronenmikroskopische und molekularbiologische Untersuchungen an HSV-1 und PrV gut untersucht. Eine PrV-Infektion beginnt mit der Adsorption (attachment) des Virus an die Oberfläche der Wirtszelle. Dabei erfolgt die erste Interaktion mit einer schwachen reversiblen Bindung des nicht essentiellen viralen Glykoproteins C an Heparansulfat tragende Proteoglykane der Wirtszelle (Karger &

Mettenleiter, 1993; Mettenleiter et al., 1990; Sawitzky et al., 1990). Diese Bindung wird unter Beteiligung von gD stabilisiert, was die Fusion von Viruspartikel und Zytoplasmamembran inhibiert (Karger & Mettenleiter, 1993; Mettenleiter et al., 1990; Rauh & Mettenleiter, 1991; Sawitzky et al., 1990). Bisher wurden 5 verschiedene gD-spezifische zelluläre Rezeptoren (herpesvirus entry mediators, Hve) für HSV-1 identifiziert: HveA (Tumor Nekrose Faktor Rezeptor Superfamilie 14) (Montgomery et al., 1996), HveB (Nektin-2) (Warner et al., 1998), HveC (Nektin-1) (Geraghty et al., 1998; Krummenacher et al., 1999) und HveD (CD155) (Cocchi et al., 1998; Geraghty et al., 1998), sowie eine Form der 3-O-sulfatierten Heparansulfate (Xu et al., 2005) (Spear et al., 2000). Als PrV-gD-spezifische Rezeptoren wurden 2 Vertreter der Nektine beschrieben: Nektin-1 (HveC) (Geraghty et al., 1998) und Nektin-2 (HveB) (Warner et al., 1998) und ein dem Poliovirus-Rezeptor verwandtes Protein (HveD, CD155) (Nixdorf et al., 1999). Neben den genannten gD-spezifischen PrV- Rezeptoren wird die Existenz weiterer Rezeptoren postuliert, die die Infektion gD-negativer PrV-Mutanten vermitteln (Karger et al., 1998; Nixdorf et al., 1999; Schmidt et al., 1997). Nach der Adsorption fusioniert die virale Hülle unter Beteiligung der PrV-Glykoproteine B, D und dem gH/gL-Komplex (Klupp et al., 1997; Klupp et al., 2000; Rauh & Mettenleiter, 1991) in einem pH-unabhängigen Prozess mit der Plasmamembran (Penetration) und das hüllenlose Pr-Virion wird in das Zytoplasma entlassen. Ein Großteil der Tegumentproteine verteilt sich danach innerhalb des Zytoplasma, während pUL36, pUL37, sowie die Proteinkinase pUS3 mit dem Kapsid assoziiert bleiben (Granzow et al., 2005), das entlang der Mikrotubuli zur Kernpore wandert (Granzow et al., 1997; Granzow et al., 2005). Für HSV-1 wurde eine Beteiligung der zellulären Motor-ATPasen Dynein, Kinesin-1 und deren Kofaktor Dynaktin an diesem gerichteten Transport beschrieben (Dohner et al., 2002; Radtke et al., 2010; Sodeik et al., 1997). An dem Kernporenkomplex angedockt, entlässt das PrV-Nukleokapsid das virale Genom in den Zellkern (Granzow et al., 1997). Im Kern bildet die lineare virale DNA eine episomale zirkuläre Form und die Transkription der viralen Gene beginnt. Es folgt die Replikation der viralen DNA und die Neubildung von Nukleokapsiden im Zellkern. Für die Kapsidmorphogenese wird zunächst ein sphärisches Prokapsid aus den Gerüstproteinen pUL26 und pUL26.5 und dem Hauptkapsidprotein pUL19, den Triplexproteinen pUL18 und pUL38, dem kleinem Kapsidprotein pUL35 und dem Portalprotein pUL6 gebildet (Newcomb et al., 1996). Während des Einbaus des Virusgenoms in das Prokapsid werden die gerüstbildenden Proteine proteolytisch abgebaut und durch Konformationsänderung entsteht die ikosaedrische Kapsidform (Cardone et al., 2007; Trus et al., 1996). Die Kapside werden anschließend in einem Knospungs- und Fusionsprozess (envelopment-deenvelopment- reenvelopment) durch die Kernmembranen in das Zytosol transferiert (Granzow et al., 2001; Skepper et al., 2001). Die reifen Nukleokapside knospen (budding) dabei an der inneren Kernmembran und gelangen als primär umhüllte (primary envelopment) Virionen in den

perinukleären Spalt (Granzow et al., 1997; Mettenleiter, 2004; Mettenleiter et al., 2009). An diesem Vorgang sind unter anderem die viralen Proteine pUS3, pUL25 und die beiden innerhalb der Herpesviridae konservierten Proteine pUL31 und pUL34 beteiligt (Fuchs et al., 2002b; Klupp et al., 2006; Santarelli et al., 2008; Schnee et al., 2006). Während der Infektion mit HSV-1 kommt es unter Beteiligung des pUS3 und der zellulären Proteinkinase C zur Hyperphosphorylierung von Laminen (Lamin A/C und Lamin B1), die zur Auflockerung des Laminnetzwerkes führt (Mou et al., 2008; Park & Baines, 2006), was die Interaktion der Kapside mit der Kernmembran erleichtert (Mettenleiter et al., 2009). Nach der Knospung verlieren die primär umhüllten Kapside ihre Hülle durch Fusion mit der äußeren Kernmembran und gelangen als unbehüllte Kapside in das Zytoplasma. Der molekulare Mechanismus dieser zweiten Fusion (de-envelopment) ist bisher unklar (Mettenleiter, 2004). Der konservierten und nicht essentiellen pUS3 Kinase kommt bei diesem Vorgang eine besondere Bedeutung zu, da in deren Abwesenheit primär umhüllte Virionen im perinukleären Spalt akkumulieren (Klupp et al., 2001; Reynolds et al., 2002). Nach dem Durchtritt aus dem Kern in das Zytoplasma erhält das Kapsid seine endgültige Hülle durch einen zweiten Umhüllungsschritt (secondary envelopment) am Trans-Golgi-Netzwerk (Mettenleiter, 2006). Diese durch vielfältige Protein-Protein-Interaktionen vermittelten komplexen Schritte beinhalten die Anlagerung der Tegumentproteine an das Kapsid und die darauf folgende Knospung in glykoproteinhaltige Vesikel (Granzow et al., 1997; Mettenleiter et al., 2006; Whealy et al., 1991) des Trans-Golgi-Netzwerks. Die umhüllten Virionen werden in diesen Vesikeln zur Plasmamembran transportiert und anschließend durch Exozytose freigesetzt (Granzow et al., 2001; Mettenleiter, 2004). Über die Mechanismen dieses Prozesses ist bisher wenig bekannt, lediglich die Beteiligung der Proteine pUL20 und des Glykoproteins K konnte nachgewiesen werden (Baines et al., 1991; Dietz et al., 2000; Foster et al., 2008; Fuchs et al., 1997; Guggemoos et al., 2006; Klupp et al., 1998).

I.2.6 Die Expression herpesviraler Gene

Die Expression herpesviraler Gene erfolgt mit Hilfe der zellulären DNA-abhängigen RNA- Polymerase II in einer kaskadenartigen Regulation (Alwine et al., 1974; Costanzo et al., 1977; Feldman et al., 1979; Honess & Roizman, 1975), die durch virale Transkriptionsaktivatoren und negative Rückkoppelungsmechanismen gesteuert wird. Sie wird in eine sehr frühe (immediate-early, IE, α), eine frühe (early, E, β) und eine späte (late, L, γ) Phase unterteilt, wobei die späte Phase zusätzlich in die early-late und true-late Phasen eingeteilt wird (Honess & Roizman, 1974). Zirka 40 Minuten nach PrV-Infektion werden die beiden einzigen „immediate-early“ Gene, das IE180 (HSV-1 ICP4 (infected cell protein 4) homolog) und US1 (RSp40; HSV-1 ICP22 homolog), exprimiert, deren Transkription durch 6

Cycloheximid oder Puromycin gehemmt werden kann (Cheung, 1989; Fuchs et al., 2000). Für HSV-1 wurden sechs IE-Proteine beschrieben: ICP0 (Sacks & Schaffer, 1987), ICP4 (Preston, 1979), ICP22 (Post & Roizman, 1981), ICP27 (Sacks et al., 1985), ICP47 und pUS 1.5 (Ogle & Roizman, 1999). Die Expression der immediate-early Gene wird von dem viralen Transaktivator VP16 (virus protein 16) (α-TIF/pUL48), der im infizierenden Viruspartikel enthalten ist und unabhängig vom Kapsid in den Zellkern wandert, induziert und verstärkt (Fuchs et al., 2002a;Herr, 1998;Kwong & Frenkel, 1989;Wysocka & Herr, 2003). Das bis ca. 2,5 Stunden nach Infektion synthetisierte PrV IE180 Protein, bei HSV-1 die immediate-early Proteine ICP4, ICP0, ICP27, ICP22 und pUS1.5, fungieren als effiziente Transaktivatoren der early-Gene, die noch vor der viralen DNA Replikation (1 bis 4 Stunden nach PrV- Infektion) transkribiert und synthetisiert werden (Feldman et al., 1979). Die early-Gene kodieren für Enzyme, die am Nukleotidmetabolismus und an der DNA-Synthese beteiligt sind, so für die Thymidin Kinase (pUL23), die Uracil-DNA Glykosylase (UL2), die Ribonukleotid Reduktase (UL39/UL40), die Desoxyuridin-Triphosphatase (UL50), die DNA- Polymerase-assoziierten Faktoren (UL30/UL42), den Helikase-Primase-Komplex (UL5/UL8/UL52), das Einzelstrang-DNA-bindende Protein (UL29), das ori-bindende Protein (UL9), sowie für die beiden Proteinkinasen US3 und UL13 (Mettenleiter, 2000; Mettenleiter, 2008). Zusätzlich hemmen die early-Genprodukte die IE-Genexpression (Honess & Roizman, 1974). Die Replikation der viralen DNA erfolgt nach dem rolling circle Prinzip innerhalb von nukleären Replikationskompartimenten (Ben-Porat, 1981). Dabei werden mit Hilfe der essentiellen Replikationsproteine (pUL9, pUL29, pUL5, pUL8, pUL52, pUL30 und pUL42) zunächst Genomkonkatemere gebildet, die durch den viralen Terminasekomplex (pUL15/pUL28/UL32) an spezifischen Erkennungssignalen zu Genomeinheiten gespalten werden (Wilkinson & Weller, 2004), von denen jeweils eine Kopie in ein Kapsid eingebaut wird (Wu et al., 1986). Die Transkription der späten (true-late) Gene beginnt bereits 1,5 Stunden nach Infektion, aber die Synthese findet ihren Höhepunkt nach der DNA-Replikation (4-9 Stunden nach Infektion) und kann somit von Inhibitoren der DNA-abhängigen DNA- Polymerase wie Phosphonoessigsäure oder Acyclovir gehemmt werden. Zu den späten Genprodukten gehören im Wesentlichen virale Strukturproteine, die für den Aufbau der Kapside, des Teguments und der Glykoproteine der Membranhülle verantwortlich sind (Ben- Porat, 1981). Diese werden im Zytosol synthetisiert und in den Kern transportiert, wo Kapsidsynthese und DNA-Verpackung stattfinden (Newcomb et al., 1999). Die Einteilung in die Genexpressionsklassen immediate-early, early und late gilt nicht strikt. So kann die Expression einiger early-Gene durch Hemmung der viralen DNA-Synthese vermindert werden (Uprichard & Knipe, 1996), wohingegen einige late-Gene (z.B. UL27) früh exprimiert und nur minimal durch DNA-Inhibitoren gehemmt werden können (Rafield & Knipe, 1984).

I.2.7 Einfluss der Infektion mit Herpesviren auf die Expression und posttranslationale Modifikation zelleigener Proteine

Herpesviren kodieren für eine Vielzahl von Proteinen, die die Expression viraler aber auch zelluläre Proteine im Sinne einer effektiven Virusinfektion beeinflussen und damit zu Veränderungen des Proteoms im Verlauf einer Infektion beitragen. Hierzu zählen insbesondere Proteine, die in die Synthese und Prozessierung der Boten-RNA (mRNA) eingreifen, wie pUL41 und ICP27 und virale Kinasen wie pUS3 und pUL13.

pUL41 Das unter den Alphaherpesviren konservierte Tegumentprotein pUL41, auch als virion-host- shutoff-Protein (vhs) bezeichnet, ist an der Abschaltung der Synthese von Zellproteinen nach Infektion (shutoff) beteiligt. Es besitzt Endoribonukleaseaktivität und pUL41 von HSV-1 ähnelt in der Substratspezifität der zellulären Ribonuklease A (RNase A) (Elgadi et al., 1999; Lin et al., 2004; Taddeo et al., 2006; Taddeo & Roizman, 2006), wobei die RNase Aktivität des PrV vhs geringer als die des HSV-1-Proteins ist (Elgadi et al., 1999; Lin et al., 2004). Als Tegumentprotein gelangt es nach Infektion unmittelbar in das Zytoplasma, wo es im Falle des HSV-1 sofort (Kwong et al., 1988; Strom & Frenkel, 1987) und nach Infektion mit PrV mit einer zeitlichen Verzögerung (Ambagala et al., 2003) mit der Degradation der mRNA beginnt. Auf Grund der nur begrenzten Anzahl von Studien zum vhs des PrV, sollen hier die weiteren detaillierten Eigenschaften des HSV-1 pUL41 beschrieben werden. Die ribonukleolytische Aktivität des pUL41 ist auf mRNAs beschränkt und begrenzt sequenzspezifisch (Krikorian & Read, 1991; Kwong & Frenkel, 1987). So konnte gezeigt werden, dass bevorzugt mRNA mit AU-reichen Elementen (AREs) vom 3’- zum 5′-Ende abgebaut wird (Taddeo & Roizman, 2006). Trotz fehlender Spezifität für den Abbau zelleigener mRNA (Esclatine et al., 2004a; Esclatine et al., 2004b; Kwong & Frenkel, 1987; Oroskar & Read, 1989) kommt es aufgrund der schnelleren und vermehrten Transkription viraler mRNAs zur Akkumulation viraler Proteine (Taddeo et al., 2004). Interaktionen des pUL41 mit den zellulären Translations- Initiationsfaktoren eIF4A, elF4H, eIF4B und eIF4F (Doepker et al., 2004; Feng et al., 2001; Page & Read, 2010) deuten darauf hin, dass pUL41 in den Initiationsprozess bei der Proteinsynthese eingreift (Page & Read, 2010). Die RNase-Aktivität des pUL41 von HSV-1 wird zu späten Zeitpunkten der Infektion durch pUL48 (VP16) gehemmt (Lam et al., 1996; Smibert et al., 1994; Taddeo et al., 2010). Ob dies auch für das vhs des PrV gilt, ist unklar, denn PrV pUL41 fehlt die entsprechende Bindungsstelle für pUL48 (Berthomme et al., 1993).

ICP27 (pUL54)

Das ICP27 des HSV-1 ist ein essentielles, konserviertes und multifunktionelles IE-Protein. Im Gegensatz dazu ist das homologe pUL54 des PrV nicht essentiell und wird mit einer early Kinetik exprimiert (Baumeister et al., 1995; Huang & Wu, 2004; Schwartz et al., 2006). Beide Proteine können RNA binden und besitzen unter anderem das RNA-Bindungsmotiv RGG (Huang & Wu, 2004; Huang et al., 2005; Mears & Rice, 1996). Gut charakterisiert ist das ICP27 des HSV-1. Neben seinen Funktionen als Transaktivator und Transrepressor der viralen Genregulation (Jean et al., 2001; Rice & Knipe, 1988; Spencer et al., 1997) ist es vor allem bei der Polyadenylierung der prä-mRNA (Cheung et al., 2000; McGregor et al., 1996; McLauchlan et al., 1992), der Stabilisierung von labilen Transkripten (Brown et al., 1995) und dem Transport von viralen mRNAs aus dem Zellkern in das Zytosol beteiligt (Sandri-Goldin, 1998). Eine wesentliche Funktion des ICP27 ist die Hemmung der Synthese von zellulären Proteinen (host-cell-shutoff), der durch die Hemmung des Spleißosoms realisiert wird (Hardwicke & Sandri-Goldin, 1994; Hardy & Sandri-Goldin, 1994; Lindberg & Kreivi, 2002). Interaktionen mit dem spliceosome-associated protein 145 (SAP145) (Bryant et al., 2001) und einigen snRNPs (small nuclear ribonucleoproteins) (Phelan et al., 1993; Sandri-Goldin et al., 1995) wurden nachgewiesen. Für die Expression von PrV-Genen ist der Ausfall des Spleißapparates weniger relevant, da bis auf drei (US1 (Fuchs et al., 2000), „Large Latency Transcript” (LLT) (Cheung, 1991), und UL15 (Klupp et al., 2004)) alle viralen Gene intronlos sind, bei HSV-1 sind die Gene für ICP0, ICP4, ICP27 und pUL15 ebenfalls ohne Intron (Perry et al., 1986). Auch der Transport der intronlosen viralen mRNAs aus dem Kern in das Zytosol wird durch ICP27 reguliert (Chen et al., 2002; Phelan & Clements, 1997). Dazu interagiert es mit zellulären Transportfaktoren (nuclear RNA export factor 1 (Johnson & Sandri-Goldin, 2009) und dem poly(A)-bindenden zytoplasmatischen Protein 1 (Dobrikova et al., 2010)). Das ICP27 des HSV-1 stimuliert die Translation viraler Proteine durch Bindung von Komponenten des Translations-Initiationskomplexes (Eukaryotische Translations- Initiationsfaktoren (eIF) 3B, 3F und 4G1) (Fontaine-Rodriguez et al., 2004). Daneben wirkt es auf Signaltransduktionskaskaden wie den p38 MAP Kinase Weg (Gillis et al., 2009) und dem c-Jun N-terminal kinase (JNK) Weg (Hargett et al., 2005). Untersuchungen am pUL54 des PrV konnten die Lokalisation des Proteins im Kern infizierter Zellen bestätigen, wie sie auch für HSV-1 beschrieben wurde (Huang & Wu, 2004; Mears & Rice, 1998). In Abwesenheit des pUL54 von PrV kommt es nach Infektion zu einer verminderten Virusreplikation, die durch Komplementierung mittels des homologen ICP27 des HSV-1 aufgehoben werden kann (Schwartz et al., 2006). Weiterhin konnte mit einer pUL54 Deletionsmutante eine erhöhte Expression des gB, gE und des pUS9 gezeigt werden, wohingegen die Expression des gC vermindert und die des gK nicht mehr nachweisbar war (Schwartz et al., 2006).

Virale Kinasen

Die Phosphorylierung ist eine der häufigsten und bedeutendsten posttranslationalen Modifikationen (PTM). Durch Phosphorylierung und Dephosphorylierung von Proteinen kann die Zelle schnell und reversibel auf veränderte Bedingungen reagieren. So ist es nicht verwunderlich, dass auch Viren eigene Kinasen besitzen, mit denen sie den Zellstoffwechsel beeinflussen können. PrV kodiert insgesamt für vier Kinasen mit unterschiedlicher Substratspezifität, drei Serin/Threonin-Kinasen (pUS3, pUL13, pUL39) und eine Thymidinkinase (pUL23) (de Wind et al., 1992; de Wind et al., 1994; Katan et al., 1985; Klupp et al., 2004; Purves et al., 1987; Van Minnebruggen et al., 2003; van Zijl et al., 1990; Zhang et al., 1990). Die Serine/Threonin-Kinasen sind innerhalb der Alphaherpesvirinae konserviert, aber nicht essentiell (de Wind et al., 1992; de Wind et al., 1994; Purves et al., 1987; Smith & Smith, 1989; Van Minnebruggen et al., 2003; van Zijl et al., 1990; Zhang et al., 1990). Das pUS3 von PrV ist ein Tegumentprotein und wird aufgrund zweier unterschiedlicher Transkriptionsstarts in zwei unterschiedlich großen Isoformen exprimiert (van Zijl et al., 1990), einer 53 kDa großen und einer N-terminalen verkürzten 41 kDa kleinen Form (Van Minnebruggen et al., 2003; van Zijl et al., 1990). Die US3-Proteine des PrV und des HSV-1 sind hochgradig multifunktionell (Deruelle & Favoreel, 2010). Genannt seien hier pUS3-abhängige Veränderungen des Zytoskeletts, die zum Abbau von Stressfasern oder zur Bildung von aktinhaltigen Zellprojektionen führen (Favoreel et al., 2005; Murata et al., 2002; Van den Broeke et al., 2009a; Van Minnebruggen et al., 2003), Phosphorylierungen von Bestandteilen der Kernlamina (Lamin A/C, B1, Emerin (Leach et al., 2007; Mou et al., 2007; Mou et al., 2008)) durch das pUS3 des HSV-1, die Regulation der Genaktivität über die Beeinflussung der Histon-Deacetylase 2 durch pUS3 (PrV) (Walters et al., 2010), sowie zusätzlich der Histon-Deacetylase 1 (Poon et al., 2006) durch pUS3 (HSV-1), sowie die Modulation von zellulären Signaltransduktionswegen (p21-activated kinase (Van den Broeke et al., 2009b)) nach PrV-Infektion. Die anti-apoptotische Wirkung des pUS3 wird durch die pUS3-abhängige Phosphorylierung von Effektoren der Apoptose wie BAD (Bcl-2-Antagonist of Cell Death) (Benetti et al., 2003; Kato et al., 2005; Munger & Roizman, 2001),(Deruelle et al., 2007), Bid (BH3 interacting domain death agonist) (Cartier et al., 2003) oder Procaspase 3 (Benetti & Roizman, 2007) vermittelt. Transkriptom-Studien nach Infektion mit US3- negativen Mutanten des HSV-1 und HSV-2 belegen die US3-abhängige Beeinflussung weiterer zellulärer Funktionen wie Immunantwort oder Zellzykluskontrolle (Kamakura et al., 2008). Die Akkumulation primär umhüllter Virionen im Kernspalt nach Infektion mit US3-negativen Mutanten des PrV und HSV-1 weisen auf eine Funktion des pUS3 während der primären Umhüllung hin (Klupp et al., 2001; Reynolds et al., 2002). Das essentielle Membranprotein pUL34 ist ein Substrat des HSV-1 pUS3 (Purves et al., 1991; Purves et al., 1992; Ryckman 10

& Roller, 2004), allerdings ist die Phosphorylierung von PrV pUL34 nicht pUS3-abhängig (Klupp et al., 2001). Als weitere virale Substrate des pUS3 von HSV-1 konnten durch in-vitro Phosphorylierungsstudien pUS1 (ICP22) (Kato et al., 2005), pUS9 (Kato et al., 2005), pUL31 (Mou et al., 2009), gB (Kato et al., 2009) sowie für pUS3 von HSV-2 das pUL12 (Daikoku et al., 1995) identifiziert werden. Darüber hinaus konnte in Zellkultur eine pUS3-abhängige Phosphorylierung des pUL31 (Mou et al., 2009) und des gB (Kato et al., 2009) sowie eine Autophosphorylierung des pUS3 von HSV-1 (Kato et al., 2008;Sagou et al., 2009) nachgewiesen werden. Die pUL13-Kinase ist ein bei allen Herpesviridae konserviertes nicht essentielles Tegumentprotein (de Wind et al., 1992; Purves & Roizman, 1992). Neben den viralen HSV-1 Substraten pUS3 (Kato et al., 2006), ICP22 (Purves et al., 1993), ICP0 (Ogle et al., 1997) den Glykoproteinen gE/gI (Ng et al., 1998), dem pUL49 (VP22) (Asai et al., 2007; Coulter et al., 1993) und dem pUL41 (Asai et al., 2007; Overton et al., 1994), sind bisher pUL13-abhängige Phosphorylierungen bei nur wenigen zellulären Proteinen beschrieben worden, obwohl sich die Substratspezifitäten des pUL13 und der zellulären Protein Kinase 2 (cdc2) zum Teil überschneiden (Advani et al., 2000; Kawaguchi & Kato, 2003). So konnte bisher nur die pUL13-abhängige Phosphorylierung des Translations- Elongationsfaktors-1-Delta (Kawaguchi et al., 1998) und, ähnliche wie für pUS3 beschrieben, von Lamin A/C und Lamin B1 (Cano-Monreal et al., 2009) in HSV-1-infizierten Zellen nachgewiesen werden.

Ubiquitinylierung Neben der Phosphorylierung spielt die Ubiquitinylierung als reversible posttranslationale Modifikation eine wichtige Rolle in der Zellphysiologie. Sie ist am Abbau der Proteine (protein turnover), Membrantransport und an der Regulation der Transkription beteiligt. Im Tegumentprotein pUL36 wurde eine innerhalb der Familie der Herpesviridae konservierte Deubiquitinylierungsdomäne nachgewiesen (Bottcher et al., 2006; Kattenhorn et al., 2005; Schlieker et al., 2005). Die biologische Bedeutung einer herpesviralen Deubiquitinylierungsaktivät im pUL36, die bisher für die pUL36 von HSV-1, Marek´s disease virus, Murinem sowie Humanem Cytomegalovirus, Epstein-Barr-Virus und Murinem Gammaherpesvirus 68 belegt worden ist, ist bisher noch ungeklärt (Gredmark et al., 2007; Jarosinski et al., 2007; Kattenhorn et al., 2005; Schlieker et al., 2005; Wang et al., 2006). Die Deletion der Domäne im PrV führt zu einer unvollständigen Virusmorphogenese, die durch Akkumulationen von nicht umhüllten Nukleokapsiden im Zytoplasma gekennzeichnet ist (Bottcher et al., 2008). Neben dem pUL36 scheinen auch ICP0 des HSV-1 und EP0 des PrV am Ubiquitin-Proteasom-Pfad als eine Ubiquitin-Ligase beteiligt zu sein (Boutell et al., 2002; Everett et al., 2010).

I.3 Das Newcastle Disease Virus I.3.1 Taxonomische Einordnung und Pathogenese

Das Newcastle Disease Virus (NDV), auch als Aviäres Paramyxovirus Serotyp 1 (APMV-1) bezeichnet, gehört dem Genus Avulavirus in der Unterfamilie Paramyxovirinae an. Die Paramyxovirinae werden der Familie der Paramyxoviridae innerhalb der Ordnung Mononegavirales zugeordnet (Fauquet & Fargette, 2005; Mayo, 2002). Die ersten Ausbrüche der Krankheit, die als atypische Geflügelpest bezeichnet wird, wurden zeitgleich im Jahr 1926 in der Nähe von Newcastle (England) (Doyle, 1927) und auf Java in Indonesien (Kraneveld, 1926) beschrieben. NDV verursacht weltweit schwere systemische Erkrankungen in allen bisher untersuchten Vogelspezies (Alexander, 1997; Kaleta & Baldauf, 1988), vor allem aber in Puten- und Hühnerbeständen (Alexander, 1997; Alexander & Allan, 1974), wobei die Letalitätsrate 100 Prozent erreichen kann. Durch seine hohe Kontagiosität verbreitet sich das Virus schnell und die Symptome treten je nach Virusstamm, Wirtsspezies und Immunstatus des Wirtes unterschiedlich intensiv und in verschiedenen Kombinationen auf (Piacenti et al., 2006). Aus diesem Grund werden die NDV-Stämme in Abhängigkeit von der Schwere der Krankheit, die sie bei Hühnern hervorrufen, anhand des intrazerebralen Pathogenitätsindex (ICPI) in die Pathotypen lentogen (schwach virulent), mesogen (mittlere Virulenz) und velogen (hoch virulent) eingeteilt (Alexander & Allan, 1974). Die NDV-Infektion erfolgt über die Schleimhäute des respiratorischen Systems und des Legeapparates mit einer anschließenden Verbreitung in das Zentralnervensystem, die Lunge und den Darm. Die Symptome der Krankheit sind vielfältig und reichen von drastischem Rückgang der Legeleistung, Atemnot, Konjunktivitis, Durchfall, Ödeme an Kopf und Kopfanhängen bis zu Apathie und neurologischen Symptomen (Alexander & Allan, 1974; Hanson, 1974).

I.3.2 Morphologie und Genomorganisation des NDV

Das Newcastle Disease Virus besitzt eine Größe von 150-200 nm, ist von pleomorpher Gestalt und wird von einer Lipidhülle zellulären Ursprungs umgeben, in der die beiden viralen Transmembran-Glykoproteine, das Fusionsprotein (F) und das Hämagglutinin- Neuraminidase-Protein (HN), eingebettet sind. Diese beiden Oberflächenglykoproteine sind für die Virus-Zell-Fusion verantwortlich und stellen darüber hinaus die wichtigsten Antigene für eine schützende Immunantwort dar (Alexander, 1997). An der Innenseite der Membranhülle ist das Matrixprotein (M) zur Stabilisierung der Virushülle lokalisiert. Die virale RNA bildet zusammen mit dem Nukleoprotein (NP) und der RNA-abhängigen RNA- Polymerase („Large-Protein“, L), die mit dem Phosphoprotein (P) assoziiert ist, den Ribonukleoproteinkomplex (RNP), der die kleinste infektiöse Einheit darstellt. Das Genom liegt als nicht segmentierte, einzelsträngige RNA negativer Polarität (Lamb und Kolakofsky 12

1974) von ca. 15 kb Länge vor (de Leeuw & Peeters, 1999). Aufgrund seiner negativen Polarität kann das Genom nicht als mRNA dienen und ist somit auch nicht infektiös. Am 3’- Ende des Genoms besitzt das NDV eine 55 Nukleotide lange, nicht gecappte und nicht polyadenylierte Leadersequenz und am 5’-Ende eine 114 lange Trailersequenz. Das virale Genom kodiert für sechs Proteine. In 3'- 5'-Richtung sind dies das Nukleoprotein (55 kDa), das Phosphoprotein (53 kDa), das Matrixprotein (40 kDa), das Fusionsprotein (67 kDa), das Hämagglutinin-Neuraminidase-Protein (74 kDa) und die RNA-abhängige RNA-Polymerase (200 kDa) (Abb. 2). Zwischen den einzelnen offenen Leserahmen befinden sich nicht kodierende (intergenische) Sequenzen, die eine Länge zwischen einem und 47 Nukleotiden aufweisen (de Leeuw & Peeters, 1999; Krishnamurthy & Samal, 1998). Zusätzlich befinden sich virusspezifische Genstart- oder Genstop-Sequenzen neben den nicht kodierenden Bereichen. Neben dem Phosphoprotein werden noch die Proteine V und W von dem P-Gen kodiert. Diese werden nach RNA-Editing durch Einfügen einer bzw. zweier zusätzlicher Guanosinnukleotide in die mRNA translatiert (Steward et al., 1993).

Abb. 2: Aufbau und Genomorganisation des Newcastle Disease Virus Partikels

Der Aufbau eines NDV-Partikels ist anhand einer elektronenmikroskopischen Aufnahme (rechts und links) und einer schematischen Übersicht (Mitte) dargestellt (Dr. H. Granzow, M. Jörn).

Das untere Schema zeigt die Genomorganisation und den mRNA-Transkriptionsgradienten.

I.3.3 Die Transkription der NDV-Gene

Nach Infektion der Wirtszelle erfolgt die Transkription der subgenomischen viralen mRNAs durch den aus dem L- und P-Protein bestehenden Polymerase-Komplex im Zytoplasma. Dazu wird am 3’-Terminus des Genoms eine kurze zur Leadersequenz komplementäre RNA synthetisiert, die als Promotor und als einziger Bindungsort für die RNA-abhängige RNA- Polymerase dient. Während der RNA-Synthese werden die Virusgene sukzessive in einem Stop-Start Mechanismus vom 3‘-Terminus zum 5‘-Terminus abgelesen. Die dabei entstehenden monocistronischen RNAs werden am 3’-Ende polyadenyliert und am 5’-Ende mit einem 7-Methylguanosin-Rest (Cap) versehen. Während der Transkription pausiert die Polymerase an den Genstop-Signalen kurz vor den nicht zu transkribierenden intergenischen Sequenzen, um eine Oligo-U-Sequenz in einen Poly-A-Schwanz umzuschreiben und beginnt nach dem Überspringen der intergenischen Sequenzen mit der weiteren Transkription am nächstgelegenen Genstart des weiter abwärts liegenden kodierenden Gens. Dabei kann der Polymerasekomplex gelegentlich von der Matrize dissoziieren. Dies führt dazu, dass die Menge der synthetisierten mRNAs zum 5‘-Terminus kontinuierlich abnimmt und der für die Vertreter der nichtsegmentierten Negativstrang-RNA-Viren (Mononegavirales) typische Transkriptionsgradient entsteht (Abb. 2). Ein Transkriptionsgradient ist außer beim NDV (Collins et al., 1978; Collins et al., 1980) für das Vesicular Stomatitis Virus (VSV) (Abraham & Banerjee, 1976; Iverson & Rose, 1981; Villarreal et al., 1976), Borna Disease Virus (Briese et al., 1994), das Humane Respiratorische Synzytial Virus (Collins & Wertz, 1983), Sendai Virus (Glazier et al., 1977; Homann et al., 1990), Masernvirus (Cattaneo et al., 1987a; Cattaneo et al., 1987b), das Hendra Virus (Wright et al., 2005) und einigen weiteren durch Quantifizierung der mRNA nachgewiesen worden. Der Transkriptionsgradient muss nicht linear verlaufen. Iverson und Rose zeigten für VSV, dass die Expressionsrate in Richtung 3’- Terminus um 29- bis 33-Prozent pro Gen sinkt (Iverson & Rose, 1981). Die Transkriptmenge korrelierten dabei sehr gut mit den entsprechenden Proteinmengen (Villarreal et al., 1976). Bestätigt wurden diese Ergebnisse durch Studien mit einem an verschiedenen Stellen des VSV-Genoms eingebauten N-Gen (Wertz et al., 1998). Im Gegensatz dazu zeigten quantitative mRNA Analysen von Viren der Familie der Paramyxoviridae, wie dem Sendai Virus (Homann et al., 1990), Masern Virus (Cattaneo et al., 1987b) und dem Hendra Virus (Wright et al., 2005) keinen linearen Transkriptionsgradienten.

I.3.4 NDV als viraler Vektor für rekombinante Impfstoffe

Mit Hilfe der reversen Genetik können Fremdgene in rekombinanten NDV exprimiert werden (Krishnamurthy et al., 2000; Peeters et al., 1999; Romer-Oberdorfer et al., 1999), was zur Entwicklung bivalenter Impfstoffe genutzt wird. Einige Charakteristika des NDV machen es als Vektor für Impfstoffe interessant. So infiziert NDV eine Vielzahl von Zellen, löst eine starke zelluläre Immunantwort in vivo aus und repliziert ausschließlich im Zytoplasma ohne DNA Zwischenprodukte zu bilden, wodurch die Vakzine eine besondere biologische Sicherheit bietet, denn das Problem des Einbaus von viraler DNA in das Wirtszellgenom entfällt (Huang et al., 2003). Im Veterinärbereich wurde die Eignung von NDV als Vektor für die Expression von Proteinen anderer viraler Krankheitserreger bereits belegt, so z.B. für das Glykoprotein D des Bovinen Herpesvirus 1 (Khattar et al., 2010), das Glykoprotein Gn des Rifttalfieber Virus (Rift Valley Fever Virus) (Kortekaas et al., 2010b; Kortekaas et al., 2010a), das Gruppenspezifische Antigen (Gag) des Immundefizienz-Virus des Affen (Nakaya et al., 2004), das Virus Protein 2 (VP2) des Virus der Infektiösen Bursitis (Huang et al., 2004), das Hämagglutinin des Masernvirus (Kim et al., 2010) und der Hämagglutinin-Neuraminidase (HN) des Aviären Paramyxovirus Typ 4 (Peeters et al., 2001). Darüber hinaus wurden auch Hämagglutinin-Gene (HA) verschiedener Influenzaviren unterschiedlicher Subtypen an verschiedenen Orten des NDV-Genoms inseriert, um bivalente Impfstoffe gegen NDV und das Aviäre Influenza Virus (AIV) zu entwickeln. AIV gehört zum Genus Influenza A Virus in der Familie der Orthomyxoviridae. Es besitzt ein in 8 Segmente unterteiltes Negativstrang- RNA Genom, das für 11 Proteine kodiert. Auf Grund antigener Unterschiede der beiden Oberflächenglykoproteine, Hämagglutinin (HA) und Neuraminidase (NA) werden diese in 16 verschiedene HA (H1-H16) und 9 verschiedene NA (N1-N9) Subtypen unterteilt. Die Symptome der durch das AIV im Nutzgeflügel ausgelösten Erkrankung sind in Abhängigkeit vom Virusstamm sehr variabel und reichen von klinisch inapparent über verminderte Legeleistung bis zu schweren Erkrankungen mit bis zu 100-prozentiger Mortalität. Eine weitere Unterteilung der AIV-Stämme erfolgt darum nach verschiedenen Pathotypen, den niedrig pathogen (LP) und hoch pathogen (HP), wobei letztere nur bei den Subtypen H5 und H7 auftreten. Rekombinante NDV, in denen das AIV-HA der Subtypen H1N1 (Nakaya et al., 2001), H5N1 (DiNapoli et al., 2007b; DiNapoli et al., 2010; Ge et al., 2007; Nayak et al., 2009), H7N7 (Park et al., 2006), H7N2 (Swayne et al., 2003), H5N2 (Lozano-Dubernard et al., 2010; Sarfati-Mizrahi et al., 2010) und H9N2 (Ge et al., 2010) zwischen den NDV-Genen P und M inseriert wurde, liegen vor. Aber auch Insertionen zwischen den NDV-Genen F und HN sind für HA der Subtypen H5N2 (Romer-Oberdorfer et al., 2008; Veits et al., 2006) und H7N1 (Schroer et al., 2009) beschrieben. Die Expression der AIV-Gene in den NDV- Rekombinanten wurde in der Regel nach Northern Blot Analyse der Transkripte und nach Western Blot oder Immunfluoreszenzanalyse der Proteine beurteilt. Dabei zeigten alle 15

Rekombinanten nach einmaliger Verabreichung eine hohe Schutzwirkung gegen eine NDV- und AIV-Infektion im Tier. Mit einem Schutz von lediglich 40-Prozent, traf dies nur auf eine Mutante nicht zu, die ein Hämagglutinin des Typs H7 aus einem H7N2-Isolats exprimierte (Swayne et al., 2003). Daneben wurden auch NDV-Vektorimpfstoffe mit Insertionen aus humanpathogenen Viren, wie z.B. dem Humanen Respiratorischen Synzytial-Virus (Fusion Glykoprotein) (Martinez-Sobrido et al., 2006), dem Humanen Immundefizienz-Virus (Gag) (Carnero et al., 2009), dem Humanen Parainfluenza Virus Typ 3 (HN) (Bukreyev et al., 2005) und dem Schweren Akuten Respiratorischen Syndrom-assoziiertem Coronavirus (S Glykoprotein) (DiNapoli et al., 2007a) beschrieben. Erfolgversprechend scheint in diesem Zusammenhang auch eine mögliche Anwendung von rekombinanten NDV-Vektoren in der humanen Tumortherapie, bei der das Virus selektiv die Tumorzellen infiziert und therapeutisch wirksame Fremdproteine wie z.B. den Granulozyt/Makrophagen Kolonie- stimulierenden Faktor (Janke et al., 2007), Interleukin-2 (Vigil et al., 2007) oder tumorverwandte Antikörper (Puhler et al., 2008) exprimiert.

I.4 Proteomanalyse Das Proteom bezeichnet die Proteinausstattung einer Zelle zu einem bestimmten Zeitpunkt unter definierten Bedingungen (Wilkins et al., 1996). Seine Zusammensetzung hängt von vielen internen Parametern, wie zum Beispiel der Wachstums- oder Entwicklungsphase der Zelle ab, aber auch von externen Faktoren wie der Temperatur, den osmotischen Verhältnissen, der Exposition gegenüber Fremdstoffen oder anderen physiologisch wirksamen Substanzen, und schließlich auch von einer möglichen Virusinfektion. Im Gegensatz zum statischen Genom ist das Proteom qualitativ und quantitativ äußerst dynamisch. So unterliegen die Biosynthese der Proteine, und deren ko- und posttranskriptionale Modifikation einer intensiven Kontrolle, die sich auf die Zusammensetzung des Proteoms auswirkt. Aufgrund der hohen Komplexität der Probe und deren extremer Dynamik, die je nach Gewebetyp Konzentrationsunterschiede über bis zu 12 Zehnerpotenzen umfassen kann (Corthals et al., 2000), stellen Proteomuntersuchungen hohe Anforderungen an die verwendeten analytischen Verfahren, zumal die möglichst vollständige Erfassung des gesamten Proteoms das, wenn auch kaum zu erreichende, Ziel einer erfolgversprechenden Proteomstudie sein muss. Die Grundlage der massenanalytischen Identifizierung von Proteinproben ist die möglichst vollständige Verfügbarkeit der Genomsequenz der zu untersuchenden Spezies. Einen klassischen Zugang zur Proteomanalyse stellt die zweidimensionale Gelelektrophorese (2DE) (Klose, 1975; O'Farrell, 1975) mit immobilisierten pH-Gradienten (Gorg et al., 1983; Westermeier et al., 1983) und anschließender massenspektrometrischer Proteinidentifizierung, vorzugsweise 16

durch MALDI-Peptidmassenfingerabdruck (peptide mass fingerprint, PMF) (Pappin et al., 1993; Perkins et al., 1999), dar. Bei der zweidimensionalen Gelelektrophorese werden die Proteine entsprechend ihres isoelektrischen Punktes und ihrer Masse durch sequenzielle isoelektrischer Fokussierung und SDS-Polyacrylamid-Gelelektrophorese getrennt. Dieses leistungsfähige orthogonale Trennverfahren erlaubt sowohl die mikropräparative Darstellung verschiedener Proteine, als auch die Analyse von posttranslationalen Modifikationen eines bestimmten Proteins, wenn sie sich in ihrer Masse oder Ladung oder beidem unterscheiden. Die 2DE leidet aber auch unter einigen technischen Einschränkungen. So ist die Analytmenge beschränkt und die Präparation von sehr großen, sehr kleinen, sehr basischen oder sehr sauren Proteinen erweist sich als problematisch. Abundanz-Unterschiede schränken den dynamischen Bereich bei der Detektion ein. Aus diesen und anderen Gründen erfreuen sich gelfreie Methoden steigender Beliebtheit und ersetzen die 2DE zunehmend. Allerdings gibt es auch Möglichkeiten, die Einschränkungen der 2DE zu umgehen. So kann eine Probe parallel auf mehreren Gelen mit unterschiedlichem und vorzugsweise engerem pH-Bereich analysiert werden, oder eine komplexe Probe kann vor der 2DE in besser analysierbare Teilproben vorfraktioniert werden (Righetti et al., 2005). Auch eine Vorfraktionierung durch präparative isoelektrische Fokussierung vor der konventionellen mikropräparativen 2DE wurden beschrieben (Gorg et al., 1995). Weitere Ansätze zur Vorfraktionierung sind die Zellfraktionierung (Fountoulakis et al., 2002; Fountoulakis & Takacs, 2002), die Affinitätschromatographie (Azarkan et al., 2007), oder die gruppenspezifische Markierung oder Färbung von strukturell definierten Subproteomen, wie den Phosphoproteinen oder den Glykoproteinen (Patton, 2002; Steinberg et al., 2003).

I.4.1 Quantitative Proteomics

Eukaryotische Zellen reagieren auf verschiedenen Ebenen auf Umweltreize, von der Geninduktion über die Prozessierung der mRNA bis hin zu posttranslationalen Modifizierungen (PTM), von denen die Phosphorylierung das markanteste Beispiel ist. Für eine Proteomstudie an eukaryotischen Zellen bedarf es daher einer Analysentechnik, die Variationen in den PTM erfasst und eine einfache und genaue Quantifizierung erlaubt, da sich Proteinprofile eukaryotischer Zellen oft weniger qualitativ als quantitativ unterscheiden. Zur Durchführung von quantitativen Proteomstudien stehen eine Reihe von Analyseverfahren bzw. -plattformen zur Verfügung. Grundsätzlich ist eine Quantifizierung einzelner Proteine während der Analyse, also z.B. während einer chromatographischen oder nach einer elektrophoretischen Trennung, oder bei der späteren massenspektrometrischen Identifizierung des analysierten Proteins oder Peptids möglich. Aufgrund der generell eher mäßigen Reproduzierbarkeit von 2DE Gelen haben sich hier vor allem solche Verfahren 17

durchgesetzt, die einen globalen internen Standard verwenden, der vor der 2DE der Probe zugesetzt wird. Zu nennen wäre hier vor allem die 2D-DIGE-Technik (difference in-gel electrophoresis) (Shaw et al., 2003; Unlu et al., 1997). Dabei werden Proteinextrakte der zu vergleichenden Zellen durch kovalente Kopplung an verschiedenfarbige Fluoreszenzfarbstoffe markiert und auf einem einzigen Gel getrennt. Eine dritte Probe, die aus den zu vergleichenden Extrakten besteht und mit einem weiteren Farbstoff markiert ist, dient als Referenz. Scans des resultierenden Gels werden unter Bedingungen, die für die einzelnen Farbstoffe selektiv sind, erstellt und die relativen Mengen der verschiedenen Proteine als Fluoreszenzintensitätsverhältnisse angezeigt. Das Verfahren ist zum Vergleich von verschiedenen Zellzuständen sehr gut geeignet. Durch die späte Einführung der probenspezifischen Markierung im Analysengang ist sie aber eventuell gegen Artefakte bei der Extraktion, Fraktionierung oder der Markierung selbst anfällig. In dieser Hinsicht ist das von Ong und Kollegen (Ong et al., 2002) eingeführte Verfahren des „stable isotope labelling by amino acids in cell culture“ (SILAC) ideal. Hier wird die probenspezifische Markierung als Massendifferenz durch stabil isotopenmarkierte essentielle Aminosäuren vor dem eigentlichen Versuch in die Zellkultur und damit in alle Proteine der Referenz, oder der Probe metabolisch eingeführt. Nach der Infektion (oder anderweitiger Belastung bzw. Exposition) der Probenzellkultur können Probe und Referenz vereinigt werden und der globale interne Standard ist hergestellt. Alle folgenden Analysen wie Vorfraktionierungen oder elektrophoretische Trennungen sind somit bereits standardisiert und beeinflussen die spätere massenspektrometrische Quantifizierung nicht. Nachteilig ist, dass alle Proben einer massenspektrometrischen Analyse zugeführt werden müssen, was z.B. beim DIGE- Verfahren nur bei noch nicht identifizierten Proteinen notwendig ist. Für die vorliegenden Untersuchungen an PrV-infizierten Zellen wurde dennoch das SILAC-Verfahren gewählt, da zur Erstellung der angestrebten 2D-elektrophoretischen Kartierung des Proteoms der verwendeten Rindernierenzelllinie MDBK (Madin & Darby, 1958) alle isolierbaren Proteine ohnehin identifiziert werden sollten. In den letzten Jahren wurden auch Varianten der SILAC- Methode entwickelt, um gewisse Einschränkungen des Verfahrens zu überwinden. So sind inzwischen auch Hefen (Gruhler et al., 2005a), Pflanzen (Gruhler et al., 2005b) und sogar ganze Tiere wie Mäuse (Kruger et al., 2008) und Fliegen (Sury et al., 2010) metabolisch markiert und dem Verfahren zugänglich gemacht worden. Daneben kann SILAC als dynamic oder pulsed SILAC zur Quantifizierung der de-novo Proteinsynthese eingesetzt werden (Doherty et al., 2009; Pratt et al., 2002). Weite Verbreitung findet es darüber zur Quantifizierung von posttranslationalen Modifikationen wie z.B. der Phosphorylierung (Ibarrola et al., 2003) oder Methylierung (Ong et al., 2004), sowie bei Protein-Protein (Blagoev et al., 2003), Protein-DNA (Mittler et al., 2003) oder Protein-RNA (Butter et al., 2009) Interaktionen. Multiplexmarkierungen mit unterschiedlichen Markermassen erlauben in

gewissem Maß auch den Vergleich mehrerer Zustände in einer Probe in einer einzigen Messung (Molina et al., 2009).

Abb. 3 Vergleichende Darstellung zur Generierung eines globalen inneren Standards durch quantitative Massenmarkierung.

Die schematische Darstellung stellt die zu vergleichenden Proben (blau und gelb) und die Zeitpunkte ihrer Vereinigung während des Analysenganges dar. Gestrichelte Linien zeigen den Teil des Experiments an, in dem Probe und Referenz einzeln, also ohne internen Standard, prozessiert werden. Das Schema verdeutlicht, dass das SILAC-Verfahren mit der metabolischen Markierung und der Probenvereinigung zum frühest möglichen Zeitpunkt am robustesten gegen Analyseartefakte ist (Bantscheff et al., 2007).

I.4.2 Proteomanalysen von Viruspartikeln

Proteomanalysen von Herpesvirus Partikeln liegen für die Alphaherpesviren Pseudorabiesvirus (PrV) und Herpes Simplex Virus Typ-1 (HSV-1) (Loret et al., 2008; Michael et al., 2006b; Michael et al., 2006a; Michael et al., 2007; Padula et al., 2009), die Betaherpesviren HCMV und MCMV (Baldick, Jr. & Shenk, 1996; Kattenhorn et al., 2004; Varnum et al., 2004) und die Gammaherpesviren Epstein-Barr-Virus (Johannsen et al., 2004), Kaposi-Sarkom-assoziierten Herpesvirus (KSHV; HHV-8) (Bechtel et al., 2005; Zhu et al., 2005), Rhesusaffen Rhadinovirus (O'Connor & Kedes, 2006), sowie murines Gammaherpesvirus 68 (Bortz et al., 2003) vor. Neben den erwarteten viralen Strukturproteinen wurde auch der Einbau zahlreicher zellulärer Proteine in Herpesvirionen beobachtet (Bechtel et al., 2005; del Rio et al., 2005; Johannsen et al., 2004; Kattenhorn et 19

al., 2004; Loret et al., 2008; Michael et al., 2006b; Varnum et al., 2004). Bei diesen Proteinen zellulären Ursprungs handelt es sich oft um strukturbildende Komponenten wie Aktin (Bechtel et al., 2005; del Rio et al., 2005; Johannsen et al., 2004; Kattenhorn et al., 2004; Loret et al., 2008; Michael et al., 2006b; Varnum et al., 2004), Annexin (Kattenhorn et al., 2004; Loret et al., 2008; Michael et al., 2006b; Varnum et al., 2004), Tubulin (Bechtel et al., 2005; Johannsen et al., 2004; Varnum et al., 2004), Cofilin (Johannsen et al., 2004; Kattenhorn et al., 2004; Loret et al., 2008; Varnum et al., 2004), Myosin (Varnum et al., 2004), aber auch Proteine des Vesikeltransports (Clathrin) (Varnum et al., 2004), und Hitzeschockproteine (Hsp70 (Bechtel et al., 2005; Johannsen et al., 2004; Loret et al., 2008; Michael et al., 2006b; Varnum et al., 2004), Hsp90 (Bechtel et al., 2005; Johannsen et al., 2004; Varnum et al., 2004)), die für die Stabilisierung und korrekte Faltung von Proteinen verantwortlich sein könnten. In quantitativen Proteomstudien von PrV-Deletionsmutanten (Michael et al., 2006b; Michael et al., 2006a; Michael et al., 2007) konnten Wechselwirkungen zwischen den einzelnen Strukturkomponenten nachgewiesen werden.

I.4.3 Proteomanalysen und Expressionsstudien mit PrV-infizierten Zellen

Expressionsstudien an Herpesvirus-infizierten Zellen liegen vor allem als Transkriptanalysen in Form von Mikroarraystudien vor. Neben Studien an humanpathogenen Vertretern wie dem HSV-1 (Kamakura et al., 2008; Khodarev et al., 1999; Mossman et al., 2001; Paludan et al., 2002; Pasieka et al., 2006; Ray & Enquist, 2004; Taddeo et al., 2002), dem HCMV (Browne et al., 2001) und dem VZV (Jones & Arvin, 2003) wurden auch PrV-infizierte Zellen untersucht (Blanchard et al., 2006; Brukman & Enquist, 2006; Flori et al., 2008; Hsiang et al., 1996; Paulus et al., 2006; Ray & Enquist, 2004; Yuan et al., 2009). Allerdings ist bekannt, dass Transkriptom- und Proteomdaten nicht immer korrelieren (Gygi et al., 1999), sodass zur vollständigen funktionellen Charakterisierung infizierter Zellen Proteomstudien erforderlich sind. Dies gilt umso mehr, als posttranslationale Modifikationen wie Ubiquitinylierung (Shackelford & Pagano, 2005), Phosphorylierung, oder Glykosylierung von zellulären Proteinen durch Transkriptanalysen nicht erfasst werden, aber eine erhebliche Rolle spielen können. Aufgrund des höheren Aufwandes bei der Proteomanalyse liegen Daten von virusinfizierten Zellen nur in begrenzter Zahl und vorzugsweise für humanpathogene Erreger vor (Tabelle 1). Quantitative Resultate sind bis heute lediglich in 27 Studien erhalten worden, wovon neun mit der SILAC-Technik, neun mit DIGE, drei mit ICAT (isotope-coded affinity tag), zwei mit iTRAQ (isobaric tag for relative and absolute quantitation), drei mit 18O/16O Markierungen und vier als markierungsfreie (label free) Quantifizierung durchgeführt worden sind (Literatur siehe Tabelle 1). In zwei Studien wurden verschiedene Quantifizierungsmethoden verglichen (DIGE/ICAT bzw. ICAT/18O/markierungsfreie 20

Quantifizierung) (Go et al., 2006; Jiang et al., 2005). Umfang und Analysentiefe der vorliegenden Studien unterscheiden sich beträchtlich, entsprechend heterogen sind die Ergebnisse. So wurden beispielsweise in Studien mit dem Humanen Immundefizienz-Virus Typ-1 (HIV-1) in einer Studie mit 1D-Gelelektrophorese/SELDI-MS (surface-enhanced laser desorption/ionization- Massenspektrometrie) lediglich 33 Proteine identifiziert (Toro-Nieves et al., 2009), mit 2D-Gelelektrophorese/LC-MS (Flüssigkeitschromatographie- Massenspektrometrie-Kopplung)/MALDI-TOF-MS (matrix assisted laser desorption/ionisation time-of-flight- Massenspektrometrie) ergaben sich 1275 identifizierte Proteine (Zhang et al., 2010b), wohingegen die Untersuchung unter Verwendung einer LC-MS-Kopplung 3255 Proteine identifizierte Proteine ergab (Chan et al., 2007). Analysen mit einer vorgeschalteten Vorfraktionierung zeigten durchweg eine höhere Anzahl von identifizierten Proteinen gegenüber Studien mit Rohextrakten. Eine Studie mit SARS-Coronavirus infizierten Zellen zeigte auch eine Abhängigkeit der Ergebnisse von der verwendeten analytischen Plattform: Quantifizierung durch 2D-DIGE/ESI-MS/MS ergab 63 identifizierte Proteine von denen 30 signifikant moduliert waren, Analyse durch ICAT/2D-LC-MS/MS erkannte 322 Proteine, von denen 167 virusbedingt moduliert waren (Jiang et al., 2005). Bei den meisten Proteomanalysen virusinfizierter Zellen wurden quantitative Veränderungen vor allem bei Hitzeschockproteinen, ribosomalen Proteinen, Proteinen des Transkriptions- oder Translationsapparates und Bestandteile des Zytoskelettes gefunden. Dies lässt die Vermutung zu, dass es sich dabei um eine relativ unspezifische Antwort der Wirtszelle auf eine Virusinfektion handelt. Allerdings sind diese Proteine auch in vielen anderen Proteomstudien auffällig, die nicht an virusinfizierten Zellen durchgeführt wurden (Petrak et al., 2008), und somit zumindest teilweise als generelle Stressantwort gelten können. In einer frühen Studie mit PrV-infizierten MDBK-Zellen (Hsiang et al., 1996) waren eine Stunde nach Infektion 70 der 1400 untersuchten Transkripte verändert, überwiegend betroffen waren Gene, die für Proteine der Signaltransduktion und der Translation kodieren. In einer vergleichenden Studie mit PrV und HSV-1 fanden Ray und Enquist (Ray & Enquist, 2004) zu verschiedenen Zeitpunkten zwischen 0 und 12 Stunden nach der Infektion von embryonalen Rattenfibroblasten Veränderungen bei 1549 von 4626 untersuchten Transkripten. Von diesen reagierten nur 498 auf beide Viren. Dabei handelte es sich vor allem um Gene die an Signaltransduktionswegen beteiligt sind, wie z.B. dem PI3K/Akt Signalweg. Auch virusspezifische Regulationen wurden beobachtet. Nach Infektion mit PrV waren vor allem Gene von Transkriptionsfaktoren, der Stressantwort, von ribosomalen Proteinen und RNA- bindenden Proteinen auffällig, wohingegen nach Infektion mit HSV-1 vor allem Zelladhäsion, Immunität, intrazelluläre Transport- und Wachstumsfaktoren betroffen waren. Blanchard und Kollegen verwendeten in einer kinetischen Untersuchung einen humanen Mikroarray mit 9850 Proben, um den Zeitverlauf der Veränderung des Transkriptoms nach einer PrV-

Infektion von humanen embryonalen Nierenzellen zu analysieren (Blanchard et al., 2006). In den ersten drei Stunden nach Infektion wurden nur wenige zelluläre Gene reguliert, wohingegen nach sechs Stunden mehr als 1000 Gene und nach neun Stunden mehr als 2400 Transkripte eine signifikante Änderung aufwiesen. Die herunter-regulierten Transkripte kodierten hauptsächlich für Proteine, die an der DNA-, RNA- oder Proteinsynthese und dem Zellzyklus beteiligt sind, hochreguliert wurden vorzugsweise Transkripte, die die Regulation der DNA-Transkription, Entwicklungsprozesse (Entwicklung des zentralen Nervensystems, Neurogenese, Angiogenese), Zelladhäsion und den Kaliumtransport beeinflussen. In einer in-vivo Studie von Paulus und Kollegen wurden die Effekte einer Infektion mit dem attenuierten PrV-Stamm Bartha (Bartha, 1961) im zentralen Nervensystem von infizierten Nagern zu verschiedenen Zeitpunkten untersucht (Paulus et al., 2006). Dabei zeigten 182 von mehr als 7000 Genen eine erhöhte Expression, von denen sechzig Prozent an der Immunabwehr, an proinflammtorischen und anderen zellulären Abwehrmechanismen beteiligt waren, darüber hinaus wurden Gene der zellulären Stressantwort aktiviert. Flori und Kollegen analysierten in einer kinetischen Microarray-Untersuchung an PrV-infizierten Schweinezellen sowohl virale als auch zelluläre Transkripte analysiert (Flori et al., 2008). Von den 13297 Proben des Array, die 8541 Genen entsprachen, wiesen 6693 eine signifikante Veränderung 6 Stunden nach Infektion auf. Dabei wurden quantitative Veränderungen vor allem bei Proteinen der Apoptose, des Nukleinsäuremetabolismus, des Zytoskeletts und der Interferon-vermittelten antiviralen Antwort festgestellt. Überraschenderweise konnte der Beginn eines generellen Verlustes zellulärer mRNAs erst 4 Stunden nach Infektion beobachtet werden, wobei 2756 Proben moduliert vorlagen.

Die Anwendung der typischen „Proteomics“ Techniken in der Familie der Herpesviren beschränkte sich bisher noch im Wesentlichen auf die Identifizierung von Virusprotein- Interaktionen, zellulären Interaktionspartnern herpesviraler Proteine in infizierten Zellen (Fontaine-Rodriguez et al., 2004; Holowaty et al., 2003; Schlee et al., 2004; Taylor & Knipe, 2004), oder die Erstellung von Proteinprofilen von Zellen, die einzelne virale Proteine exprimierten (Bartee et al., 2006; Greco et al., 2001; Kessler et al., 2007). Auf diese Weise konnten z.B. Interaktionen der VP19C und VP26 Proteine des HSV-1 mit dem Ribosom in infizierten HeLa-Zellen (Greco et al., 2001) und des Kernantigen-I des Epstein-Barr-Virus u.a. mit HAUSP/USP7 nachgewiesen werden (Holowaty et al., 2003). In weiteren Studien wurden durch eine Kombination von Immunpräzipitation, Gelelektrophorese und Massenspektrometrie mehr als fünfzig zelluläre Interaktionspartner des Einzelstrang-DNA- bindenden Proteins ICP8 (Taylor & Knipe, 2004) und mehrere Interaktionen mit dem ICP27 des HSV-1 (Fontaine-Rodriguez et al., 2004) identifiziert. Dabei wurden für das ICP27 vor allem Interaktionen mit Translations-Initations-Faktoren gefunden und für das ICP8 Proteine,

die an der Replikation, der Transkription, der Chromatin-Modellierung und als Strukturproteine beteiligt sind (Fontaine-Rodriguez et al., 2004; Taylor & Knipe, 2004).

In einer quantitativen Studie wurden die Proteomveränderungen analysiert, die ein einzelnes herpesvirales Protein in transfizierten Zellen auslöst (Bartee et al., 2006). Dabei wurden Proteomveränderungen durch die virale K5-RING Ubiquitin-Ligase des HHV-8 mittels SILAC- Quantifizierung und LC-MS-Kopplung in verschiedenen Zellfraktionen (Zytoplasma, Golgi- Apparat, Endoplasmatisches Retikulum) erfasst. Bei vier Proteinen (MHC I, bone marrow stromal antigen 2, activated leukocyte cell adhesion molecule und Syntaxin-4) konnte eine verminderte Abundanz in den K5-exprimierenden Zellen nachgewiesen werden. Subproteomstudien auf Basis von 2DE an HSV-1-infizierten Zellen zur Untersuchung spezieller Fragestellungen wie dem Einfluss der Infektion auf die Zusammensetzung des Ribosoms (Diaz et al., 2002) oder dem Einfluss des shutoff auf eine kleinere Auswahl zellulärer Proteine (Greco et al., 2000) liegen ebenfalls vor. Dabei konnten Diaz und Kollegen an einer Ribosomenfraktion zeigen, dass die HSV-1 Infektion zu einer nicht reversiblen Phosphorylierung des ribosomalen Proteins S6 führt, sowie dass auch virale Proteine mit der Ribosomenfraktion assoziiert sind (Diaz et al., 2002). Vollständigere Proteomanalysen von Herpesvirus-infizierten Zellen liegen von Epstein-Barr-Virus-infizierten Zellen vor, einem Vertreter der Gammaherpesviren (Toda et al., 2000). Dabei wurden unterschiedlich lang passagierte transformierte humane B Lymphozyten verglichen. Systematische und umfassende Proteomanalysen nach Infektion mit Alphaherpesviren standen zu Beginn der vorliegenden Arbeit noch aus. Allerdings wurden nach der Veröffentlichung von Teilen dieser Arbeit auch Studien an mit HSV-1 (Antrobus et al., 2009; Santamaria et al., 2009) und Marek's disease Virus (Ramaroson et al., 2008; Thanthrige-Don et al., 2009) infizierten Zellen vorgelegt.

modulierte Vorfrakti- Genom Virus Technik MS Quantifiziert Literatur Proteine onierung Arenavirus MALDI-TOF/TOF- (-) ss RNA 123 2D ja (Bowick et al., 2009) Pichindé Virus MS/MS Aviäres Influenza MALDI-TOF/TOF- (-) ss RNA 31 2D (Zou et al., 2010) Virus (H5N1) MS/MS Aviäres Influenza 2D-LC-LTQ ion trap (-) ss RNA 400 LC label free (Brown et al., 2010b) Virus (H5N1) MS/MS Aviäres Influenza MALDI-TOF-MS / nano- (-) ss RNA 22 2D (Liu et al., 2008) Virus (H9N2) LC-ESI-Q-TOF-MS/MS Hepatitis Delta (-) ss RNA 23 2D MALDI-TOF-MS (Mota et al., 2009) Virus Humanes Influenza nanoHPLC-nanoESI- (-) ss RNA 22 2D DIGE (Vester et al., 2010) A Virus (H1N1) QqTOF-MS/MS Humanes Influenza 2D-RP-HPLC-Q-TOF- (Coombs et al., (-) ss RNA 280 LC SILAC ja A Virus (H1N1) MS/MS 2010) Humanes Influenza nano-LC-LTQ-Orbitrap (Emmott et al., (-) ss RNA A Virus (H1N1) und 23 1D/LC SILAC ja MS/MS 2010b) (H3N2) 23

modulierte Vorfrakti- Genom Virus Technik MS Quantifiziert Literatur Proteine onierung Humanes Influenza nanoHPLC-nanoESI- (-) ss RNA 16 2D DIGE (Vester et al., 2009) A Virus (H1N1) Qq-TOF-MS/MS Humanes nano-LC-LTQ-Orbitrap (-) ss RNA Respiratorisches 431 LC SILAC ja (Munday et al., 2010) MS/MS Synzytial-Virus Influenza Virus A RPLC-ESI-LTQ-ion (-) ss RNA - LC ja (Baas et al., 2006) (H1N1) trap-MS/MS mehrere 21+18+19+ nano-HPLC-Q-ion trap- (van Diepen et al., (-) ss RNA respiratorische 2D DIGE 17 spots FT-ICR 2010) Viren MALDI-TOF/TOF- (-) ss RNA Nipah Virus 6 2D (Chang et al., 2007) MS/MS nanoHPLC-ESI-Q-TOF- (-) ss RNA Rabies Virus 5 2D (Zandi et al., 2009) MS/MS

(-) ss RNA Rabies Virus 45 2D MALDI-TOF-MS/MS (Dhingra et al., 2007)

Respiratorisches MALDI-TOF-MS/ LC- (-) ss RNA 24 2D ja (Brasier et al., 2004) Synzytial-Virus ESI-Q-TOF-MS/MS Coronavirus (Emmott et al., (+) ss RNA Infektiöse- 179 LC LC-MS/MS SILAC ja 2010a) Bronchitis-Virus (Rassmann et al., (+) ss RNA Coxsackievirus B3 >230 2D MALDI-TOF/TOF-MS 2006) MALDI-TOF-MS/MS/ nano-HPLC-ESI-LTQ- (Hammer et al., (+) ss RNA Coxsackievirus B3 136 2D DIGE FT-MS /nanoUPLC- 2010) LTQ-Orbitrap-MS/MS LC-Q-TOF-MS/MS + (+) ss RNA Dengue Virus 59 1D/2D (Higa et al., 2008) MALDI-TOF-MS/MS

(+) ss RNA Dengue Virus 15 2D Q-TOF-MS/MS (Kanlaya et al., 2009)

(+) ss RNA Dengue Virus 38 2D Q-TOF-MS/MS (Kanlaya et al., 2010)

ICAT/18O/label (+) ss RNA Flock house virus 216 LC LC-MS/MS* (Go et al., 2006) free

(+) ss RNA Hepatitis C Virus - MudPIT LC-Q-ion-trap MS/MS label free ja (Jacobs et al., 2005)

LC-linear ion trap- (Diamond et al., (+) ss RNA Hepatitis C Virus 210 LC 18O MS/MS/ LC-FT-ICR-MS 2007)

(+) ss RNA Hepatitis C Virus 179 2D MALDI-TOF/TOF-MS (Fang et al., 2006)

nanoLC-ESI-Q-TOF- (Mannova et al., (+) ss RNA Hepatitis C Virus 150 1D/2D MS/MS LC-LTQ-FT- SILAC ja 2006) MS/MS Porcine reproductive and MALDI-TOF/TOF- (+) ss RNA 23 2D (Zhang et al., 2009a) respiratory MS/MS syndrome virus Porcine reproductive and (+) ss RNA 45 2D MALDI-TOF/TOF-MS DIGE (Xiao et al., 2010) respiratory syndrome virus MALDI-TOF/TOF- Rice yellow mottle (Ventelon-Debout et (+) ss RNA 24+40 2D MS/MS + nano-LC-Q- virus al., 2004) TOF-MS/MS SARS-assoziiertes ESI-MS/MS + 2D-LC- (+) ss RNA 186 2D DIGE/ICAT (Jiang et al., 2005) Coronavirus MS/MS MALDI-TOF-MS (Casado-Vela et al., (+) ss RNA Tabakmosaikvirus 17 2D nanoHPLC-QQQ-IT- 2006) MS/MS

modulierte Vorfrakti- Genom Virus Technik MS Quantifiziert Literatur Proteine onierung Taura syndrome nano-LC-ESI-Q-TOF- (Chongsatja et al., (+) ss RNA 13 2D virus MS/MS 2007) unbekanntes Virus (später als HPLC-ESI-LCQ-ion (+) ss RNA wenige 2D (Cooper et al., 2003) Kartoffelvirus X trap-MS/MS identifiziert) Virus der (+) ss RNA klassischen 15 spots 2D MALDI-TOF-MS/MS (Li et al., 2010) Schweinepest Virus der (+) ss RNA klassischen 21 2D MALDI-TOF-MS/MS (Sun et al., 2008) Schweinepest nano-LC-Q-TOF- (Pastorino et al., (+) ss RNA West Nile Virus 54 2D MS/MS 2009)

(+) ss RNA West Nile Virus 55 2D MALDI-TOF-MS DIGE (Dhingra et al., 2005)

HPLC-ESI-Q-TOF- ds DNA Adenovirus 24 1D/2D SILAC ja (Lam et al., 2010) MS/MS Afrikanisches ds DNA 12 2D MALDI-TOF-MS (Alfonso et al., 2004) Schweinpest Virus

ds DNA Epstein-Barr-Virus 20 2D MALDI-TOF-MS (Schlee et al., 2004)

nano-ESI- Q-TOF- ds DNA Epstein-Barr-Virus 1 2D (Toda et al., 2000) MS/MS LC-LTQ-linear IT- ds DNA Hepatitis B Virus 27 2D (Ding et al., 2009) MS/MS 2D-nanoLC-ESI-Q-TOF- ds DNA Hepatitis B Virus 14/16 LC iTRAQ Zhang, 2009 MS/MS 2D-nano-LC-Q-TOF- ds DNA Hepatitis B Virus 15 LC iTRAQ (Niu et al., 2009) MS/MS ESI(MALDI)-Q-TOF- ds DNA Hepatitis B Virus 61 2D (Tong et al., 2008) MS/MS Hepatitis B Virus X ESI(MALDI)-Q-TOF- ds DNA 50 2D (Tong et al., 2009) Protein MS/MS Herpes-Simplex- (Antrobus et al., ds DNA 103 2D LC-Q-TOF-MS/MS Virus-1 2009) Herpes-Simplex- nano-LC-Q-TOF- (Santamaria et al., ds DNA 16 2D DIGE ja Virus-1 MS/MS 2009) Herpes-Simplex- ds DNA - 2D MALDI-TOF-MS ja (Diaz et al., 2002) Virus-1 Herpes-Simplex- ds DNA 28 2D nicht benutzt ja (Greco et al., 2000) Virus-1 Humanes nanoLC-ESI-Q-TRAP- ds DNA Papillomavirus Typ 20 2D DIGE (Akgul et al., 2009) MS/MS 8 Kaposi sarcoma– ds DNA assoziertes 4 LC LCQ-ion-trap-MS/MS SILAC ja (Bartee et al., 2006) Herpesvirus Marek's disease µrpLC-Q-ion-trap- (Ramaroson et al., ds DNA 312 MudPIT ja virus MS/MS 2008) Marek's disease 1D-LC ESI-LCQ-ion- (Thanthrige-Don et ds DNA 48 2D virus trap MS/MS al., 2009) Marek's disease µrpLC-Q-ion-trap- ds DNA - MudPIT ja (Liu et al., 2006) virus MS/MS Marek's disease ds DNA 26 2D MALDI-TOF/TOF-MS (Lu et al., 2010) virus nano-HPLC-LTQ- ds DNA Monkeypox virus 614 LC (Brown et al., 2010a) Orbitrap-MS/MS

modulierte Vorfrakti- Genom Virus Technik MS Quantifiziert Literatur Proteine onierung (Skiba et al., MALDI-TOF/TOF- ds DNA Pseudorabiesvirus 109 2D SILAC ja 2008;Skiba et al., MS/MS 2010)

ds DNA Tiger Frosch Virus 10 2D MALDI-TOF-MS (Luo et al., 2009)

White spot ds DNA 53 2D LC-nanoESI-MS/MS (Wang et al., 2007) syndrome virus White spot MALDI-TOF/TOF- ds DNA 60 2D (Chai et al., 2010) syndrome virus MS/MS White spot MALDI-TOF/TOF- ds DNA 12 LC ICAT ja (Wu et al., 2007) syndrome virus MS/MS Bursal Disease ds RNA 51 2D MALDI-TOF/TOF-MS (Zheng et al., 2008) Virus Humanes ss RNA-RT Immundefizienz- 26 1D LC-ESI-Q-TOF-MS/MS SILAC ja (Pathak et al., 2009) Virus Typ-1 Humanes SELDI-TOF / LTQ LC- (Toro-Nieves et al., ss RNA-RT Immundefizienz- - 1D ja MS/MS 2009) Virus Typ-1 Humanes ss RNA-RT Immundefizienz- 168 LC LC-ESI-Orbitrap-MS/MS label free (Chan et al., 2009) Virus Typ-1 Humanes LC-LTQ-ion trap-MS 18 ss RNA-RT Immundefizienz- 687 LC O ja (Chan et al., 2007) /LC- FT-ICR-MS Virus Typ-1 Humanes high capacity ion trap- ss RNA-RT Immundefizienz- 13 2D MS / MALDI-TOF/TOF- (Zhang et al., 2010b) Virus Typ-1 MS/MS Humanes ss RNA-RT Immundefizienz- 58 Chip SELDI-TOF ja (Carlson et al., 2004) Virus Typ-1 Humanes ss RNA-RT Immundefizienz- 19 2D nanoLC-ESI-MS/MS (Zhang et al., 2010a) Virus Typ-1 Porcine Circovirus MALDI-TOF/TOF- ss DNA 34 2D (Zhang et al., 2009b) Typ 2 MS/MS

Tabelle 1: Proteomanalysen von virusinfizierten Wirtszellen

(-) ssRNA - Einzelstrang RNA negativer Polarität; (+) ssRNA - Einzelstrang RNA positiver Polarität; dsRNA - Doppelstrang RNA, dsDNA - Doppelstrang DNA; 1D - eindimensionale Gelelektrophorese; 2D - zweidimensionale (Gelelektrophorese); DIGE - difference in-gel electrophoresis; ESI - Elektrospray-Ionisation; FTICR - Fouriertransformation-Ionenzyklotronresonanz; HPLC - Hochleistungsflüssigkeitschromatographie; iTRAQ - isobaric tag for relative and absolute quantitation; IT - Ionenfalle; LC - Flüssigkeitschromatographie; LTQ - linear trap quadrupole; MALDI - Matrix- unterstützte Laser-Desorption/Ionisation (Matrix-assisted laser desorption/ionization); MS - Massenspektrometrie; MS/MS - Tandem-Massenspektrometrie; MudPIT - multidimensional protein identification technology; SILAC - Stable isotope labelling by amino acids in cell culture; SELDI - surface-enhanced laser desorption/ionization; TOF - time-of-flight; Q (q) - Quadrupol

II. Zielstellung

In dieser Arbeit wurde das Verfahren des „stable isotope labelling by amino acids in cell culture“ (SILAC) an virologische Fragestellungen angepasst. Die metabolische Markierung von Proteinen der Probe und der Referenz mit stabilen Isotopen in Gewebekultur erlaubt zur relativen Quantifizierung von Proteinen Analysengänge mit sehr früh eingeführtem globalem internem Standard, die äußerst robust gegen Analysenartefakte sind. Daraus ergibt sich ihre exzellente Eignung für typische quantitative Proteomics-Anwendungen, aber auch für die präzise Quantifizierung von einzelnen Proteinen. Diese herausragenden Charakteristika der SILAC-Quantifizierung sollten zur Bearbeitung virologischer Fragestellungen genutzt werden.

II.1 Erstellung einer Proteomkarte PrV-infizierter MDBK Zellen Viren manipulieren den Stoffwechsel ihrer Wirtszellen in vielfacher Weise, um ihre eigene Replikation zu gewährleisten. Zusammen mit den Abwehrreaktionen der Zelle führt dies zu einer im Vergleich zum physiologischen Zustand veränderten Stoffwechsellage, deren Charakterisierung die Grundlage des Verständnisses der Pathogenese einer Virusinfektion ist. Um die molekularen Grundlagen der PrV-Infektion besser verstehen zu können, war die möglichst vollständige und quantitative Darstellung des Proteoms PrV-infizierter Zellen das primäre Ziel dieser Arbeit. Dazu sollte mittels hochauflösender zweidimensionaler Gelelektrophorese eine quantitative Proteomkarte PrV-infizierter Rindernierenzellen (MDBK) erstellt werden, die auch Veränderungen bei posttranslationalen Proteinmodifikationen umfasst. Bekannte technische Einschränkungen der zweidimensionalen Gelelektrophorese (2DE) wie Begrenzungen bezüglich der Analytmenge und die daraus resultierende Unterrepräsentanz weniger abundanter Proteine sollten durch die Etablierung einer der 2DE vorgeschalteten Vorfraktionierungsstrategie egalisiert oder zumindest abgemildert werden. Die Effizienz der Vorfraktionierung sollte durch Auswertung der in den einzelnen Fraktionen identifizierten Proteine beurteilt werden. Die bei der isoelektrischen Fokussierung verwendeten pH-Bereiche sollten hinsichtlich der Zahl der zu identifizierenden und zu quantifizierenden Proteine optimiert werden. Da zur Bewertung der relativen Mengen der einzelnen Proteine nach PrV-Infektion ein statistisch abgesicherter Grenzwert (cutoff) notwendig ist, sollte die Streuung der Versuchsanordnung in einem gesonderten Versuch empirisch ermittelt werden.

II.2 Proteomanalysen an Zellfraktionen nach Infektion mit PrV- Wildtyp und einer Kinase-negativen Deletionsmutante Im zweiten Teil sollten die Ergebnisse der zuvor durchgeführten quantitativen Proteomstudie an PrV-infizierten Zellen mit denen einer Transkriptom-Studie verglichen werden, um die Mechanismen der Regulation der Wirtszelle nach einer vierstündigen PrV-Infektion zu verstehen. Darüber hinaus sollte eine ebenfalls auf der SILAC-Methode basierende quantitativen Proteomstudie mit einer US3-Kinase-negativen Deletionsmutante des PrV erstellt werden, um pUS3-abhängige Modulationen des Wirtszellproteoms, aber auch der viralen Proteine zu erfassen. Aufgrund der besonderen Bedeutung des Zellkerns für die Replikation des PrV sollte das SILAC-Verfahren mit einem Zellfraktionierungsprotokoll kombiniert und die Effizienz mit der im ersten Teil der Arbeit etablierten Vorfraktionierung durch Affinitätsfestphasenextraktion verglichen werden.

II.3 Quantitative Analyse der Proteinexpression von Fremdgenen in rekombinanten Newcastle Disease Viren Im dritten Teil dieser Arbeit sollte das SILAC-Verfahren zur Quantifizierung der Proteinexpression von Fremdgenen in viralen Vektoren eingesetzt werden. In dieser Studie wurden rekombinante Newcastle Disease Viren (NDV) verwendet, die das Hauptimmunogen des Aviären Influenza Virus, das Hämagglutinin, exprimieren und im Rahmen der Entwicklung von bivalenten Impfstoffen zur Bekämpfung der Newcastle-Krankheit und der Vogelgrippe generiert wurden. Hohe Expressionsraten der Immunogene sind die Voraussetzung für eine effiziente Immunisierung und damit ein Qualitätsmerkmal rekombinanter Vakzinen. Die präzise Quantifizierung der Immunogene ermöglicht eine Beurteilung einer potenziellen Vakzine in Zellkultur. Die Voraussetzungen für den Einsatz der SILAC-Technik waren gegeben: die Infektion erfolgte in Zellkultur, die NDV-Rekombinanten exprimierten identische Fremdgene, nur der Ort der Insertion im Genom war unterschiedlich. Aufgrund des beim NDV beschriebenen, vom 3’- zum 5’-Ende abnehmenden Transkriptionsgradienten der NDV-spezifischen Gene wurde eine Korrelation der Expressionsstärke auch des HA-Gens mit der Lage der Insertion im Genom erwartet. Um diese Arbeitshypothese zu prüfen, sollten in den Rekombinanten die relativen Abundanzen der Transkripte und der Proteine bestimmt werden. Zur Quantifizierung des HA schien das SILAC Verfahren aufgrund seiner hohen Genauigkeit und der Unabhängigkeit von immunologischen Reagenzien besonders geeignet.

III. Zusammenfassende Darstellung und Diskussion der Ergebnisse

III.1 “Quantitative whole-cell proteome analysis of pseudorabies virus-infected cells.” Der Erreger der Aujeszky’schen Krankheit beim Schwein, das Pseudorabiesvirus (PrV), ist ein Vertreter der Unterfamilie der Alphaherpesvirinae. Die Expression herpesviraler Gene im Verlauf der Infektion erfolgt kaskadenartig und ist gut untersucht. Systematische Untersuchungen zur Expression von Wirtszellproteinen in PrV-infizierten Zellen standen jedoch noch aus. In der vorliegenden Arbeit wurde diese vier Stunden nach PrV-Infektion boviner Nierenzellen (MDBK) durch eine quantitative Proteomstudie untersucht. Zur Verbesserung der Analysentiefe wurde zunächst der sehr komplex zusammengesetzte Gesamtzellextrakt durch Affinitäts-Festphasenextraktion an drei verschiedenen Matrices (Cibacron Blau F3G-A, Heparin und Phosphoprotein spezifisches Metallchelat) vorfraktioniert. Die erhaltenen fünf Fraktionen (Eluate dreier Affinitätsmatrices, kombinierter Durchfluss der Cibacron Blau F3G-A- und Heparinmatrices und der Durchfluss der Phosphoprotein-spezifischen Matrix) wurden daraufhin durch klassische zweidimensionale Gelelektrophorese (2DE) in vier pH-Bereichen (pH 3-6, 4-7 und 6-9, sowie 3-10) analysiert. Alle sichtbaren Proteine wurden, soweit möglich, massenspektrometrisch identifiziert und mit Hilfe des SILAC-Verfahrens quantifiziert. Die Leistungsfähigkeit der Affinitätsfraktionierung wurde durch Abgleich der in den Fraktionen identifizierten Proteine mit den in der Gene Ontology Datenbank niedergelegten biochemischen Eigenschaften beurteilt. Sie erwies sich als sehr spezifisch und trennscharf. Die Zusammensetzung der Fraktionen entsprach weitgehend den Affinitäten der verwendeten Matrices und die Überschneidungen zwischen den einzelnen Fraktionen waren gering. Der pH-Bereich 3-6 erwies sich im Vergleich zu den anderen untersuchten Bereichen als relativ proteinarm. Zur statistisch unterlegten Beurteilung der quantitativen Daten war die Kenntnis der Streuung des Verfahrens notwendig. Dazu wurde der vollständige Analysengang auch mit nicht infizierten Zellen durchgeführt. Die dabei empirisch erhaltenen 99% Quantilen von 0,63 und 1,63 wurden als Schwellenwert (cutoff) zur Beurteilung von infektionsbedingten quantitativen Schwankungen („Modulationen“) der relativen Proteinmengen verwendet. Da für PrV-Infektionen ein virion- host-shutoff beschrieben ist, der die Neusynthese von Wirtsproteinen effizient verhindert, war die hohe Stabilität der überwältigenden Mehrheit der zelleigenen Proteine während der Infektion überraschend. Von den 2600 identifizierten Proteinen zeigten nur 109 Modulationen

jenseits der Schwellenwerte. Diese waren im Wesentlichen der Kernlamina, dem Translationsapparat, dem intrazellulären Transport, sowie der Stressantwort zuzuordnen. Eine Reihe von Proteinen trat in der zweidimensionalen Gelelektrophorese in mehreren Ladungsvarianten auf, die gegensinnig moduliert wurden. Dabei handelte es sich um die Lamine A/C und B2, das 60S Saure Ribosomale Protein P0, das Hitzeschock-27 Protein 1, das Heterogene Nukleäre Ribonukleoprotein K (hnRNP K), das Sorting Nexin-9 und den Eukaryotischen Translations-Initiations Faktor 4B. Auf Grund der bei diesen Proteinen durchgängig beobachteten Mengenverschiebungen zu den saureren Isoformen wird eine infektionsbedingte Phosphorylierung durch virale oder zelluläre Kinasen vermutet. In einer quantitativen Proteomstudie an Herpes-Simplex Virus Typ-1 (HSV-1) infizierten humanen Epithelzellen (HEp-2) (Antrobus et al., 2009) wurden sechs Stunden nach Infektion 103 modulierte Proteine gefunden. Unter diesen waren mit Ausnahme des Lamin A/C und des hnRNP K keine weiteren Vertreter, die nach PrV-Infektion signifikant moduliert waren. In einer weiteren Proteomstudie an HSV-1 infizierten humanen Hepatoma Zellen (Huh7) wiesen sechzehn Proteine quantitative Veränderungen auf, darunter ebenfalls das nach PrV- Infektion modulierte hnRNP K, das Macrophage-capping Protein und das RuvB-like 2 protein (Santamaria et al., 2009). Die geringen Übereinstimmungen zwischen den genannten Studien und der vorliegenden Arbeit könnten neben der Verwendung unterschiedlicher Viren (HSV-1 bzw. PrV), auch durch die unterschiedlichen Infektionszeitpunkte (6 h (Antrobus et al., 2009) und 4 h sowie 8 h (Santamaria et al., 2009) bzw. 4 h bei PrV), Zellen (humane HEp-2 (Antrobus et al., 2009) und Huh7 (Santamaria et al., 2009) bzw. bovinen MDBK) oder analytischen Techniken (DIGE/LC-MS (Antrobus et al., 2009; Santamaria et al., 2009) zu SILAC/MALDI-MS) begründet sein. Trotz der nur geringen Übereinstimmung der infektionsbedingt modulierten Proteine weisen die jeweils betroffenen zellulären Prozesse und Strukturen (Stressantwort, Proteinexpression, Zytoskelett, Ribosom, Proteasom) auf Gemeinsamkeiten bezüglich der durch Alphaherpesviren bedingten Proteomveränderungen hin, die sich eventuell auch auf Gammaherpesviren wie das Epstein-Barr-Virus (Toda et al., 2000) ausdehnen lassen. Ähnlich wie in dieser Studie wurden auch nach Infektion mit HSV-1 quantitative Verschiebungen zwischen verschiedenen Ladungsvarianten desselben Proteins festgestellt (Antrobus et al., 2009; Santamaria et al., 2009), was auf die virusbedingte Beeinflussung posttranslationaler Modifikationen hinweist.

III.2 “Gene expression profiling of Pseudorabies virus (PrV) infected bovine cells by combination of transcript analysis and quantitative proteomic techniques.”

Aufbauend auf der zuvor genannten Studie, waren Ziele dieser Arbeit, den Mechanismus der Regulation der Wirtszellproteinexpression durch eine PrV-Infektion zu ergründen, die Eignung von Zellfraktionierungsprotokollen für die SILAC-Quantifizierung zu prüfen und eine Kinase-negative Mutante des PrV mit in die Studien einzubeziehen und somit auch die Expressionsstärken viraler Proteine in verschiedenen Virusmutanten für die SILAC-Technik zugänglich zu machen. Eine Transkriptanalyse wurde mit einem bovinen Mikroarray durchgeführt. Unter den 55 Genen, deren Genprodukte laut Proteomstudie signifikant moduliert waren, fanden sich nur zwei, Lamin A/C und RuvB-like 2 Protein, die auch auf Transkriptebene quantitative Veränderungen aufwiesen. In beiden Fällen sanken die Transkriptniveaus. Eine Korrelation zwischen der Transkript- und Proteinmenge konnte somit nicht festgestellt werden, was einen weiteren Hinweis auf die Bedeutung posttranslationaler Ereignisse bei der Wirtszellantwort auf die PrV-Infektion darstellt. Im zweiten Teil war die Eignung der SILAC-Methode für die Bestimmung der Expressionsstärke viraler Proteine nach Infektion mit Deletionsmutanten zu prüfen. Dabei galt der multifunktionellen, innerhalb der Alphaherpesviren konservierten, Kinase pUS3 aus einer Reihe von Gründen ein besonderes Interesse. Sie beeinflusst das Rearrangement des Aktinskeletts, die virale Genexpression, die Virusmorphogenese (Ausschleusung der Kapside aus dem Kern) und wirkt antiapoptotisch. Daher wurde das SILAC-Verfahren entsprechend abgewandelt: als globaler interner Standard dienten Zellextrakte nach Infektion mit PrV-Wildtyp des Stammes Kaplan (Kaplan & Vatter, 1959), die Proben bestanden aus Zellextrakten nach Infektion mit einer US3-negativen Mutante. Aufgrund der besonderen Bedeutung des Zellkerns als Ort der Replikation des Genoms, der Verpackung der viralen DNA und des Zusammenbaus der Kapside, sowie der Hauptlokalisation der viralen Kinase pUS3, wurde die zur Vorfraktionierung entwickelte Affinitäts-Festphasenextraktion um eine organellenspezifische Vorfraktionierung erweitert. Um Kernproteine spezifisch zu analysieren, aber auch um die Gesamtanzahl zugänglicher Proteine im Sinne einer möglichst vollständigen Proteomkartierung der MDBK Zellen weiter zu erhöhen, wurden Kern- und Zytosolfraktionen präpariert und mittels zweidimensionaler Gelelektrophorese in zwei verschiedenen pH- Bereichen (4-7 und 6-9) analysiert. Nach Identifizierung der erhaltenen Proteine wurden diese hinsichtlich ihrer Effizienz und Selektivität untersucht und mit dem Protokoll zur Affinitäts-Festphasenextraktion verglichen. Ein Großteil der Proteine aus der Zellfraktionierung wird durch die, allerdings zeitintensivere, Affinitäts-Festphasenextraktion ebenfalls erfasst. Einige wichtige kernlokalisierte Proteine, wie z.B. Spleißfaktoren, wurden

jedoch ausschließlich in der Kernfraktion identifiziert. Ein Abgleich der identifizierten Proteine mit der Gene Ontology Datenbank zeigte, dass Kontaminationen von mitochondrialen oder endoplasmatischen Proteinen in den Zellfraktionen enthalten sind. Die SILAC- Quantifizierung von fünfzehn viralen Proteinen ergab signifikante quantitative Veränderungen beim DNA-bindenden Protein (pUL29), der großen Untereinheit der Ribonukleotid Reduktase (pUL39), sowie bei einer Untereinheit der viralen Polymerase (pUL42). Während die Proteinmengen von pUL29 und pUL39 in Abwesenheit von pUS3 erhöht waren, war pUL42 reduziert. Im Gegensatz zu pUL39 besitzen pUL29 und pUL42 eine Konsensussequenz für eine US3-abhängige Phosphorylierung. Keines der drei Proteine, die am viralen DNA- Metabolismus beteiligt sind, wurde allerdings bisher als Substrate der pUS3 Kinase beschrieben.

III.3 “Influence of Insertion Site of Avian Influenza Virus Hemagglutinin (HA) Gene Within the Newcastle Disease Virus Genome on HA.”

NDV und AIV verursachen Infektionen im Nutzgeflügel, die weltweit zu hohen wirtschaftlichen Verlusten führen. Zur Entwicklung eines bivalenten Impfstoffes gegen NDV und die durch bestimmte Stämme des AIV ausgelöste Geflügelpest wurden rekombinante NDV Vektoren generiert, die zusätzlich zu den sechs NDV Genen das Hämagglutinin- und/oder Neuraminidase-Gen des AIV exprimieren. Da die Immunisierungseffizienz einer Vakzine wesentlich von der Expressionsstärke der Immunogene abhängt und entlang des NDV-Genoms ein Transkriptionsgradient vom 3’- zum 5’-Terminus hin beschrieben wurde, sollte die Frage geklärt werden, ob dieser sich zur Verbesserung der Expression von Fremdgenen in einem NDV-Vektor nutzen lässt. Dazu wurden NDV-Rekombinante generiert, in die das AIV-Hämagglutinin-Gen des hoch pathogenen AIV A/chicken/Vietnam/P41/05 (Subtyp H5N1) an verschiedenen Stellen des NDV-Genoms inseriert wurde. Als Fremdgen wurde das Hämagglutinin verwendet. Die Insertion erfolgte zwischen den Genen P und M, M und F, oder F und HN des als Vektor dienenden attenuierten NDV-Stammes Klon 30. Die Quantifizierung erfolgte durch Northern Blot-Analyse der Transkripte und massenspektrometrisch mit der SILAC-Technik nach Mikropräparation des HA Proteins durch zweidimensionale Elektrophorese. Als interner Standard wurden Zellextrakte nach Infektion mit der Rekombinante mit dem höchsten zu erwartenden HA-Expressionsniveau verwendet, bei der das HA-Gen zwischen den NDV-Genen P und M lag. RNA- und Proteinmessungen ergaben nur moderate quantitative Unterschiede zwischen den Mutanten, die Korrelation der relativen RNA-Mengen mit den relativen Proteinmengen war gut. Die 32

stärkste Expression wurde in der Rekombinante beobachtet, die die HA-Insertion zwischen dem F und HN Gen des NDV trug. Sie war etwa doppelt so hoch wie in der Rekombinante, in der das Gen zwischen M und F inseriert wurde. Dies zeigt deutlich, dass nach dem Einbau eines Fremdgens in das NDV Genom die Synthese der Fremdproteine nicht mit dem Transkriptionsgradienten korreliert. Obwohl die verzeichneten Unterschiede bezüglich der Expression des HA nur moderat waren, können sie immunologisch oder auch in einem Produktionsprozess von Bedeutung sein. Das SILAC-Verfahren ist in der Lage, auch solche eher geringen Unterschiede mit hoher Genauigkeit zu erfassen.

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V. Quantitative whole-cell proteome analysis of pseudorabies virus-infected cells.

Quantitative Whole-Cell Proteome Analysis of Pseudorabies Virus-Infected Cellsᰔ Martin Skiba, Thomas C. Mettenleiter, and Axel Karger* Institute of Molecular Biology, Friedrich-Loefﬂer-Institut, Su¨dufer 10, 17493 Greifswald-Insel Riems, Germany

Received 13 May 2008/Accepted 16 July 2008

A quantitative proteome study using the stable isotope labeling with amino acids in cell culture technique was performed on bovine kidney cells after infection with the alphaherpesvirus pseudorabies virus (PrV), the etiological agent of Aujeszky’s disease. To enhance yields of proteins to be identified, raw extracts were fractionated by affinity solid-phase extraction with a combination of a cibacron blue F3G-A and a heparin matrix and with a phosphoprotein-specific matrix. After two-dimensional gel electrophoresis in different pH ranges between pH 3 and pH 10, 2,600 proteins representing 565 genes were identified by mass spectrometry and screened for virus-induced changes in relative protein levels. Four hours after infection, significant quantitative variations were found for constituents of the nuclear lamina, representatives of the heterogeneous nuclear ribonucleoproteins, proteins involved in membrane trafficking and intracellular transport, a ribosomal protein, and heat shock protein 27. Several proteins were present in multiple charge variants that were differentially affected by infection with PrV. As a common pattern for all these proteins, a mass shift in favor of the more acidic isoforms was observed, suggesting the involvement of viral or cellular kinases.

Herpesviral genes are generally expressed in three kinetic However, alphaherpesvirus infection has an impact not only on classes (18, 19), which are regulated sequentially by a number the transcription of cellular genes but also on posttranslational of positive and negative feedback mechanisms exerted by virusprotein metabolism. Alphaherpesviruses encode several gene encoded proteins. However, herpesvirus infection also influ- products with enzymatic functions to alter proteins by post- ences the expression of cellular genes, e.g., by host cell shutoff translational modification. These include the protein kinases mechanisms that target the integrity of cellular mRNA and pUL13 and pUS3 as well as pUL36, a large structural protein thus block the synthesis of cellular proteins. Herpes simplex that mediates deubiquitination (21, 23). Examples for post- virus type 1 (HSV-1), the prototypical alphaherpesvirus, ex- translational modifications of cellular proteins induced by in- presses two shutoff proteins, ICP27 and pUL41. Whereas fection with HSV-1 are the phosphorylation of lamins A/C and ICP27 interferes with mRNA splicing (16, 17), pUL41 de- B (35, 40), the ICP27-stimulated phosphorylation of heteroge- grades mRNA by virtue of its endoribonucleolytic activity (10, neous nuclear ribonucleoprotein (hnRNP) K by casein kinase 29, 54), with specificity for mRNAs containing AU-rich ele- 2 (CK2) (25), and the block of histone deacetylase 1 by pUS3 ments (11). Homologs of both proteins are also present in and viral ICP0 (43). Infection with HSV-1 also induces the pseudorabies virus (PrV), an alphaherpesvirus causing Aujesz- proteasome-dependent degradation of a number of proteins ky’s disease. PrV, whose main host is the pig, infects numerous like CD83 (26), the ND10-related proteins PML and Sp100 mammalian species except higher primates including humans. (8), or the catalytic subunit of the DNA-dependent protein In contrast to HSV-1 ICP27, the PrV homolog pUL54 is dis- kinase (41). However, a systematic examination of the impact pensable for virus replication in cell culture (47, 49). Transcript of alphaherpesvirus infection on the protein composition of analyses of PrV-infected rat (44), human (6), and porcine (12) the infected cell has not yet been performed. Thus, the objec- cells demonstrated alterations in the abundance of individual tive of this study was to establish a quantitative protein expres- cellular transcripts, which resulted in the depletion or accumu- sion profile of PrV-infected cultured cells, which includes post- lation of specific mRNAs. Of the 9,850 genes examined in one translationally modified isoforms of individual proteins. study (6), the number of significantly up- or downregulated genes increased from approximately 1,000 to over 2,400 between 6 and 9 h after infection. Evaluation of the functions MATERIALS AND METHODS annotated for highly regulated cellular genes shows that PrV Cells and viruses. Madin-Darby bovine kidney cells (31) were provided by the infection influences numerous cellular pathways and that genes Collection of Cell Lines in Veterinary Medicine, Insel Riems, Germany. PrV strain Kaplan (22) was used. involved in protein and nucleic acid metabolism, signaling, Stable isotope labeling. The original stable isotope labeling procedure (38) transport, cell cycle control, adhesion, transcription, the stress was adapted as follows. Dulbecco’s modified Eagle (DME)/F12 medium (D- response, and innate immunity are affected most frequently. 9785; Sigma-Aldrich, Taufkirchen, Germany) was supplemented with 5% dia- lyzed fetal calf serum and all missing amino acids (Sigma-Aldrich) except L- leucine. Medium was then divided and supplemented with conventional or

deuterated L-leucine (L-leucine-5,5,5-D3 [99 atom% D]) (catalog number * Corresponding author. Mailing address: Institute of Molecular 486825; Sigma-Aldrich) to produce PROLeu-DME/F12 or DEULeu-DME/F12 Biology, Friedrich-Loeffler-Institut, Su¨dufer 10, 17493 Greifswald-Insel medium, respectively. MDBK cells were passaged in parallel in both media at a Riems, Germany. Phone: 49-38351-7251. Fax: 49-38351-7175. E-mail: 1:10 ratio every 3 days. After four passages, aliquots of the cell cultures were axel.karger@fli.bund.de. lysed, and proteins were separated by gel electrophoresis. The efficiency of the ᰔ Published ahead of print on 23 July 2008. exchange of normal by deuterated leucine was controlled by mass spectrometry.

9689 9690 SKIBA ET AL. J. VIROL.

The incorporation of L-leucine-5,5,5-D3 increases the mass of leucine-containing Fingerprint and up to four tandem mass (MS/MS) spectra per sample were peptides by 3 mass units per leucine residue. registered on a Bruker Ultraflex I tandem time-of-flight instrument (Bruker) and Infection. Deuterium-labeled cells were used as mock-infected controls, and processed by flexAnalysis 2.0 software (Bruker). For the quantitation of mass- cells grown on the conventional amino acid source were infected. Cell batches tagged peptide peak pairs, it was crucial to choose the “SNAP” option as the passaged in the two media were seeded in 75-cm2 or 150-cm2 cell culture flasks peak detection algorithm in the flexAnalysis software, which is robust with and inoculated with virus stock corresponding to a multiplicity of infection of 10 respect to overlapping isotope patterns. A batch database search (MASCOT (cells grown in PROLeu-DME/F12 medium) or mock inoculated (cells grown in Server 2.0.0 software; Matrix Science Ltd., London, United Kingdom) (42) was DEULeu-DME/F12 medium) on ice for 1 h and then incubated at 37°C for 4 h. launched by Biotools 2.2 (Bruker) using the bovine International Protein Index Extraction of phosphoproteins. Phosphoproteins of infected and mock-in- (IPI) database (www.ebi.ac.uk) (24) or an in-house database covering the PrV fected cells were purified with the PhosphoProtein purification kit (catalog num- proteome as compiled from the Swiss-Prot database (www.expasy.org) (13). ber 37101; Qiagen, Hilden, Germany). In short, 1 ϫ 107 cells were extracted with Carbamidomethylation was set as a fixed modification for cysteine residues, the the buffer supplied with the kit, containing 0.24% 3-[(3-cholamidopropyl)-di- significance level was set to 95%, and mass tolerance was set to 50 ppm for the methylammonio]-1-propansulfonate (CHAPS), and unsolubilized material was fingerprint spectra. Proteins were considered as identified and selected for quan- removed by centrifugation. The protein concentration in the clarified extract was titative evaluation if significant molecular weight search (MOWSE) scores (39) determined with the BCA protein assay (Pierce, Rockford, IL), and equal were obtained with the fingerprint spectrum or in a combined search of the amounts of proteins were mixed and applied onto the column supplied with the fingerprint and MS/MS spectra. kit. The eluate containing mainly phosphoproteins and the flowthrough contain- Quantitation and data processing. Quantitation was carried out by in-house ing mainly the nonphosphorylated proteins were concentrated by ultrafiltration software (AMaDEuS) based on Visual Basic for applications (Microsoft, Red- in Nanosep tubes equipped with a 10-kDa-cutoff membrane. Protein concentra- mond, WA) macros. For identified samples, peaks representing the protein with tions were assayed with the BCA protein assay (Pierce). Total eluates, usually the highest MOWSE score were selected, and the masses of the expected iso- containing approximately 200 ␮g of protein and aliquots of 1 mg of the concen- tope-labeled peaks within error margins of 15 ppm were calculated on the basis trated flowthrough, were precipitated (2D clean-up kit, catalog number 80-6484- of the number of leucine residues present in the peptide and the mass shift of ϩ3 51; GE Healthcare, Braunschweig, Germany), and the pellets were dissolved in Da per leucine residue. If the spectrum contained the mass of the expected 200 ␮l of rehydration buffer (RHB) (7) with mild sonication and either used deuterated peak, the peak pair was selected for quantitative evaluation, and the immediately or stored at Ϫ20°C. The resulting fractions are referred to as the intensity ratio of the peaks was calculated. If more than four peak pairs were phosphoprotein fraction and nonphosphoprotein fraction (flowthrough). Both obtained from one spectrum, outliers were eliminated by the symmetrical trun- fractions were tested for the phosphorylation status of the included proteins by cation of intensity ratios. Truncation was limited to a maximum of three rounds Western blotting with antibodies directed against phosphoserine, phosphothreo- or the number of rounds that would leave a minimum of three values. Truncated nine, or phosphotyrosine, and the efficiency of the separation for HeLa cells as mean values and standard deviations of the intensity ratios of qualified peak pairs given by the manufacturer (PhosphoProtein purification kit user manual) was representing the identified protein were calculated. Relative standard deviations confirmed for the MDBK cells used. rarely exceeded 15%. The database query was then repeated with a fixed mod- ASPE with cibacron blue F3G-A-Sepharose and heparin-Sepharose. Two 150- ification of ϩ3 Da for leucine residues to ensure the identification of proteins cm2 flasks each of labeled and unlabeled cells corresponding to approximately 8 ϫ that were strongly downregulated after infection. Quantitative evaluation was 7 Ϫ 10 cells in total were harvested in 10 ml extraction buffer (10 mM Na2HPO4- again carried out as described above, with the only difference being that a 3-Da

KH2PO4, 150 mM NaCl, 1% CHAPS [pH 7.0]) supplemented with protease shift was used to calculate the expected mass of the unlabeled peptide peak in the inhibitors (Complete Mini, 1 tablet/10 ml; Roche), extracted for 60 min on ice AMaDEuS software. Results of both queries and calculations were combined with occasional shaking, and centrifuged (15 min at 4°C at 4,000 ϫ g) to remove and compared. Data sets resulting from the same sample (protein spot) yielding unsoluble material. Extracts from unlabeled and isotope-labeled cells were higher MOWSE scores were preferred to those with lower scores. The final mixed at a 1:1 protein ratio. HiTrap columns (1-ml column volume) (catalog output for every sample was the truncated mean of the ratios (unlabeled over numbers 17-0412-01 and 17-0406-01; GE Healthcare) were washed with 10 labeled, that is, infected over mock [IOM]) of qualified peak pairs, which will be column volumes each of water, elution buffer (10 mM Na2HPO4-KH2PO4,2M abbreviated as IOM ratios. IOM ratios greater than 1.0 indicate higher relative NaCl, 1% CHAPS [pH 7.0]), and extraction buffer before the extract was applied levels, and IOM ratios below 1.0 indicate lower relative levels of the respective with a peristaltic pump and recirculated for 30 min. The columns were washed proteins after infection with PrV. The average number of qualified peptides in with 10 column volumes of extraction buffer, and bound material was eluted with identified proteins was 3.3, and the maximum number was 18. 5 ml elution buffer. Samples containing 1 mg of the eluates or the flowthrough were precipitated with trichloroacetic acid, and precipitates were resuspended in RHB and stored at Ϫ20°C. The resulting fractions are referred to as the cibacron RESULTS fraction, the heparin fraction, and the affinity solid-phase extraction (ASPE) flowthrough in the text. Protein yields were approximately 11% in the cibacron Experimental design. The strategy for the fractionation and fraction and 17% in the heparin fraction, and the rest was found in the analysis of raw cell extracts is depicted in Fig. 1. The design of flowthrough. Protein recoveries approximated 100%. the experiment aimed at two goals. First, the very complex Two-dimensional (2D) gel electrophoresis. Phosphoproteins were analyzed on protein mixture present in whole-cell extracts was prefraction- 11-cm ReadyStrips (Bio-Rad, Munich, Germany) with a nonlinear pH range of ated into well-defined fractions in order to facilitate further 3 to 10, nonphosphoproteins were analyzed on 24-cm strips with a nonlinear pH range of 3 to 10, and all other fractions were analyzed on 24-cm strips with linear analysis (45); second, proteins with interesting features like pH ranges of 3 to 6, 4 to 7, and 6 to 9. Precipitated samples were resuspended phosphorylation as well as nucleotide-binding capacities (ciba- in RHB, briefly sonicated on ice, and extracted for2hat20°C with intensive cron blue F3G-A-Sepharose) or DNA- and RNA-binding ca- shaking. Undissolved material was removed by centrifugation (20°C at 10 min at pacities (heparin-Sepharose) were enriched by affinity purifi- 14,000 ϫ g), and sample proteins were allowed to diffuse into ReadyStrips and focused in an IEF cell (Bio-Rad) according to the guidelines provided by the cation. Quantitation was carried out with the stable isotope manufacturer. Focused strips were frozen at Ϫ80°C, thawed, and sequentially labeling with amino acids in cell culture (SILAC) technique equilibrated in buffers containing dithiothreitol and iodoacetamide as recom- (38), which makes use of the high resolution of mass spectrom- mended by the manufacturer. The second dimension was run on hand-cast etry to allow the differentiation and quantitation of isotopically full-size 12% acrylamide gels in a Dodeca cell electrophoresis chamber (Bio- modified peptides that are chemically identical. Thus, the ma- Rad) with two 11-cm strips or one 24-cm strip loaded per gel. After the electrophoretic run, gels were fixed, stained overnight with colloidal Coomassie brilliant jor steps for quantitation by SILAC are the metabolic labeling blue (36), and scanned. of a sample with stable isotopes, mixing with an unlabeled Peptide mass fingerprint (PMF) analysis. After evaluation of the gel scans reference (or vice versa), purification of individual proteins with Delta2D software (version 3.4; Decodon, Greifswald, Germany), lists of from the mix, digestion of the proteins to peptides, and calcu- protein spots to be picked were used to operate a Proteineer SPII Spotpicker (Bruker Daltonics, Bremen, Germany). Tryptic digestion (46) was carried out in lation of intensity ratios of protein-specific peptides in a pep- 96-well V-bottom polypropylene microtiter plates for 3 h with 30 ng trypsin tide mass fingerprint spectrum. Ratios of isotope-labeled pep- (catalog number V5111; Promega, Mannheim, Germany) per sample at 37°C. tides reflect the relative levels of the protein in sample and VOL. 82, 2008 PROTEOME OF PrV-INFECTED CELLS 9691

FIG. 1. Flowchart of sample preparation and analysis. Note that all separation and analytical procedures (affinity extractions, 2D gel electrophoresis, tryptic digestion, and mass spectrometric analysis) were carried out with a 1:1 protein mixture of extracts of conventional and heavy-isotope-labeled cells. After extraction with three different affinity matrices and separation in different pH ranges, a total of 11 2D electrophoretic gels were analyzed for each experiment. FT, flowthrough.

reference with high accuracy. In the experiments reported In the course of this study, over 4,000 proteins were identi- here, the intensity ratios of unlabeled over labeled peptide fied, and 2,374 proteins were quantified from a minimum num- peaks were calculated to result in IOM ratios given in the text ber of three qualified peptide pairs. Of the 1,490 spots quan- and figures. Cell cultures metabolically labeled with deuterated tified in infection experiments, IOM ratios of 109 samples L-leucine and unlabeled cultures served as starting material. (7.3%) representing 55 genes (Table 1) were beyond the em- After infection with PrV or mock infection and incubation for pirical 1% and 99% quantiles of 0.63 and 1.63. Thus, approx- 4 h, a mix of raw extracts from labeled (mock-infected) and imately 30 of the 109 samples were expected to result from the unlabeled (PrV-infected) cells at 1:1 protein ratios was then statistical variance of the experiment itself, and approximately used for the parallel extraction of phosphoproteins with a 79 samples were expected to result from virus-induced commercially available reagent kit and a two-step ASPE pro- changes. cedure with cibacron blue F3G-A-Sepharose and heparin- Performance of the ASPE. Representative gels from the Sepharose. All five resulting fractions (phosphoproteins and three fractions of the ASPE at the pH range of 4 to 7 show very nonphosphoproteins from the phosphoprotein-specific extrac- distinct protein spot patterns (Fig. 2). The distribution of iden- tion and the heparin-binding proteins, the cibacron blue-bind- tified proteins over the ASPE fractions is shown in Fig. 3A (9). ing proteins, and the flowthrough fraction from the ASPE) Overlaps among the three fractions were low and are not were then analyzed by large-format 2D gel electrophoresis in necessarily attributable to the unsatisfactory performance of different pH ranges in order to maximize the yield of separated the ASPE, since different isoforms of the same protein may proteins. Isolated proteins were identified by PMF analysis distribute into different fractions on the basis of their biochem- using matrix-assisted laser desorption–ionization mass spectrom- ical properties. Yields of identified proteins in the range of pH etry with the MS/MS option and quantitated from the PMF spec- 3 to 6 (12%) were lower than those in the ranges of pH 4 to 7 tra. Three repetitions of the experiment were performed to screen (51%) and pH 6 to 9 (37%). Protein compositions of the for proteins with significantly altered relative protein levels. Can- cibacron and heparin fractions of the ASPE differ markedly didate proteins were then evaluated by additional experiments from the composition of the phosphoprotein fraction (Fig. 3B), focusing on the fractions and gel regions of interest. indicating that phosphoprotein extraction and the ASPE pro- The time point of 4 h after infection was chosen to monitor cedure are largely complementary, and a combination of both early effects of infection under conditions where cellular pro- significantly enhances the yield of identified proteins from a tein synthesis is already effectively blocked by viral shutoff single sample. The specificity of the ASPE procedure was es- mechanisms (data not shown), but the cell is still morpholog- timated by statistical evaluation (http://gostat.wehi.edu.au) (3) ically intact and not massively damaged by the release of prog- of the Gene Ontology (GO) annotations (“molecular function” eny virions. branch at www.geneontology.org) (2) characterizing the pro- 9692 SKIBA ET AL. J. VIROL.

TABLE 1. Proteins up- or downregulated after infection with PrV-Kad

IPI accession Genea Descriptionb IOM SDd number 540272 IPI00695524 Proliferation-associated 2G4 0.18 0.04 505686 IPI00706431 Ladinin 1 0.29 0.08 511512 IPI00690613 RNA binding protein 14 0.36 0.01 509771 IPI00688922 RNA binding motif protein 3 0.36 0.04 281165 IPI00692328 Filamin A 0.40 0.45 527471 IPI00725795 hnRNP D (AU-rich element RNA binding protein 1) 0.42 0.09 282419 IPI00707334 CK2 subunit alpha 0.47 0.04 505242 IPI00706906 EBP50 0.48 0.06 404098 IPI00695802 Staphylococcal nuclease domain-containing protein 1 0.48 0.13 533874 IPI00705755 Proteasome subunit beta type 3 0.49 0.16 510041 IPI00837986 Proteasome activator subunit 1 0.49 0.28 511048 IPI00726962 RuvB-like 2 0.50 0.15 507345 IPI00710727 Transitional endoplasmic reticulum ATPase 0.53 0.02 539060 IPI00827112 Serpine 1 mRNA binding protein 1 0.53 0.05 533851 IPI00829551 Enigma protein 0.54 0.23 353121 IPI00693691 Macrophage-capping protein 0.55 0.18 530409 IPI00688006 Prohibitin 0.55 0.03 511475 IPI00685691 RuvB-like 1 0.57 0.09 327682 IPI00700792 Guanine-nucleotide-binding protein subunit beta-2-like 1 0.58 0.15 615447 IPI00686420 Nucleoside diphosphate kinase B 0.59 0.21 281574 IPI00694641 Ezrin 0.59 0.20 515646 IPI00693645 Chloride intracellular channel protein 1 0.59 0.19 513410 IPI00691068 hnRNP AB 0.60 0.16 281615 IPI00692627 Retinal dehydrogenase 1 0.60 0.09 541202 IPI00734138 hnRNP H 0.62 0.10 282485 IPI00695563 Sulfotransferase 1A1 0.62 0.09 535119 IPI00689197 Cytokine-induced apoptosis inhibitor 1 0.63 0.13 514355 IPI00700509 T-complex protein 1 subunit beta 0.63 0.02 507197 IPI00702566 Serine hydroxymethyltransferase, mitochondrial precursor 1.65 0.42 282689 IPI00706002 Annexin A2 1.67 0.01 539218 IPI00716493 Calumenin precursor 1.68 0.23 533746 IPI00698338 Dihydropyrimidinase-related protein 2 1.73 0.4 513793 IPI00715791 eIF-3F 1.73 0.29 505968 IPI00704474 Acyl-coenzyme A dehydrogenase, C-4 to C-12 straight chain 1.73 0.23 506059 IPI00699601 Acyl-coenzyme A synthetase long-chain family member 6 1.75 0.02 540643 IPI00701266 Lamin B1 1.77 0.21 281544 IPI00708322 Tropomyosin alpha-1 chain 1.79 0.27 512584 IPI00711352 Small nuclear ribonucleoprotein polypeptide A 1.79 0.15 510201 IPI00698102 Serine/threonine kinase receptor-associated protein 1.85 0.32 ENSBTAG00000033117 IPI00842846 Histone H3 1.88 0.17 504912 IPI00711479 Target of Myb1 2.04 0.50 281660 IPI00686760 L-Caldesmon 2.31 0.27 281181 IPI00713814 GAPDH 2.32 0.3 732539 IPI00697070 Cytokine-induced protein 29 kDa 2.64 0.66 539060 IPI00827112 Serpine 1 mRNA binding protein 1 2.67 0.66 507564 IPI00691167 hnRNPs A2 and B1 2.69 1.50 782669 IPI00690667 hnRNP A3 2.80 0.23 281961 IPI00717623 Osteoclast-stimulating factor 1 4.57 1.02 404144 IPI00689750 Lamin A/C 0.72–Ͼ10c 528135 IPI00696554 hnRNP K 0.43–3.03c 506218 IPI00697355 SNX-9 0.53–1.99c 505850 IPI00700182 eIF-4B 0.32–3.19c 286868 IPI00703564 60S acidic ribosomal protein P0 0.61–2.32c 516099 IPI00704836 Heat shock 27-kDa protein 1 0.42–Ͼ10c 516326 IPI00713660 Lamin B2 0.38–2.01c

a Gene identifiers referenced in the IPI database from the Entrez Gene (33) or the Ensembl (12) database are given. b Descriptions given in the IPI data set or the referenced gene in the Entrez Gene database. c See the text for details. d In three rounds of screening. One hundred nine of the 1,490 quantified protein spots representing 55 genes showed IOM ratios beyond the empirical cutoff values of 1.63 and 0.63. teins of each fraction. In both affinity fractions (cibacron and fraction and 82/340 in the heparin fraction). In the heparin heparin), the GO accession numbers with the highest frequen- fraction, GO:0008135 (“translation factor activity”) and GO: cies were GO:0000166 (“nucleotide binding”) (78/233 in the 0003743 (“translation initiation factor activity”) followed, cibacron fraction and 98/340 in the heparin fraction) and GO: whereas in the cibacron fraction, GO:0003676 (“nucleic acid 0017076 (“purine nucleotide binding”) (59/233 in the cibacron binding”) and GO:0017111 (“nucleoside-triphosphatase activ- VOL. 82, 2008 PROTEOME OF PrV-INFECTED CELLS 9693

FIG. 2. Scans of 2D electrophoretic gels representing the two affinity-purified fractions (BLUE, cibacron fraction; HEP, heparin fraction) and the flowthrough (FT) of the ASPE separation procedure. Differing protein patterns reflect an efficient separation into three well-defined protein fractions. Boxes indicate gel regions that were analyzed in more detail (Fig. 6 and 7) for modifications of eIF-4B and SNX-9 (box A), lamin B2 (box B), 60S acidic ribosomal protein P0 (box C), Hsp27 (box D), and hnRNP K (box in panel FT). 9694 SKIBA ET AL. J. VIROL.

FIG. 3. Graphic representation of separation results. Overlaps were calculated on the basis of the number of entries of identified proteins found in the IPI database. Shown are overlaps between the different fractions of the ASPE (BLUE, cibacron fraction; HEP, heparin fraction; FT, flowthrough) (A) and overlaps between the cibacron, heparin, and phosphoprotein (PP) fractions (B) demonstrating a high selectivity of the ASPE procedure (A) and a good complementarity of the ASPE with phosphoprotein extraction (B). FIG. 4. Distribution of isotope ratios of proteins identified in a control experiment with labeled and unlabeled mock-infected cells. Isotope ratios were calculated as described in Materials and Methods. ity”) were the next most numerous. All mentioned annotations The frequency of isotope ratios was registered in steps of 0.01. As were significantly overrepresented in the affinity fractions com- expected, the distribution centers around 1.0. Quantiles are given in percentages, and the corresponding isotope ratios are shown as plain pared to the raw extract on the basis of P values of 0.05. Thus, numbers. both affinity fractions contained a high abundance of proteins with molecular functions in accordance with the properties of cibacron blue F3G-A (nucleotide binding) and heparin (DNA binding, with affinity for translation factors). erable fraction of available Hsp27 had undergone modification Determination of a cutoff value. In a pilot experiment, a mix at 4 h after infection. Spot A was not detectable by Coomassie of unlabeled and labeled cell extracts, both originating from staining of gels from mock-infected cell extracts (not shown) mock-infected cells, was analyzed. The purpose of this exper- and contains only very little material from mock-infected ref- iment was to determine the precision and the variation of our erence cells used in the SILAC procedure (see spectra in Fig. quantitation. As expected, most of the isotope ratios were close 6B). Other stress-related proteins that had been identified and to 1.0, and the frequency distribution of isotope ratios was very quantified (alpha crystalline B chain; heat shock 70-kDa pro- narrow (Fig. 4) so that empirical 1% and 99% quantiles were teins 1B, 5, and 9B; heat shock cognate 71-kDa protein; and determined for isotope ratios of 0.63 and 1.63 and were used as heat shock protein 90-alpha) did not show significant varia- cutoff values in the following infection experiments. tions. Infection experiments. In three independent infection ex- Lamins. Lamin A/C was found mainly in the phosphopro- periments, quantitative protein profiles were determined for tein fraction but was also present in the nonphosphoprotein PrV-infected MDBK cells. Figure 5 shows that the distribution and heparin fractions of the ASPE in two strings of poorly of IOM ratios in the five protein fractions is much broader than resolved protein spots representing the unprocessed lamin A the distribution of isotope ratios in the control experiment and the approximately 20-kDa-smaller proteolytic cleavage (Fig. 4), indicating that infection leads to numerous changes in product, lamin C (Fig. 7A). For both proteins, a shift to more the quantitative composition of the MDBK proteome. Up- and acidic isoforms was observed after infection. As found for the downregulated proteins were about equal in all fractions, most acidic modification of Hsp27, the protein spot represent- which was surprising, since under the experimental conditions ing the most acidic variant of lamin C contained hardly any used, the biosynthesis of cellular proteins is severely inhibited material from mock-infected cells. Lamin B2 was found in one due to PrV-mediated host cell shutoff (4, 20), and a significant major and one minor more acidic spot (Fig. 7B). Considering bias to IOM ratios smaller than 1.0 had been expected. For a the relative protein levels and the distribution of the total mass number of proteins that were identified in multiple isoforms, between the two spots, gains and losses are not nearly balanced differential or even inverse effects were found for different so that under the condition that no other lamin B2 isoforms charge variants (Fig. 6 and 7). In subsequent experiments, have been missed, a total loss of protein occurred after infec- proteins that showed relative protein levels beyond the calcu- tion. Lamin B1 was also identified in three protein spots with lated cutoff values or showed interesting differential regula- quantitative variations within the isoforms being less pro- tions were reanalyzed. All qualitative and quantitative data nounced than those for lamin B2, although for lamin B1 in- reported in the text or in figures rely on a minimum of three creasing abundances were also found with increasing acidity experiments. (IOM ratios from the most acidic spot to the least acidic spot Stress response proteins. Heat shock protein 27 was found were 1.77, 1.08, and 0.80). in five spots, two of which (spots B and D) (Fig. 6A) represent Proteins involved in translation. Although numerous ribo- over 90% of the total protein. Relative protein levels of 2.11 somal proteins and translation factors were identified and and 0.57 in spots B and D, respectively, indicate that a consid- quantified, only one ribosomal protein (60S acidic ribosomal VOL. 82, 2008 PROTEOME OF PrV-INFECTED CELLS 9695

FIG. 5. Distribution of relative cellular protein levels 4 h after infection with PrV-Ka. Error bars indicate the standard deviations of the isotope ratios of the peptides used for the calculation of the IOM ratio. PP, phosphoprotein fraction; PP-FT, flowthrough of the PhosphoProtein purification kit (nonphosphoproteins); BLUE, cibacron fraction; HEP, heparin fraction; FT, ASPE flowthrough. Cutoff values of 0.63 and 1.63 are given as horizontal lines. protein P0) (Fig. 7C) and two translation initiation factors functionally related protein, ezrin-radixin-moesin-binding showed a significant (eukaryotic initiation factor 4, subunit B phosphoprotein 50 (EBP50) was found with decreased relative [eIF-4B]) (Fig. 7D) or a moderate response (eIF-3F) (Table 1) levels (IOM of 0.48 Ϯ 0.06) in the heparin fraction. Glyceral- to PrV infection. eIF-4B was present in seven charge variants dehyde-3-phosphate dehydrogenase (GAPDH), which, apart most likely representing different phosphorylation levels. from its function in the glycolytic pathway, is involved in in- hnRNPs. hnRNPs were highly enriched in the cibacron frac- tracellular membrane trafficking (55, 57), was present in the tion with the exception of hnRNP K, which reliably separated cibacron fraction and found to be markedly upregulated (IOM into the flowthrough of the ASPE (Fig. 2). A number of of 2.32 Ϯ 0.30). Relative levels of the other glycolytic enzymes hnRNP representatives like hnRNP A3 (IOM of 2.80 Ϯ 0.23), that were identified and quantified (fructose-bisphosphate al- hnRNP D (IOM of 0.42 Ϯ 0.09), and hnRNP A2/B1 (IOM of dolase, triosephosphate isomerase, phosphoglycerate kinase, 2.69 Ϯ 1.50) showed significant variation after infection, while enolase, and pyruvate kinase) did not differ significantly be- others (hnRNP A1 and hnRNP F) remained constant or were tween infected and noninfected cells. only slightly changed (IOM of 0.62 Ϯ 0.1 for hnRNP H and IOM of 0.60 Ϯ 0.16 for hnRNP A/B). Of the six spots that were DISCUSSION identified as being hnRNP K (Fig. 7E), two migrated with a slightly smaller apparent molecular weight and decreased in The aim of this study was to systematically screen for alter- abundance after infection, whereas relative levels of the ations in the amounts of cellular proteins during infection with slightly larger proteins increased, with gains being more pro- PrV. To increase the number of cellular proteins that could be nounced for the more acidic isoforms. identified by 2D gel electrophoresis followed by mass spec- Proteins related to intracellular transport and the cytoskel- trometry, a simple prefractionation scheme was established, eton. Sorting nexin 9 (SNX-9) (Fig. 7D), enriched in the hep- which yielded three fractions with well-defined compositions. arin fraction, appeared in four distinct spots, which followed Overlaps between fractions were low, and the fractions con- the observed shift to more acidic isoforms. However, in this tained a high abundance of proteins with the expected bio- case, the mass gain was not highest for the most acidic form. A chemical properties and molecular functions. The parallel ex- 9696 SKIBA ET AL. J. VIROL.

FIG. 6. (A) Detailed view from a 2D electrophoretic gel of a heparin fraction analyzed in the pH range 4 to 7 (box D in Fig. 2). Five protein spots were identified as being Hsp27, and the relative protein levels, given in numbers, were calculated as described in the text. Details of mass spectra originating from spot A (most acidic isoform) and spot E (least acidic isoform) show two peptide peaks containing one (1,413 Da) or two (1,163 Da) leucine residues. IOM ratios were calculated from intensity ratios of peak pairs with a mass distance corresponding to the number of leucine residues present, in this case 3 Da (1,413 Da) and 6 Da (1,163 Da). The respective deuterium-labeled peaks at 1,416 Da and 1,169 Da representing peptides from mock-infected cells are marked with asterisks. Both labeled and unlabeled peptides occur in their natural isotope patterns, giving rise to multiple peaks with descending intensities in 1-Da distances. Note that reliable relative quantitation was restricted to ratios between 1:10 and 10:1 so that IOM ratios of the most acidic spots of Hsp27 and lamin C (Fig. 7A) could be assessed only as “greater than 10”. traction of phosphoproteins was shown to be highly efficient, as Nevertheless, the IOM ratios of a considerable number of both procedures yielded largely complementary protein frac- protein spots did change significantly in either direction. More- tions. Prefractionation was not detrimental for the subsequent over, in several cases, e.g., Hsp27, lamin A/C, lamin B2, mass spectrometric quantitation using the SILAC technique. hnRNP K, SNX9, eIF-4B, or 60S acidic ribosomal protein P0, The high precision of the mass spectrometric quantitation al- inverse modulations of different charge variants of the same lowed us to set cutoff values close to 1.0, which provided the protein were observed. high sensitivity of the procedure for the detection of regulated Stress response proteins. The most acidic of the five iso- protein spots. forms of Hsp27 was detected almost exclusively in infected Overall, the cellular proteome appeared to be very stable. cells, indicating that PrV infection caused a sharp rise in these The relative levels of the vast majority of proteins were unaf- isoforms. Most probably, the charge variants are the result of fected by infection with PrV, and the calculated IOM ratios differential phosphorylation, which was previously described were close to 1.0 despite the known detrimental effects of PrV for human (37, 48) and bovine (28) Hsp27. A similar shift to infection on the stability of mRNA and cell protein synthesis higher phosphorylated forms of Hsp27 has been observed after due to viral host cell shutoff functions (29, 49). Thus, the treatment of bovine cells with cadmium (28), indicating that delayed character of PrV-induced host cell shutoff indicated by this reaction is not specific for infections with herpesviruses but microarray studies in porcine (12), rat (44), and human (6) rather reflects phosphorylation of Hsp27 during cellular stress cells was confirmed on the protein level for the bovine cells (27). However, other stress-related proteins that were identi- used here. From time course studies with unfractioned mate- fied and quantified showed no significant changes in relative rial and analysis with 2D electrophoresis in the pH range of 3 protein levels (data not shown). Hsp27 is a multifunctional to 10 (data not shown), there was no indication for a global protein (1) with strong antiapoptotic properties. It is unclear so decrease of protein levels up to 8 h after infection with PrV, far if the changes in levels of Hsp27 are related to the pUS3- indicating that the degradation of cellular mRNA by viral mediated suppression of apoptosis (14), which is observed af- pUL41 and other mechanisms of host cell shutoff seem to ter infection with PrV. exclusively target the RNA metabolism but do not interfere Lamins. The observed shift to more acidic isoforms of lamin with the physiological steady-state levels of most proteins, at A/C parallels the reported changes of 2D gel electrophoretic least not at early times after infection. patterns of lamin A/C caused by the viral kinase pUS3 after VOL. 82, 2008 PROTEOME OF PrV-INFECTED CELLS 9697

FIG. 7. Proteins that were differentially regulated in different isoforms. Relative abundances of the respective isoforms after infection are given in plain numbers.

infection with HSV-1 (35). In an ongoing study with a US3- encoding the respective ribosomal proteins (15, 52). With the deleted PrV mutant, the impact of pUS3(PrV) on the phos- exception of the redistribution seen within the isoforms of the phorylation pattern of lamin A/C will be addressed. Infection 60S acidic ribosomal protein P0, we also found stable levels of with HSV-1 is also accompanied by an intracellular rearrange- ribosomal proteins after PrV infection. Likewise, significant ment (5) and loss (50) of B-type lamins, which were identified changes in relative levels of translation factors were not ob- and quantified from the heparin fraction. A shift to more acidic served, with the exception of eIF-4B and the F subunit of isoforms was observed for both, which corresponds to the pre- eIF-3. viously reported phosphorylation of B-type lamins following Proteins related to intracellular transport and the cytoskel- HSV-1 infection (40). The hyperphosphorylation of the lamins eton. So far, none of the three proteins related to intracellular precedes the disintegration of the nuclear lamina in many transport and the cytoskeleton (SNX9, EBP50, and GAPDH), physiological processes, which is presumably a prerequisite for which we found to be significantly altered in abundance, have the transfer of PrV capsids from the nucleus into the cytoplasm been correlated with any step during herpesvirus replication. for secondary envelopment (34). However, they exhibit interesting features, which may have Proteins related to translation. After infection with HSV-1, functions related to herpesvirus infections. Members of the the synthesis of several ribosomal proteins and their assembly SNX family of proteins contain a Phox domain that mediates into ribosomes continue in spite of a general inhibition of binding to membrane-anchored phospholipids. They are in- cellular protein synthesis and a concomitant loss of the mRNA volved in intracellular membrane trafficking (51), which is im- 9698 SKIBA ET AL. J. VIROL. portant for the replication of enveloped viruses. SNX9 shares K) serves to validate our experimental system. The newly es- a relative promiscuity with other SNXs with respect to the tablished protocol can be easily adapted to study virus-host cell different phosphatidylinositols that are bound by the Phox do- interactions of any virus that can be propagated in cultured main, the presence of an SH3 domain, and the presence of a cells of a species with a sequenced and annotated genome. It BAR dimerization domain. SNX9 is activated by phosphor- can be further refined by the application of additional affinity ylation (30), which correlates with the observed shift to more matrices or the use of narrow-range isoelectric focusing strips negatively charged variants, which we observed after infection to improve yields of less abundant proteins. The demonstra- (Fig. 7D). After phosphorylation, SNX9 is translocated to the tion that most of the observed changes affected the distribution plasma membrane, where it can interact with transmembrane of protein in different posttranslationally modified isoforms proteins, but also recruits dynamin to the plasma membrane rather than absolute expression levels underlines the impor- (30). It is also required for effective clathrin-mediated endo- tance of analysis at the protein level for a comprehensive cytosis (53). By virtue of its BAR domain, it may be involved in understanding of the molecular effects of virus infections on the stabilization but also in the sensoring of strongly curved the cellular metabolism. membranes (30), which occur during budding. EBP50 is a peripheral membrane protein that resides at the ACKNOWLEDGMENTS plasma membrane and supports functions of the ezrin-radixin- This study was in part supported by the Deutsche Forschungsge- moesin proteins in connecting the actin cytoskeleton to the meinschaft (DFG Me 854/8). plasma membrane. Cytomorphological alterations of PrV in- We thank Barbara Bettin for expert technical assistance. fection have been attributed to actin stress fiber breakdown mediated by the pUS3 kinase (58), which may occur via the REFERENCES phosphorylation of EBP50. 1. Arrigo, A. P. 2007. The cellular “networking” of mammalian Hsp27 and its functions in the control of protein folding, redox state and apoptosis. Adv. 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VI. Gene expression profiling of Pseudorabies virus (PrV) infected bovine cells by combination of transcript analysis and quantitative proteomic techniques.

Veterinary Microbiology 143 (2010) 14–20

Contents lists available at ScienceDirect

Veterinary Microbiology

journal homepage: www.elsevier.com/locate/vetmic

Gene expression proﬁling of Pseudorabies virus (PrV) infected bovine cells by combination of transcript analysis and quantitative proteomic techniques

Martin Skiba a, Frithjof Glowinski a, Dirk Koczan b, Thomas C. Mettenleiter a, Axel Karger a,* a Institute of Molecular Biology, Friedrich-Loefﬂer-Institut, Su¨dufer 10, 17493 Greifswald-Insel Riems, Germany b Department of Immunology, University of Rostock, Schillingallee 70, 18055 Rostock, Germany

ARTICLE INFO ABSTRACT

Keywords: Infection of target cells by alphaherpesviruses leads to extensive modulation of host cell Pseudorabies virus gene expression. To gain detailed information on the molecular pathways affected by US3 protein kinase infection of Madin–Darby bovine kidney (MDBK) cells with PrV, transcript analysis was Two-dimensional gel electrophoresis combined with a stable isotope-based quantitative proteomic approach (SILAC). Four Stable isotope labelling with amino acids in hours after infection cells were harvested and processed in parallel either for transcript cell culture (SILAC) analysis, for subcellular fractionation into nuclei and cytosol, for extraction of MALDI-TOF MS phosphoproteins, or for affinity extraction with Heparin Sepharose and Cibacron Blue F3G-A-Sepharose. All fractions were further analysed by large format two-dimensional gel electrophoresis in different pH-ranges to maximize the number of proteins to be identified and quantified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). Cell fractionation was quick and easy to perform but in comparison to affinity fractionation yielded lower numbers of identified and quantified proteins. After infection with PrV, only two of the 55 proteins with significantly modulated protein levels showed significant changes in transcript levels, indicating that posttranslational modifications may play a major role in the cellular response to PrV infection. Application of isotope labelling to cell cultures infected with wild-type PrV-Ka and a US3 protein kinase negative mutant allowed to monitor pUS3-dependent changes in the expression levels of viral proteins pUL29, pUL39 and pUL42. ß 2010 Elsevier B.V. All rights reserved.

1. Introduction homologs, which have been reported to interfere with the stability of cellular transcript RNA and thus impede Herpesviral genes are expressed in three major cellular protein biosynthesis. As the vhs effect is rather temporal classes in a regulated cascade which is controlled unspeciﬁc, it affects numerous transcripts as has been by viral transcription activators and negative feedback shown for Herpes simplex virus-1 (HSV-1), the prototypic mechanisms. But herpesviruses do not only control the alphaherpesvirus but also for PrV by microarray transcript expression of their own genes, infection impacts host cell analyses (Blanchard et al., 2006; Flori et al., 2008; Ray and protein expression as well. Most prominent mechanism for Enquist, 2004). The impact of the infection by an the interference of an alphaherpesvirus infection with alphaherpesvirus on host cell protein levels is less well cellular gene expression is the ‘virion host shutoff’ (vhs) documented. Alphaherpesviruses code for several gene mediated among other proteins by the UL41 protein products with enzymatic functions involved in posttranslational protein metabolism like the protein kinases pUL13 and pUS3 as well as pUL36, a large structural * Corresponding author. Tel.: +49 38351 7251; fax: +49 38351 7275. protein with deubiquitinating activity in HSV-1 (Katten- E-mail address: [email protected] (A. Karger). horn et al., 2005). In two recent proteome studies with

HSV-1 (Antrobus et al., 2009) and Pseudorabies virus level after infection with PrV-Ka were compared to infected cells (Skiba et al., 2008) systematic screens for elucidate the mechanisms of the regulation of host cell quantitative changes in levels of cellular proteins during protein expression by PrV-infection. the early phase of infection have been performed. Up- or Special attention was given to the US3 protein of PrV, a down-regulation was observed for 103 (HSV-1) and 109 multifunctional viral protein kinase with antiapoptotic (PrV) protein spots 6 h (HSV-1) or 4 h (PrV) after infection activity (Ogg et al., 2004) which is also involved in stress of human Hep-2 (HSV-1) or bovine MDBK (PrV) cells while fibre breakdown (Van Minnebruggen et al., 2003) and in the expression levels of the vast majority of host cell the egress of primary PrV particles from the nucleus into proteins was unaltered at these early times after infection. the cytoplasm (Klupp et al., 2001). Therefore cell fractio- In both studies a number of proteins showed quantitative nation experiments were carried out with extracts from shifts within charge variants (Skiba et al., 2008) or mobility cells that had been infected with the wild-type PrV strain shifts (Antrobus et al., 2009) upon infection, indicating that Kaplan and a US3-negative mutant in order to assess pUS3- infection by either virus may lead to alterations in the post dependent modulation of expression of nuclear proteins. translational modifications of host cell proteins. With the exception of Lamin A/C and the heterogeneous nuclear ribonucleoprotein (hnRNP) K, the lists of modulated 2. Materials and methods proteins from both studies are mutually exclusive, which might result from the different cells and viruses used but 2.1. Experimental setup also could be a consequence of the different analytical approaches, which may have lead to different fractions of The workflow of the proteome analysis is depicted in the proteome that were analysed. Fig. 1. The strategy was to maximize the number of In this study, we have analysed the cellular response identified and quantitated proteins by combination of two to PrV infection by combination of microarray transcript independent prefractionation techniques, cell fractiona- analysis with proteome analysis. For the latter, the tion and affinity enrichment of phosphoproteins, heparin- published affinity solid phase extraction (ASPE) protocol binding and nucleotide-binding proteins. All fractions (Skiba et al., 2008) for the prefractionation of the were then analysed by high-resolution large size 2DE in proteome was complemented by subcellular fractiona- one (phosphoproteins and non-phosphoproteins, pH 3–10) tion into a cytosolic and a nuclear fraction with a or two pH ranges (all other fractions, pH 4–7 and pH 6–9). commercially available kit. The objective of this com- All experiments were repeated four times with 2 technical bined fractionation scheme was to yet increase the replicates each. Data was combined in a joint database, number of quantified proteins but also to specifically from which the results were then extracted. Mass target nuclear proteins as PrV, like all herpesviruses, spectrometric quantitation relies on the SILAC approach replicates in the nucleus. Protein fractions prepared with (Ong et al., 2002), which requires two cell batches with both protocols were then analysed by two-dimensional isotope labelled and unlabelled proteins as starting gel electrophoresis (2DE) and all visible proteins were material. Depending on the type of experiment, labelled processed for mass spectrometric identification and and unlabelled cells were infected with PrV-Ka and mock quantitation using the SILAC (stable isotope labelling infected to assess the effect of infection on the expression with amino acids in cell culture) approach (Ong et al., levels of host cell proteins or they were infected by PrV-Ka 2002). Relative protein levels and relative transcript and PrV-DUS3 to study the effect of US3 on the expression levels of proteins significantly modulated on the protein levels of host cell proteins and other viral proteins.

Fig. 1. Schematic representation of the workflow used for the proteome analysis. Before infection, two batches of cell cultures were passaged in standard cell culture medium or a medium in which L-leucine was exchanged for a heavy isotope labelled counterpart. Depending on the type of experiment cells were simultaneously infected with PrV-Ka and mock infected or infected by PrV-Ka and PrV-DUS3. Four hours after infection cell extracts were then mixed at a 1:1 protein ratio and processed for affinity fractionation with a phosphoprotein-specific matrix, a sequential affinity solid phase extraction for nucleotide- binding proteins with Cibacron Blue F3G-A-Sepharose and heparin binding proteins with Heparin-Sepharose, and for subcellular fractionation with NE-PER kit into a cytosolic and a nuclear protein fraction. All fractions were further analysed by large-format 2D electrophoresis in the pH range 3–10 (phosphoproteins and non-phosphoproteins) or in the pH ranges 4–7 and 6–9. From all resulting gels, proteins visible by Coomassie staining were excised and processed for mass spectrometric identification and quantification by virtue of the stable isotope labels inserted into the proteins prior to infection. 16 M. Skiba et al. / Veterinary Microbiology 143 (2010) 14–20

2.2. Cells and viruses (Bruker, Bremen, Germany). Identification was performed using an in-house database compiled from all Bos taurus Madin–Darby bovine kidney cells (Madin and Darby, specific entries from the International Protein Index (IPI, 1958) were provided by the Collection of Cell Lines in Kersey et al., 2004) and all PrV-specific entries from the Veterinary Medicine, Insel Riems, Germany. PrV strain UniProt database (The UniProt Consortium, 2009). Quan- Kaplan (Kaplan and Vatter, 1959) and the US3-negative titation was carried out with an in-house software. Major deletion mutant PrV-DUS3 (Klupp et al., 2001) were used. data processing steps were the screening of the peak lists for leucine containing peptide peak pairs, calculation of the 2.3. Preparation of protein fractions isotope ratios of individual peptides, correction for outliers by robust statistical analysis and calculation of the mean Affinity solid phase extraction (ASPE) fractionation of isotope ratios, which very accurately reflect the relative whole cell extracts was described earlier (Skiba et al., 2008). protein levels in the samples to be compared. Isotope ratios In short, two cell batches were passaged in the presence of >1 reflect protein levels higher after infection with PrV-Ka conventional L-leucine or deuterated L-leucine to incorpo- than after mock infection. For experiments with mixtures rate a mass label of 3 Da for every leucine in the complete of cell extracts obtained after infection with PrV-Ka and complement of cellular proteins. Isotope labelled cells were PrV-DUS3, ratios >1 indicate higher levels of protein used as mock infected controls, whereas the unlabelled cell expression after infection with the mutant and vice versa. batch was infected with PrV-Ka. Four hours after infection, Based on an empirical cutoff value determined in earlier cells were harvested, extracted, and aliquots from the experiments (Skiba et al., 2008), relative expression levels extracts of an infected culture were mixed at a protein ratio corresponding to more than 1.63-fold up or les than 1.59- of 1:1. The mixture was then extracted by affinity matrices fold down were considered to be significant. specific for phosphoproteins (Phosphoprotein Purification Kit, Qiagen, Hilden, Germany) or nucleotide-binding pro- 2.6. Microarray analysis teins (Cibacron Blue F3G-A-Sepharose, GE Healthcare, Braunschweig, Germany) and heparin-binding proteins RNA was extracted from duplicate cell cultures of 106 (Heparin-Sepharose, GE Healthcare). cells each of PrV-infected and mock infected cells. Array Cell fractionation was carried out with the NE-PER hybridization on the Affymetrix GeneChip1 Bovine Gen- Nuclear and Cytoplasmatic Extraction Kit (Pierce Biotech- ome Array was performed according to the supplier’s nology, Rockford IL, USA). These experiments were carried instructions using the GeneChip1 Expression 3‘Amplifica- out with deuterium labelled cells infected with PrV-Ka and tion One-Cycle Target Labeling and Control reagents unlabelled cells infected with PrV-DUS3 to directly assay (Affymetrix, St. Clara, CA). In detail, the first strand cDNA the effects of the US3 protein on the relative expression was synthesized using 5 mg whole RNA sample and levels of virus and host cell proteins. Deuterium labelled superscript II reverse transcriptase (RNaseH minus) and unlabelled cells were mixed at a 1:1 DNA ratio and introducing a T7-(dT)24 primer. The second strand synth- then processed according to the protocol provided by the esis was done as strand replacement reaction using the E. manufacturer, which was scaled up 5-fold. Before 2DE coli DNA-polymerase I complex, hybrid strand specific RNA aliquots of the nuclear extract containing 600 mg and degrading RNaseH, a ligase reaction (E. coli DNA ligase) and aliquots of the cytosolic extract containing 1 mg protein finally by polishing with recombinant T4-polymerase. were concentrated and desalted by precipitation with Biotin-16-UTP was introduced by a linear amplifying in- trichloroacetic acid. vitro transcription reaction using T7 polymerase over night (16 h). Hybridisation was carried out over night (16 h) at 2.4. Two-dimensional gel electrophoresis 45 8C in the GeneChip1 Hybridisation Oven 640 (Affyme- trix, St. Clara, CA). Subsequent washing and staining Protein mixtures were subjected to 2DE essentially as protocols were performed with the Affymetrix Fluidics described previously (Skiba et al., 2008). Due to the low Station 450. For a signal enhancement an antibody yield of proteins in the pH range 3–6 that was observed in amplification was carried out using a biotinylated anti- this study, analysis was confined to the pH ranges 4–7 and streptavidin antibody (Vector Laboratories, U.K.), which 6–9 for the cell fractionation experiments. In short, protein was cross-linked by a goat IgG (Sigma, Germany) followed samples were focused on 24 cm isoelectric focusing strips by a second staining with streptavidin-phycoerythrin of the desired pH range and subjected to standard SDS conjugate (Molecular Probes, Invitrogen). The microarray electrophoresis on 12% acrylamide gels in a Dodeca Cell was scanned with a GeneChip Scanner 3000 (Affymetrix, apparatus (Biorad, Munich, Germany). St. Clara, CA) at 1.56 mm resolution. The data analysis was performed with the MAS 5.0 2.5. Mass spectrometry and data processing (Microarray Suite statistical algorithm, Affymetrix) software, probe level analysis with GeneChip Operating Identification and quantification of proteins after ASPE Software (GCOS 1.4) and the final data extraction with was carried out essentially as described (Skiba et al., 2008). DataMining Tool 3.1 (Affymetrix, St. Clara, CA). In short, all proteins detectable by colloidal Coomassie Transcripts were considered as ‘upregulated’ or ‘down- staining (Neuhoff et al., 1988) were excised from the gel, regulated’ when all four crosswise comparisons were digested with trypsin and analysed by MALDI-TOF/TOF classified as ‘increased’ or ‘decreased’ by MAS 5.0 software mass spectrometry with an Ultraflex I TOF/TOF instrument and the respective p-values were below 0.01. M. Skiba et al. / Veterinary Microbiology 143 (2010) 14–20 17

2.7. Gene ontology (GO) analysis

For all fractions lists of the Entrez Gene (Maglott et al., 2007) accession numbers of identiﬁed proteins were generated and used for the construction of Venn diagrams (http://www.cmbi.ru.nl/biovenn, Hulsen et al., 2008). For GO analysis bovine gene identiﬁers were translated into homologous human genes (http://david.abcc.ncifcrf.gov, Dennis et al., 2003) which were then analysed with the human GO dataset (http://www.genetools.microarray. ntnu.no, Beisvag et al., 2006).

3. Results

3.1. Fractionation efﬁciency

The specificity of the ASPE procedure has been documented earlier (Skiba et al., 2008). Representative gels from the cell fractionation procedure are shown in Fig. 2. Although 2D-electrophoretic protein spot patterns from both cell fractions seemed quite different, a number of corresponding protein spots were found in both fractions indicating some overlap of the nuclear and cytosolic Fig. 3. Venn diagram representing the number of genes of which gene proteome. After mass spectrometric identification of the products were identified after ASPE fractionation or cell fractionation into cytosolic (Cyt) and nuclear (Nuc) proteins. proteins using the IPI database, fractionation efficiency was analysed on the basis of the genes for which products were found in the respective fraction (Fig. 3). GO-analysis of the presence in either fraction in the GO-database. In the nuclear genes represented in the different fractions could partially fraction, the presence of mitochondrial (28) or endoplasmic explain the observed overlap, as 92 of the 135 proteins found reticulum (12) proteins indicated a considerable contam- simultaneously in both fractions were indeed annotated for ination with these organelles.

Fig. 2. 2D-electrophoretic protein patterns of nuclear (upper gels) and cytosolic (lower) fractions in the pH ranges 4–7 (left) and 6–9 (right). Rectangles indicate regions in which viral proteins pUL42 (A), pUL29 (B) and pUL39 (C) were identiﬁed. 18 M. Skiba et al. / Veterinary Microbiology 143 (2010) 14–20

In Fig. 3 the number of proteins identified after ASPE and cell fractionation and the respective overlaps are given. Although the majority of the proteins identified after cell fractionation were also found after ASPE fractionation, a number of important nuclear proteins were identified only after cell fractionation, among them Matrin-3, and a number of splicing factors.

3.2. Comparison of transcript and proteome analysis

In a previous study (Skiba et al., 2008) levels of 109 protein spots representing 55 genes had been found to be significantly modulated after infection with PrV-Ka in comparison to mock infected cells. All of them were represented on the Affymetrix Bovine Gene array used for transcript analysis but only for two, Lamin A/C and RuvB- like protein 2, significant downregulation was observed on the transcript level. For RuvB-like 2, this corresponds to the downmodulation found on the protein level. For Lamin A/ C, inverse modulation of different isoforms of the protein had been found. Generally, no correlation between changes in protein levels and transcript levels was Fig. 5. Details of 2D-electrophoretic gels from nuclear fractions as observed (Fig. 4) but the majority of transcript levels indicated in Fig. 2 representing viral proteins pUL29 (region B), pUL39 were distributed within margins of 1.5-fold up or down. A (region C) and pUL42 (region A). Fold changes of relative protein levels of linkage between transcript and protein levels was not individual protein spots after infection with PrV-DUS3 in comparison to observed suggesting that the observed changes in protein infection with PrV-Ka are given. Positive values indicate higher relative protein levels after infection with the mutant and vice versa. Each value levels are the consequence of posttranslational events, represents the mean of a minimum of three independent experiments. which may play a major role in cellular response to PrV infection. pUL29, pUL26, pUL25, pUL21, pUL19, pUL17, pUL8, pUS3), 3.3. Significantly modulated viral proteins levels of pUL29, pUL39 and pUL42 were found to be significantly altered depending on the presence of pUS3 Application of the SILAC approach (Fig. 1) to a mixture (Fig. 5). Neither of these proteins has been reported to be a of extracts obtained after infection with PrV-Ka and PrV- substrate of pUS3 kinase and only the pUL29 and pUL42 DUS3 allowed to assess the effects of pUS3 not only on the proteins contain a consensus sequence for phosphoryla- relative expression levels of host cell proteins but also on tion by pUS3 (Leader et al., 1991; Purves et al., 1986). Four relative levels of other viral proteins. Of the 15 PrV-specific protein spots with apparently the same molecular weight proteins that were identified in the course of this study but differing in charge were identified as pUL29, three of (pUL50, pUL49, pUL42, pUL39, pUL38, pUL37, pUL34, which could be quantitated. pUL42 also was identified in 4 distinct protein spots, three of which appeared to be charge variants of the same molecular weight and one with a slightly lower molecular weight than the other three. Levels of pUL29 and pUL39, were higher and level of pUL42 was lower in the absence of US3 than in its presence. Relative abundances of the three isoforms of pUL29 and the four isoforms of pUL42 were very similar (Fig. 5).

4. Discussion

Expansion of the ASPE prefractionation scheme devel- oped earlier (Skiba et al., 2008) by a commercially available cell fractionation kit allowed to directly target nuclear proteins for quantitative proteome analysis. Although the number of proteins that were identified and quantitated Fig. 4. Relative abundance of 55 proteins that have been found to be significantly modulated after infection with PrV-Ka in comparison to after cell fractionation was small compared to the analysis mock-infected cells after ASPE fractionation and 2D-electrophoretic of the ASPE fractions, the procedure will be useful for analysis (x-axis, fold change) were compared to the relative transcript further investigations. Cell fractionation was very rapid levels (y-axis, fold change) of the corresponding genes by microarray compared to the cumbersome but more far comprehensive analysis. Only for Lamin A/C (L) and the RuvB-like protein 2 (R) transcript ASPE fractionation and it enriched nuclear proteins, which levels were uniformly classified as ‘decreased’ by MAS 5.0 software as described in materials and methods. No correlation between protein and represent a preferential subproteome for the analysis of transcript levels was observed. cells infected by herpesviruses, as these replicate in the M. Skiba et al. / Veterinary Microbiology 143 (2010) 14–20 19 nucleus. Also, a number of proteins that had escaped observed indicating that the host cell response to PrV- analysis by ASPE were identified only after cell fractiona- infection is at least in part determined by post-translation. On the other hand, a considerable contamination of tional events. Application of this approach to cells infected the nuclear fraction with mitochondrial or endoplasmic with wild-type PrV-strain Kaplan and a US3-negative proteins was observed. mutant allowed to study the effect of the viral US3 kinase No correlation of protein and transcript levels was on the expression levels of other viral proteins and of host observed for the overwhelming majority of proteins that cell proteins on the background of an ongoing infection. were significantly modulated on the protein level after Expression levels of three viral proteins, pUL29, pUL39 and infection with PrV-Ka. We conclude that part of the cellular pUL42 were shown to be significantly influenced by the response to PrV-infection is mediated by post-transla- presence of pUS3. tional mechanisms and will require further in-depth protein analysis. Conflict of interest statement The experimental setup that is presented also allows the characterisation of proteome alterations triggered by The authors declare no conflict of interest. single viral proteins as was demonstrated with a US3- deleted mutant of PrV. It is remarkable that of the three viral proteins that were significantly modulated in a US3- Acknowledgements dependent manner, two (pUL29, the major DNA-binding protein and pUL39, the ribonucleoside-diphosphate reduc- This study has been supported by the Deutsche tase large subunit) were increased and one (pUL42, the Forschungsgemeinschaft (SPP 1175, DFG Me 854/8-2). DNA polymerase processivity factor) was decreased in the The expert technical assistance of Barbara Bettin and nuclear fraction in the absence of pUS3. All three proteins Stefanie Jachmann is acknowledged. are involved in the viral DNA metabolism and a possible role for pUS3 in this respect remains to be elucidated. Only References pUL29 and pUL42 carry a consensus sequence for phosphorylation by US3 but neither of the three proteins Antrobus, R., Grant, K., Gangadharan, B., Chittenden, D., Everett, R.D., has so far been functionally related to the kinase. pUL29 Zitzmann, N., Boutell, C., 2009. Proteomic analysis of cells in the early stages of herpes simplex virus type-1 infection reveals widespread and pUL42 were present in the nuclear fraction (Fig. 2) and changes in the host cell proteome. Proteomics 9, 3913–3927. in the phosphoprotein fraction of the ASPE (not shown) Beisvag, V., Junge, F.K., Bergum, H., Jolsum, L., Lydersen, S., Gunther, C.C., where they produced essentially the same protein spot Ramampiaro, H., Langaas, M., Sandvik, A.K., Laegreid, A., 2006. Gene- Tools-application for functional annotation and statistical hypothesis pattern and relative protein levels after infection with PrV- testing. BMC Bioinformatics 7, 470. Ka and PrV-DUS3. pUL29 and pUL42 appeared as a string of Blanchard,Y.,LeMeur,N.,LeCunff,M.,Blanchard,P.,Leger,J.,Jestin,A., charge isomers relative levels of which were on the 2006. Cellular gene expression survey of PseudoRabies Virus (PRV) infected Human Embryonic Kidney cells (HEK-293). Vet. Res. 37, average approximately 2.4-fold higher for pUL29 and 1.7- 705–723. fold lower for pUL42 in the absence of pUS3. As all isoforms Conner, J., 1999. The unique N terminus of herpes simplex virus type 1 within one protein showed very similar relative levels of ribonucleotide reductase large subunit is phosphorylated by casein modulation and there is no indication that individual kinase 2, which may have a homologue in Escherichia coli. J. Gen. Virol. 80 (Pt 6), 1471–1476. isoforms represent different post translational modifica- Dennis Jr., G., Sherman, B.T., Hosack, D.A., Yang, J., Gao, W., Lane, H.C., tions induced by US3 but rather that presence of pUS3 Lempicki, R.A., 2003. DAVID: database for annotation, visualization, decreased and increased total amounts of pUL29 and and integrated discovery. Genome Biol. 4, 3. Flori, L., Rogel-Gaillard, C., Cochet, M., Lemonnier, G., Hugot, K., Chardon, pUL42, respectively. The mechanisms by which this is P., Robin, S., Lefevre, F., 2008. Transcriptomic analysis of the dialogue achieved remain to be determined. In the fractionation between Pseudorabies virus and porcine epithelial cells during infec- scheme presented here, pUL39 was present in the nuclear tion. BMC Genomics 9 (March), 123. Hulsen, T., de Vlieg, J., Alkema, W., 2008. BioVenn—a web application for fraction (Fig. 2) and in the nucleotide-binding fraction of the comparison and visualization of biological lists using area-pro- the ASPE (not shown), but not in the phosphoprotein portional Venn diagrams. BMC Genomics 9, 488. fraction. In contrast to the homologous protein of HSV-1, Kaplan, A.S., Vatter, A.E., 1959. A comparison of herpes simplex and pseudorabies viruses. Virology 7, 394–407. which can be phosphorylated by casein kinase 2 (Conner, Kattenhorn, L.M., Korbel, G.A., Kessler, B.M., Spooner, E., Ploegh, H.L., 1999), pUL39 of PrV has not been reported to be a 2005. A deubiquitinating enzyme encoded by HSV-1 belongs to a phosphoprotein, maybe because it lacks the N-terminal family of cysteine proteases that is conserved across the family Herpesviridae. Mol. Cell 19, 547–557. part, which carries the phosphorylation site of the HSV-1 Kersey, P.J., Duarte, J., Williams, A., Karavidopoulou, Y., Birney, E., Apwei- homolog. ler, R., 2004. The International Protein Index: an integrated database for proteomics experiments. Proteomics 4, 1985–1988. 5. Conclusion Klupp, B.G., Granzow, H., Mettenleiter, T.C., 2001. Effect of the pseudorabies virus US3 protein on nuclear membrane localization of the UL34 protein and virus egress from the nucleus. J. Gen. Virol. 82, 2363– The combination of affinity solid phase extraction, cell 2371. fractionation, 2DE and SILAC-based protein quantitation is Leader, D.P., Deana, A.D., Marchiori, F., Purves, F.C., Pinna, L.A., 1991. Further definition of the substrate specificity of the alpha-herpesvirus a powerful and promising tool for the analysis of the protein kinase and comparison with protein kinases A and C. Biochim. cellular response to virus infection. For host cell proteins of Biophys. Acta 1091, 426–431. which relative levels had been found to be significantly Madin, S.H., Darby Jr., N.B., 1958. Established kidney cell lines of normal adult bovine and ovine origin. Proc. Soc. Exp. Biol. Med. 98, 574–576. modulated after infection with PrV-Ka no correlation of Maglott, D., Ostell, J., Pruitt, K.D., Tatusova, T., 2007. Entrez gene: gene- relative transcript levels with relative protein levels was centered information at NCBI. Nucleic Acids Res. 35, D26–D31. 20 M. Skiba et al. / Veterinary Microbiology 143 (2010) 14–20

Neuhoff, V., Arold, N., Taube, D., Ehrhardt, W., 1988. Improved staining of with herpesviruses: studies with synthetic substrates [corrected] proteins in polyacrylamide gels including isoelectric focusing gels indicate structural requirements distinct from other protein kinases. with clear background at nanogram sensitivity using Coomassie Biochim. Biophys. Acta 889, 208–215. Brilliant Blue G-250 and R-250. Electrophoresis 9, 255–262. Ray, N., Enquist, L.W., 2004. Transcriptional response of a common Ogg, P.D., McDonell, P.J., Ryckman, B.J., Knudson, C.M., Roller, R.J., 2004. permissive cell type to infection by two diverse alphaherpesviruses. The HSV-1 Us3 protein kinase is sufficient to block apoptosis induced The Journal of Virology 78, 3489–3501. by overexpression of a variety of Bcl-2 family members. Virology 319, Skiba, M., Mettenleiter, T.C., Karger, A., 2008. Quantitative whole-cell 212–224. proteome analysis of pseudorabies virus-infected cells. J. Virol. 82, Ong, S.E., Blagoev, B., Kratchmarova, I., Kristensen, D.B., Steen, H., Pandey, 9689–9699. A., Mann, M., 2002. Stable isotope labeling by amino acids in cell The UniProt Consortium, 2009. The Universal Protein Resource (UniProt) culture, SILAC, as a simple and accurate approach to expression 2009. Nucleic Acids Res. 37 D169–D174. proteomics. Mol. Cell Proteomics 1, 376–386. Van Minnebruggen, G., Favoreel, H.W., Jacobs, L., Nauwynck, H.J., 2003. Purves, F.C., Deana, A.D., Marchiori, F., Leader, D.P., Pinna, L.A., 1986. The Pseudorabies virus US3 protein kinase mediates actin stress fiber substrate specificity of the protein kinase induced in cells infected breakdown. J. Virol. 77, 9074–9080.

VII. Influence of Insertion Site of Avian Influenza Virus Hemagglutinin (HA) Gene Within the Newcastle Disease Virus Genome on HA Expression.

JGV Papers in Press. Published November 10, 2010 as doi:10.1099/vir.0.027268-0

Influence of Insertion Site of Avian Influenza Virus Hemagglutinin (HA) Gene within the Newcastle Disease Virus Genome on HA Expression

5 Kristina Ramp1, Martin Skiba1, Axel Karger1, Thomas C. Mettenleiter1 and Angela Römer- Oberdörfer1*

10 1 Institute of Molecular Biology, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Südufer 10, D-17493 Greifswald-Insel Riems, Germany

Running title: AIV HA expression from NDV genome 15 Keywords: NDV, expression, AIV, hemagglutinin

Corresponding author: Angela Römer-Oberdörfer 25 Südufer 10 D 17493 Greifswald-Insel Riems Tel. + 49 38351 71107 Fax + 49 38351 71275 Email: [email protected] 30

Word count abstract: 138 Word count text: 2468 Number of figures: 3

1 Summary 35 Members of the Mononegavirales express their genes in a transcription gradient from 3’ to 5’. To assess how this impacts on expression of a foreign transgene we inserted the hemagglutinin (HA) of highly pathogenic avian influenza virus (HPAIV) A/chicken/Vietnam/P41/05 (subtype H5N1) between the P and M, M and F, or F and 40 HN genes of attenuated Newcastle Disease Virus (NDV) Clone 30. In addition, the gene encoding neuraminidase of HPAIV A/duck/Vietnam/TG24-01/05 (subtype H5N1) was inserted into the NDV genome either alone or in combination with the HA gene. All recombinants replicated well on embryonated chicken eggs. Expression levels of HA-specific mRNA and protein were quantitated by northern blot analysis 45 and mass spectrometry with good correlation. HA-expression levels differed only moderately and were highest in the recombinant carrying the HA-insertion between the F and HN genes of NDV.

2 Newcastle Disease (ND) is a highly contagious infection of many avian species which 70 causes substantial economic losses in the poultry industry worldwide. The causative agent is Newcastle Disease Virus (NDV), a negative strand RNA virus which belongs to the genus Avulavirus within the family Paramyxoviridae (Fauquet et al., 2005) of the order Mononegavirales. Its genome is 15,186 nucleotides long and encodes for six structural proteins: the nucleoprotein (NP), phosphoprotein (P), matrix protein (M), 75 fusion protein (F), haemagglutinin-neuraminidase protein (HN) and the RNA dependent RNA polymerase (L). Additional proteins V and W are transcribed from the P gene by an RNA editing mechanism (Steward et al., 1993). Based on pathogenicity for chickens, NDV is categorized into lentogenic, mesogenic and velogenic pathotypes. The virulence of a NDV strain is determined by the activation 80 cleavage of the fusion protein by cellular proteases (Nagai et al., 1976). A polybasic amino acid stretch between amino acids (aa) 112 and 116 of the F-protein characterizes highly virulent NDV, whereas NDV of low virulence exhibit only single basic aa at this site (Collins et al., 1993; Glickman et al., 1988; Millar et al., 1988; Toyoda et al., 1987). To protect poultry from ND, vaccination is practiced worldwide 85 using either inactivated or live vaccines based on lentogenic viruses, such as NDV La Sota or Clone 30, which are applied by spray or drinking water.

The generation of recombinant NDV by reverse genetics (Krishnamurthy et al., 2000; Peeters et al., 1999; Römer-Oberdörfer et al., 1999) has made NDV amenable to 90 genetic manipulation. Insertion of foreign genes into the NDV genome resulted in the development of bivalent vector vaccines. For this purpose, efficient expression of an immunogenic foreign protein by the NDV vector is required. Expression levels of genes of non-segmented negative strand RNA viruses are primarily regulated by their position relative to the single promoter, and also by cis-acting sequences 95 located at the beginning and the end of each gene. For vesicular stomatitis virus (VSV) it was shown that amounts of mRNA and protein decreased following the order of transcription from the 3´proximal to the 5´distal end of the viral genome forming a transcription gradient (Wertz et al., 2002; Whelan et al., 2004). To assay whether this also applies to a foreign transgene, Zhao et al. (Zhao & Peeters, 2003) inserted the 100 gene encoding secreted alkaline phosphatase (SEAP) into different intergenic regions of the NDV genome and showed that SEAP activities were highest when the transgene was inserted between the NDV M and F genes, and not between the more

3 3’ insertion site between P and M. These results did not correlate with the proposed transcription gradient described for Mononegavirales . 105 Development of NDV vector vaccines expressing AIV immunogens has become a promising option to simultaneously fight two important virus diseases of poultry. The negative strand RNA genome of influenza A viruses (Family Orthomyxoviridae) consists of eight segments coding for eleven proteins (Chen et al., 2001; Palese & 110 Shaila, 2006). The surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) exist in different antigenic variations (H1-H16; N1-N9) which are used to classify Influenza A virus into various subtypes (Fouchier et al., 2005; Webster et al., 1992). A further classification into low pathogenic (LP), notifiable low pathogenic (NLP) and highly pathogenic (HP) pathotypes is made according to their ability to elicit disease 115 (Alexander, 1997). Outbreaks of highly pathogenic avian influenza are caused exclusively by H5 and H7 subtypes. Both have also the potential to cause zoonotic infections in humans. Therefore, the control of HPAIV infections is essential for the poultry industry and to prevent human infections. Currently available influenza vaccines, based on inactivated whole virus preparations or attenuated live virus, do 120 not allow differentiation between infected and vaccinated animals (DIVA principle) (reviewed by Fuchs et al. (Fuchs et al., 2009)). This problem could be overcome by vector vaccines. The successful use of NDV as vector for the expression of AIV hemagglutinin has been described in different studies, where the foreign gene was integrated between the P and M (DiNapoli et al., 2010; Ge et al., 2007; Nakaya et al., 125 2001; Park et al., 2006) or F and HN genes (Schröer et al., 2009; Veits et al., 2006). In both cases, recombinant viruses conveyed protection against NDV, as well as against influenza infection of the corresponding subtype. However, an improved vaccine virus which results in higher antibody levels against AIV after immunization would be desirable. Recently, it was shown that coimmunization with infectious 130 laryngotracheitis virus (ILTV) recombinants expressing H5 and N1 further increased the efficacy of an ILTV vector vaccine (Pavlova et al., 2009). Enhanced protection against lethal influenza virus infection was also achieved by other vector vaccines coexpressing HA and NA (Chen et al., 1999; Johansson, 1999; Qiao et al., 2003).

135 In this study, we created different NDV/AIV recombinants to analyze whether transcription of the foreign gene follows the NDV transcription gradient (Lamb &

4 Kolakofsky, 2001; Sakai et al., 1999; Wertz et al., 1998) or not. If so, insertion of AIV H5 gene in a more 3’ proximal position would result in higher protein yields. Therefore, H5 of HPAIV A/chicken/Vietnam/P41/05 (H5N1) was inserted into the 140 NDV genome between the P and M, M and F, and F and HN genes. In two additional recombinants, N1 of HPAIV A/duck/Vietnam/TG24-01/05 (H5N1) was inserted between F and HN genes alone, or together with a H5 insertion between P and M (Figure 1). All rescued viruses were characterized in vitro, and the expression levels of AIV HA and NA were determined by northern blot analysis. Furthermore, HA 145 protein expression was quantitated by mass spectrometry.

Lentogenic vaccine strain Clone 30 (GenBank accession no. Y18898) served as NDV backbone for all recombinant viruses. Sequences for hemagglutinin (HA) were based on HPAIV strain A/chicken/Vietnam/P41/05 (H5N1), GenBank accession no. 150 AM 183672 and for neuraminidase (NA) on HPAIV A/duck/Vietnam/TG24-01/05 (H5N1), GenBank accession no. AM183678. The sequence of H5 was identical to that of the previously described HA of recombinant NDVH5Vm (Römer-Oberdörfer et al., 2008). Recombinant NDVH5VmPM was generated by insertion of the amplified H5 gene of NDVH5Vm using primers with MluI sites, and gene end, as well as gene 155 start signal sequences. The amplified fragment consists of MluI site, gene end (GE) signal sequence of NDV P gene, nucleotide T as intergenic region, gene start (GS) signal sequence of NDV HN gene, non-coding region of NDV HN gene, open reading frame (orf) of AIV H5, non-coding region of NDV HN, and another MluI site. The NDV backbone was altered by introduction of an artificial single MluI site in front of the P 160 gene end signal sequence after backmutation of known artificial MluI sites of rNDV genome (Römer-Oberdörfer et al., 1999). NDVH5VmMF was obtained by insertion of the H5 gene flanked by MluI sites into a newly created single MluI site in front of the gene end signal sequence of M using a NDV genome without other artificial MluI sites. NDVN1FHN was generated by substitution of the H5Vm orf by the N1 orf using 165 NcoI and AflII sites. Since the AIV N1 orf contains a NcoI site, insertion was done in two steps. The resulting N1 orf sequence differs in one nucleotide from the sequence in GenBank as a result of the newly generated NcoI site which was used for cloning as previously described (Veits et al., 2006). NDVH5VmPMN1FHN expressing both AIV HA and NA was constructed by substitution of the NotI-BsiWI fragment of 170 NDVH5VmPM by that of NDVN1FHN (Figure 1). Recovery of infectious NDV from

5 cDNA was performed on BSR-T7/5 BHK cells which stably express phage T7 RNA polymerase (Buchholz et al., 1999). The in vivo replication of recovered virus was determined in ten-day old specific pathogen free (SPF) chicken eggs, which were 3 inoculated with 200 µl of 10 TCID50/ml of recombinant NDV. Virus titres in allantoic 175 fluids were determined on QM9 cells. All generated NDV/AIV recombinants replicated well, independent of the AIV H5 insertion site, however, with a delay in onset of replication compared to wild type NDV Clone 30. All generated NDV/AIV 8.0 8.5 recombinants reached a final titre (TCID50/ ml) of 10 to 10 (Figure 2). This demonstrates that neither the integration of the AIV H5 gene alone nor the 180 simultaneous integration of H5 and N1 genes into the NDV genome had a detrimental effect on virus replication despite the considerable size of the inserts of more than 3500 nt. Expression and cleavability of the HA protein of the NDV/AIV recombinants was determined by western blot analysis. Proteins of virus infected chicken embryo fibroblasts (CEF) were separated by sodium dodecylsulfate 185 polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes followed by incubation with polyclonal monospecific rabbit sera directed against AIV-H5 or AIV-N1, and incubation with the respective peroxidase (POD)- labelled secondary antibody. Three proteins of about 70 kDa , 50 kDa, and 25 kDa were detected after incubation with the AIV H5-specific antiserum, corresponding to 190 the uncleaved HA0 and the cleavage products HA1 and HA2 (Figure 3A). The HA protein with a polybasic cleavage site expressed by the recombinants NDVH5VmPM, NDVH5VmMF, NDVH5Vm, and NDVH5VmPMN1FHN was completely processed by cellular proteases. In contrast, the HA0 protein with a monobasic cleavage site expressed by NLPAIV H5N1 (A/common teal/Germany/Wv632/05), which was used 195 as a control, remained mainly uncleaved (Figure 3A). As expected, no HA protein was present in NDVN1FHN and rNDV. Expression of the N1 protein was confirmed for the recombinants NDVN1FHN, NDVH5VmPMN1FHN and NLPAIV H5N1 (A/common teal/Germany/Wv632/05) with an N1-specific antiserum (Figure 3A). Size differences of the N1 proteins expressed by the NDV recombinants 200 (A/duck/Vietnam/TG24-01/05, GenBank accession no.AM183678) and wild type AIV (A/common teal/Germany/WV632/05, GenBank accession no AM913983.1) reflect differences in lengths of the open reading frames (450 versus 470 amino acids) and different numbers of potential glycosylation sites (3 versus 7).

6 205 The relative H5- and N1-specific mRNA levels of the NDV/AIV recombinants were determined by northern blot analysis. Total RNAs were isolated from the virus- infected CEFs, separated in denaturing agarose gels, transferred to nylon membranes and hybridized with 32P-labelled antisense RNA specific for AIV H5, AIV N1 or NDV P. Signals were quantified by radioluminography using a FLA-3000 210 scanner (FujiFilm, Düsseldorf, Germany) and the Advanced Image Data Analyzer software (Raytest, Straubenhardt, Germany) for image analysis. The relative mRNA levels of HA and NA were calculated by normalization of their absolute signals to the phosphoprotein-specific signal in the same sample. For comparison of the different viruses, the relative mRNA levels of H5 as well as of N1 were again normalized to 215 the levels expressed by NDVH5VmPMN1FHN, which were set to 1.0. H5 mRNA expression of NDVH5Vm was 25 % higher compared to NDVH5VmPMN1FHN, whereas the H5 mRNA expression of NDVH5VmPM and NDVH5VmMF reached 94 % and 71 % of the level found for NDVH5VmPMN1FHN. As expected, insertion of AIV H5 in a more 3’ proximal position than N1 led to a decrease of N1-specific 220 mRNA. N1-mRNA expression from NDVH5VmPMN1FHN amounts to 53 % of that observed for NDVN1FHN (Figure 3B).

Relative HA protein expression levels in the NDV/AIV mutants were assayed by a combination of two-dimensional electrophoresis and quantitative mass spectrometry 225 using the SILAC (stable isotope labelling with amino acids in cell culture) technique (Ong et al., 2002) with some modifications (Skiba et al., 2008). This procedure is highly precise and has been used for the quantitation of cellular proteins in proteome studies but also to determine expression levels of viral proteins (Skiba et al., 2010; Skiba et al., 2008). In preceding 2D-electrophoretic analyses with cell extracts from 230 uninfected and NDV-infected cells, and cells infected with one of the recombinants, HA, the NDV-specific phosphoprotein and a number of cellular proteins were located in two-dimensional gels, and identified by mass spectrometry and in two-dimensional western blots. For the quantitation experiment, extracts from cells infected with NDVH5VmMF, NDVH5VmPM and NDVH5Vm were mixed with an extract from 235 deuterium labelled cell cultures infected with NDVH5VmPMN1FHN as internal standard. The mix was separated by 2D-electrophoresis and the relative HA-levels were determined by MALDI-TOF mass spectrometry. Like for the calculation of HA- specific relative mRNA-levels, the relative expression level of the HA protein was

7 normalized to the expression level of the NDV phosphoprotein which was determined 240 in the same manner from phosphoprotein samples isolated from the same gel. The relative HA protein levels were again normalized to the internal standard, recombinant NDVH5VmPMN1FHN, which was set to 1.0. As shown in Figure 3B, levels of HA mRNA and protein expression correlated well, indicating that HA mRNA and protein synthesis are tightly linked. Maximum HA protein expression was 245 observed after infection with recombinant NDVH5Vm which was approximately twofold higher than after infection with recombinant NDVH5VmMF in which the weakest expression was observed. As mentioned above, Zhao and colleagues constructed a panel of NDV recombinants expressing the reporter gene encoding SEAP in different positions within the genome (Zhao & Peeters, 2003). The 250 expression levels of SEAP were highest after insertion between M and F genes, and lowest between NP and P. In our study, we detected highest mRNA and protein levels of AIV H5 for NDV recombinants with the H5 insertion between F and HN. Although Zhao and Peeters (2003) did not test the insertion site between the F and HN genes, their as well as our results are in contrast to expression levels of NDV 255 proteins which decrease from the 3’ proximal to the 5’ distal end of viral genome (Lamb & Kolakofsky, 2001; Sakai et al., 1999; Wertz et al., 1998). Since the foreign H5 transgene of all our recombinants was identical, differences in H5 mRNA and protein level are caused by the different insertion sites within the NDV genome. Nevertheless, the observed differences in H5 expression levels of our NDV 260 recombinants were moderate, indicating that transgenes can be inserted at different positions in the NDV genome without severely affecting replication capacity of the recombinant virus. This result is consistent with results of Zhao & Peeters (Zhao & Peeters, 2003) for the expression of SEAP.

265 In summary, we show that NDV can accommodate HA, NA or both AIV glycoproteins increasing virus genome size by more than 3500 nt without significantly affecting virus replication. The in vitro expression levels of AIV H5 mRNA and protein were to some extent influenced by the location of the insertion site of the transgene within the NDV genome. Transgenic HA expression levels were highest when the HA gene was 270 inserted between the NDV F and HN genes. However, even then they were only twice as high as the lowest expression level detected in our assay. Animal experiments will have to demonstrate whether these moderate differences of HA

8 protein expression have an effect on the immune response after vaccination and protection of immunized chickens against HPAIV. 275 Acknowledgments We thank Martina Lange, Barbara Bettin and Stefanie Jachmann for technical assistance and Jutta Veits for academic discussions. This research was supported by European Commission (FP6; Project no. 044141). 280

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315

9 320 Figure Legend

Figure 1: Schematic representation of the genome organization of NDV and their recombinants with insertion of the hemagglutinin and/or neuraminidase genes of HPAIV H5N1. Transcription control signals are 325 marked by a triangle for transcription start and a dark gray rectangle for transcription stop sequences.

Figure 2: Replication kinetics of wild type NDV Clone 30 and recombinants expressing one or two AIV proteins in embryonated SPF chicken eggs. 3 330 Eggs were infected with 200 µl of 10 TCID50/ml of the viruses indicated and titres of progeny virus in allantoic fluids were determined at the indicated time points by titration on QM9 cells, followed by indirect immunofluorescence analysis.

335 Figure 3: A: Western blot analyses of NDV recombinants. Proteins of infected CEF cells were separated by SDS-PAGE and blotted onto nitrocellulose. Membranes were incubated with monospecific rabbit α-AIV-H5 serum and monospecific rabbit α -AIV-N1 serum. Proteins were detected by chemiluminescence after incubation 340 with suitable peroxidase labelled secondary antibodies B: Relative mRNA and protein expression levels of AIV-H5 in infected DF-1 cells. mRNA was quantitated by northern blot analysis (gray columns) and protein by quantitative mass spectrometry using the SILAC technique (black columns). Error bars indicate the standard 345 deviation of three independent experiments. Protein and mRNA levels were normalized to the NDV phosphoprotein expression levels from the same samples.

350

355

360

365

370

10 Reference List

Alexander, D. J. (1997).Newcastle disease and other avian Paramyxoviridae infections. In Diseases of poultry, 10th edn, pp. 541-569. Edited by B. W. 375 Calnek. Ames: Mosby-Wolfe iowa State University Press.

Buchholz, U. J., Finke, S. & Conzelmann, K. K. (1999).Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in tissue culture, and the human RSV leader region acts as a functional BRSV genome promoter. J Virol 73, 251-259.

380 Chen, W., Calvo, P. A., Malide, D., Gibbs, J., Schubert, U., Bacik, I., Basta, S., O'Neill, R., Schickli, J., Palese, P., Henklein, P., Bennink, J. R. & Yewdell, J. W. (2001).A novel influenza A virus mitochondrial protein that induces cell death. Nat Med 7, 1306-1312.

Chen, Z., Matsuo, K., Asanuma, H., Takahashi, H., Iwasaki, T., Suzuki, Y., 385 Aizawa, C., Kurata, T. & Tamura, S. (1999).Enhanced protection against a lethal influenza virus challenge by immunization with both hemagglutinin- and neuraminidase-expressing DNAs. Vaccine 17, 653-659.

Collins, M. S., Bashiruddin, J. B. & Alexander, D. J. (1993).Deduced amino acid sequences at the fusion protein cleavage site of Newcastle disease viruses 390 showing variation in antigenicity and pathogenicity. Arch Virol 128, 363-370.

DiNapoli, J. M., Nayak, B., Yang, L., Finneyfrock, B. W., Cook, A., Andersen, H., Torres-Velez, F., Murphy, B. R., Samal, S. K., Collins, P. L. & Bukreyev, A. (2010).Newcastle disease virus-vectored vaccines expressing the hemagglutinin or neuraminidase protein of H5N1 highly pathogenic avian 395 influenza virus protect against virus challenge in monkeys. J Virol 84, 1489- 1503.

Fauquet, C. M., Mayo, M. A., Maniloff, J., Desselberger, U. & Ball, L. A. (editors) (2005). Paramyxoviridae. In Virus Taxonomy, 8th edn, pp. 655-671. San Diego, London: Elsevier Academic Press.

400 Fouchier, R. A., Munster, V., Wallensten, A., Bestebroer, T. M., Herfst, S., Smith, D., Rimmelzwaan, G. F., Olsen, B. & Osterhaus, A. D. (2005).Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls. J Virol 79, 2814-2822.

Fuchs, W., Römer-Oberdörfer, A., Veits, J. & Mettenleiter, T. C. (2009).Novel 405 avian influenza virus vaccines. Rev Sci Tech 28, 319-332.

Glickman, R. L., Syddall, R. J., Iorio, R. M., Sheehan, J. P. & Bratt, M. A. (1988).Quantitative basic residue requirements in the cleavage-activation site

11 of the fusion glycoprotein as a determinant of virulence for Newcastle disease virus. J Virol 62, 354-356.

415 Johansson, B. E. (1999).Immunization with influenza A virus hemagglutinin and neuraminidase produced in recombinant baculovirus results in a balanced and broadened immune response superior to conventional vaccine. Vaccine 17, 2073-2080.

Krishnamurthy, S., Huang, Z. & Samal, S. K. (2000).Recovery of a virulent strain of 420 newcastle disease virus from cloned cDNA: expression of a foreign gene results in growth retardation and attenuation. Virology 278, 168-182.

Lamb, R. A. & Kolakofsky, D. (2001).Paramyxoviridae:The viruses and their replication. In Fields Virology, 4th edn, pp. 1305-1340. Edited by D. M. Knipe & P. Howley. Philadelphia: Lippincott Williams&Wilkins.

425 Millar, N. S., Chambers, P. & Emmerson, P. T. (1988).Nucleotide sequence of the fusion and haemagglutinin-neuraminidase glycoprotein genes of Newcastle disease virus, strain Ulster: molecular basis for variations in pathogenicity between strains. J Gen Virol 69 ( Pt 3), 613-620.

Nagai, Y., Klenk, H. D. & Rott, R. (1976).Proteolytic cleavage of the viral 430 glycoproteins and its significance for the virulence of Newcastle disease virus. Virology 72, 494-508.

Nakaya, T., Cros, J., Park, M. S., Nakaya, Y., Zheng, H., Sagrera, A., Villar, E., Garcia-Sastre, A. & Palese, P. (2001).Recombinant Newcastle disease virus as a vaccine vector. J Virol 75, 11868-11873.

435 Ong, S. E., Blagoev, B., Kratchmarova, I., Kristensen, D. B., Steen, H., Pandey, A. & Mann, M. (2002).Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics 1, 376-386.

Palese, P. & Shaw, M.L. (2007).Orthomyxoviridae: Virus and their replication. In 440 Fields Virology, 5th edn, pp. 1647-1689. Edited by D.M. Knipe & P.M. Howley. Philadelphia: Lippincott Williams & Wilkins.

445 Pavlova, S. P., Veits, J., Keil, G. M., Mettenleiter, T. C. & Fuchs, W. (2009).Protection of chickens against H5N1 highly pathogenic avian influenza virus infection by live vaccination with infectious laryngotracheitis virus recombinants expressing H5 hemagglutinin and N1 neuraminidase. Vaccine 27, 773-785.

450 Peeters, B. P., de Leeuw, O. S., Koch, G. & Gielkens, A. L. (1999).Rescue of Newcastle disease virus from cloned cDNA: evidence that cleavability of the fusion protein is a major determinant for virulence. J Virol 73, 5001-5009.

12 Qiao, C. L., Yu, K. Z., Jiang, Y. P., Jia, Y. Q., Tian, G. B., Liu, M., Deng, G. H., Wang, X. R., Meng, Q. W. & Tang, X. Y. (2003).Protection of chickens 455 against highly lethal H5N1 and H7N1 avian influenza viruses with a recombinant fowlpox virus co-expressing H5 haemagglutinin and N1 neuraminidase genes. Avian Pathol 32, 25-32.

Römer-Oberdörfer, A., Mundt, E., Mebatsion, T., Buchholz, U. J. & Mettenleiter, T. C. (1999).Generation of recombinant lentogenic Newcastle disease virus 460 from cDNA. J Gen Virol 80 ( Pt 11), 2987-2995.

Römer-Oberdörfer, A., Veits, J., Helferich, D. & Mettenleiter, T. C. (2008).Level of protection of chickens against highly pathogenic H5 avian influenza virus with Newcastle disease virus based live attenuated vector vaccine depends on homology of H5 sequence between vaccine and challenge virus. Vaccine 26, 465 2307-2313.

Sakai, Y., Kiyotani, K., Fukumura, M., Asakawa, M., Kato, A., Shioda, T., Yoshida, T., Tanaka, A., Hasegawa, M. & Nagai, Y. (1999).Accommodation of foreign genes into the Sendai virus genome: sizes of inserted genes and viral replication. FEBS Lett 456, 221-226.

470 Schröer, D., Veits, J., Grund, C., Dauber, M., Keil, G., Granzow, H., Mettenleiter, T. C. & Römer-Oberdörfer, A. (2009).Vaccination with Newcastle disease virus vectored vaccine protects chickens against highly pathogenic H7 avian influenza virus. Avian Diseases 53, 190-197.

Skiba, M., Glowinski, F., Koczan, D., Mettenleiter, T. C. & Karger, A. (2010).Gene 475 expression profiling of Pseudorabies virus (PrV) infected bovine cells by combination of transcript analysis and quantitative proteomic techniques. Vet Microbiol 143, 14-20.

Skiba, M., Mettenleiter, T. C. & Karger, A. (2008).Quantitative whole-cell proteome analysis of pseudorabies virus-infected cells. J Virol 82, 9689-9699.

480 Steward, M., Vipond, I. B., Millar, N. S. & Emmerson, P. T. (1993).RNA editing in Newcastle disease virus. J Gen Virol 74 ( Pt 12), 2539-2547.

Toyoda, T., Sakaguchi, T., Imai, K., Inocencio, N. M., Gotoh, B., Hamaguchi, M. & Nagai, Y. (1987).Structural comparison of the cleavage-activation site of the fusion glycoprotein between virulent and avirulent strains of Newcastle 485 disease virus. Virology 158, 242-247.

Veits, J., Wiesner, D., Fuchs, W., Hoffmann, B., Granzow, H., Starick, E., Mundt, E., Schirrmeier, H., Mebatsion, T., Mettenleiter, T. C. & Römer-Oberdörfer, A. (2006).Newcastle disease virus expressing H5 hemagglutinin gene protects chickens against Newcastle disease and avian influenza. Proc Natl Acad Sci 490 U S A 103, 8197-8202.

Webster, R. G., Bean, W. J., Gorman, O. T., Chambers, T. M. & Kawaoka, Y. (1992).Evolution and ecology of influenza A viruses. Microbiol Rev 56, 152- 179.

13 Wertz, G. W., Moudy, R. & Ball, L. A. (2002).Adding genes to the RNA genome of 495 vesicular stomatitis virus: positional effects on stability of expression. J Virol 76, 7642-7650.

Wertz, G. W., Perepelitsa, V. P. & Ball, L. A. (1998).Gene rearrangement attenuates expression and lethality of a nonsegmented negative strand RNA virus. Proc Natl Acad Sci U S A 95, 3501-3506.

500 Whelan, S. P., Barr, J. N. & Wertz, G. W. (2004).Transcription and replication of nonsegmented negative-strand RNA viruses. Curr Top Microbiol Immunol 283, 61-119.

Zhao, H. & Peeters, B. P. (2003).Recombinant Newcastle disease virus as a viral vector: effect of genomic location of foreign gene on gene expression and 505 virus replication. J Gen Virol 84, 781-788.

510

14 Figure 1 MluI MluI

rNDV NP P M F HN L

NDVH5VmPM NP P H5Vm M F HN L

MluI MluI

T H5 ORF

ncr HN ncr HN

NDVH5VmMF NP P M H5Vm F HN L

NDVH5Vm NP P M F H5Vm HN L

NDVN1FHN NP P M F N1V HN L

NDVH5VmPMN1FHN NP P H5Vm M F N1V HN L Figure 2:

8 Clone 30 /ml NDVH5VmPM 50 NDVH5VmMF 6

TCID NDVH5Vm

10 NDVN1FHN

log 4 NDVH5VmPMN1FHN

0 0244872 time p.i. (h) Figure 3:

kDa 70 ←HA0 55 ←HA1 43 -AIV H5 -AIV 24 hours p.i.

←HA2 17

70 ←N1 55 ←N1 43 -AIV N1 -AIV 24 hours p.i.

B: 1.4

1.2

0.8

0.6

0.4

0.2 relative level of expression H5 level relative 0

NDVH5VmMF NDVH5VmPM NDVH5VmPMN1FHN NDVH5Vm

VIII. Zusammenfassung der Dissertation zum Thema:

„Analyse des Wirtszellproteoms und der Expression viraler Proteine in virusinfizierten Gewebekulturen durch quantitative Massenspektrometrie mit metabolisch eingeführten stabilen Isotopen “

vorgelegt von Martin Skiba

Zur Aufklärung der molekularen Grundlagen einer Infektion mit dem Pseudorabiesvirus (PrV), dem Erreger der Aujeszky’schen Krankheit beim Schwein, wurde eine qualitative und quantitative Proteomstudie von PrV-infizierten bovinen Nierenzellen (MDBK) durchgeführt. Die Erstellung der Proteomkarten erfolgte durch hochauflösende zweidimensionale Gelelektrophorese, mit der neben zum größten Teil unmodifizierten Proteinen auch eine Reihe von Proteinen mit posttranslationalen Modifikationen nachgewiesen werden konnten. Die Identifizierung der Proteine erfolgte mit dem Peptidmassenfingerabdruck durch Matrix Assisted Laser Desorption/Ionisation Time-of-Flight (MALDI-TOF)-Massenspektrometrie. Proteine wurden massenspektrometrisch durch die stable isotope labelling by amino acids in cell culture (SILAC)-Technik quantifiziert. Um die Komplexität des Gesamtzellextraktes zu reduzieren, wurde eine Affinitätsfestphasenchromatographie aus verschiedenen Matrices, bestehend aus einer Cibacron Blau F3G-A Matrix, die Nukleotid-bindende Proteine bindet, einer Heparin Matrix, die DNA-bindende Proteine bindet und einer Phosphoprotein spezifischen Metallchelatmatrix etabliert. Dabei zeigte sich, dass sich der Gesamtzellextrakt in gut definierte Teilproteome fraktionieren ließ. Die Fraktionierung zeichnete sich durch eine hohe Trennschärfe, eine hohe Effizienz und eine spezifische Anreicherung entsprechend der Affinitäten der verwendeten Matrices aus. Zur weiteren Erhöhung der zu analysierenden Proteine wurden die Fraktionen auf mehreren Fokussierungsstreifen mit unterschiedlichen pH-Wert Bereichen (3-6, 4-7, 6-9 und 3-10) analysiert, wobei sich lediglich der pH-Bereich zwischen 3-6, als relativ proteinarm erwies. Die Streuung bezüglich der relativen Quantifizierung wurde in einem Kontrollversuch bestimmt. Sie war sehr gering, was auch die Erfassung sehr kleiner Unterschiede in den Expressionsniveaus erlaubt. Das bovine Wirtszellproteom erwies sich vier Stunden nach Infektion mit dem PrV in qualitativer und quantitativer Hinsicht, trotz des beschriebenen shutoff, der zu einem Abbau der zellulären mRNA führt, als überraschend stabil. Quantitative Unterschiede wurden bei 109

100 Wirtszellproteinen gefunden. Vorwiegend handelte es sich dabei um Proteine der Kernlamina, Bestandteile des Translationsapparates, Proteine des Membran- und intrazellulären-Transports, sowie Proteine der Stressantwort. Bei den Proteinen Lamin A/C und B2, 60S Saures Ribosomale Protein P0, Hitzeschock-27 Protein 1, Heterogenes Nukleäres Ribonukleoprotein K, Sorting Nexin-9 und dem Eukaryotischen Translations- Initiations Faktor 4B wurden Mengenverschiebungen zwischen den Isoformen hin zu den stärker negativ geladenen Varianten beobachtet, die möglicherweise auf Phosphorylierungen zurückzuführen sind.

Um den Mechanismus der Regulation der Wirtszellproteinexpression genauer zu untersuchen, wurde aufbauend auf den Ergebnissen der Proteomstudie eine Transkriptanalyse durchgeführt. Transkriptom- und Proteom-Analysen nach einer PrV- Wildtyp-Infektion zeigten eine nur geringe Korrelation, sodass die beobachteten Veränderungen der Proteinmengen die Folge von posttranslationalen Vorgängen sein dürften. Neben der Quantifizierung von Wirtszellproteinen wurde die SILAC-Methode zur Quantifizierung der Expression von viralen Proteinen in Deletionsmutanten getestet. Dazu wurde der Analysengang verändert und MDBK-Zellen mit einer PrV-US3-Deletionsmutante (PrV-∆US3) und dem PrV-Wildtyp infiziert. Die Extrakte aus PrV-Wildtyp-infizierten Zellen wurden als globaler interner Standard verwendet. Die Verwendung der SILAC-Methode erlaubte hier die Anwendung der sonst sehr Artefakt-anfälligen Zellfraktionierung in Zytosol- und Kernextrakte zur Reduktion der Probenkomplexität. Ein Vergleich zur Affinitätsfestphasenextraktion zeigte, dass ein Großteil der identifizierten Proteine auch mit letzterer erfasst wird. Einige wichtige Kernproteine wie Spleißfaktoren konnten aber nur in der Kernfraktion identifiziert werden. Vergleiche von Zellextrakten nach Infektion mit PrV- Wildtyp oder einer US3-negativen Mutante zeigten Veränderungen in den Expressionsniveaus der viralen Proteine pUL29, pUL39 und pUL42. Dabei besaßen pUL29 und pUL39 eine erhöhte und pUL42 eine verminderte relative Abundanz in Abwesenheit des pUS3. Die Proteine pUL29 und pUL42 traten in mehreren Ladungsvarianten auf, wobei das pUL42 zusätzlich eine Größenvariante aufwies.

Im dritten Teil dieser Arbeit wurde ein SILAC-gestütztes Verfahren zur Quantifizierung der Expression von Fremdgenen in viralen Vektoren etabliert. Dazu wurden Zellen mit verschiedenen rekombinanten Newcastle Disease Viren (NDV) infiziert, die als Vektoren das Hämagglutinin (HA) und/oder Neuraminidase des Aviären Influenza Virus von unterschiedlichen Stellen des NDV Genoms exprimierten. Hintergrund dieser Strategie war der für NDV beschriebene, vom 3'- zum 5'-Ende des Genoms abnehmende Transkriptionsgradient, der zur Steigerung der Expressionsstärke des Fremdgens genutzt 101 werden sollte. Die Analyse erfolgte durch zweidimensionale Gelelektrophorese und die massenspektrometrische Identifizierung und Quantifizierung. Das SILAC-Verfahren erlaubte die Bestimmung der HA-Expression in NDV-Vektoren mit hoher Genauigkeit und unabhängig von immunologischen Reagenzien. Die relativen HA-Expressionen in den verschiedenen Rekombinanten korrelierten sehr gut mit den relativen Transkriptniveaus, aber nicht mit der Lage des Fremdgens im NDV-Genom. Die gemessenen Expressionsstärken unterschieden sich nur moderat, eine genaue Erfassung mit der SILAC-Technik war aber dennoch möglich.

102

IX. Summary

“Analysis of host cell proteome and expression of viral proteins in virus- infected cell cultures by quantitative mass spectrometry using metabolically introduced stable isotopes.”

To elucidate the molecular basis of an infection by pseudorabies virus (PrV), the etiological agent of Aujeszky's disease in pigs, a qualitative and quantitative proteome study of PrV- infected bovine kidney cells (MDBK) was performed. Proteome maps were generated by high-resolution two-dimensional gel electrophoresis. A number of proteins with posttranslational modifications were identified in addition to the majority of unmodified proteins. Proteins were identified by peptide mass fingerprint analysis using matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry. Quantitation was carried out by mass spectrometry using the SILAC (stable isotope labeling by amino acids in cell culture) technique. Affinity solid-phase extraction (ASPE) with matrices specific for nucleotide-binding proteins (Cibacron Blue F3G-A), DNA and RNA-binding proteins (heparin) and phosphoproteins (a metal chelate matrix) was applied to reduce the complexity of whole cell extracts. ASPE was found to efficiently fractionate the extract into well-defined subproteomes with high selectivity and excellent accordance with the binding-specificities of the matrices used. To further enhance yields of identified proteins, fractions were analyzed on focusing strips with different pH ranges (3-6, 4-7, 6-9 and 3-10). Protein yields in the pH range between 3 and 6 were low. The variance of the procedure with respect to quantitation, which was estimated in a separate control experiment, was very low so that even small differences in the relative expression levels could be reliably detected. Four hours after infection with PrV the bovine host cell proteome was found to be surprisingly stable in spite of the viral shutoff mechanism which leads to degradation of cellular mRNA. Significant changes in relative levels were found for only 109 host cell proteins, most of them being constituents of the nuclear lamina, components of translation machinery, proteins involved in membrane and intracellular transport, and stress proteins. For the proteins lamin A/C and B2, 60S acidic ribosomal protein P0, heat shock-27 protein 1, heterogeneous nuclear ribonucleoprotein K, sorting nexin-9 and eukaryotic translation initiation factor 4B an increase of the more negatively charged forms at the loss of the more basic variants was observed, which may be the result of phosphorylations induced by PrV-infection.

103 To further investigate the mechanisms of regulation of the host cell protein expression in PrV-infected cells, a microarray study was performed with mRNA isolated from PrV-infected cells and compared to the results of the proteome study. Transcriptome and proteome analysis of PrV-infected MDBK cells showed only poor correlation, indicating that the observed changes in protein levels are most likely to result from posttranslational events.

The SILAC-approach was also applied to quantitate expression levels of viral proteins in cells infected with deletion mutants. To that purpose, MDBK cells were infected with a mutant not expressing the viral kinase pUS3 (PrV-∆US3) or with PrV wild-type. In these experiments, extracts from PrV wild-type-infected cells were used as global internal standard. The introduction of the standard at a very early stage of the experiment allowed the application of cell fractionation protocols, which otherwise are very prone to produce isolation artefacts that hamper exact quantitation. Thus, cells were fractionated into a nuclear and a cytosolic fraction, which were then analysed by two-dimensional electrophoresis and mass spectrometry. The more tedious ASPE procedure described above was shown to allow a more comprehensive analysis than the cell fractionation protocol, although a number of important nuclear proteins such as splicing factors escaped the analysis by ASPE and were only detected and quantitated after cell fractionation. Comparison of cell extracts after infection with PrV wild-type or a US3-negative mutant revealed hitherto unknown changes in expression levels of the viral proteins pUL29, pUL39 and pUL42. While levels of pUL29 and pUL39 were increased, the level of pUL42 was decreased in the absence of pUS3. Proteins pUL29 and pUL42 appeared in several charge variants, and pUL42 in an additional variant with slightly reduced molecular weight.

In the third part of this study, a SILAC-based approach to quantitate expression levels of foreign genes in viral vectors has been established. Chicken cells were infected with different recombinant Newcastle Disease Viruses (NDV), expressing the hemagglutinin (HA) and/or neuraminidase of avian influenza virus at different locations within the NDV genome. A transcription gradient declining from the 3’ to the 5’ end of the genome has been described for NDV. The intent of these experiments was to exploit this gradient for the efficient expression of foreign genes in a NDV vector by placing it in 3’-proximal positions of the genome. The analysis of the HA protein was carried out by two-dimensional gel electrophoresis and mass spectrometric identification and quantification.

The SILAC method allowed determination of HA expression in NDV vectors with high accuracy and independent of immunological reagents. Relative HA expression in different recombinants correlated very well with relative transcript levels, but not with the position of

104 HA-gene within the NDV genome. Although expression levels differed only moderately, accurate SILAC-based quantitation was possible.

105 X. Anhang

X.1 Abkürzungsverzeichnis

(-) ss RNA Einzelstrang RNA negativer Polarität (+) ss RNA Einzelstrang RNA positiver Polarität 1D eindimensional 2D zweidimensional 2DE Zweidimensionale Gelelektrohorese α immediate-early Abb. Abbildung AD Aujeszky´s disease AIHV1 Bösartiges Katarrhalfieber AIV Aviäre Influenza Virus AK Aujeszky´sche Krankheit APMV-1 Aviäres Paramyxovirus Serotyp 1 ARE AU-reiche Elemente ASPE affinity solid-phase extraction α-TIF alpha trans-inducing factor ATP Adenosintriphosphat β early BAD Bcl-2-Antagonist of Cell Death Bid BH3 interacting domain death agonist BoHV Bovines Herpesvirus bzw. beziehungsweise ca. circa Cap 7-Methylguanosin CD cluster of differentiation cdc2 Cyclin-abhängige Kinase 2 CyHV3 Koi-Herpesvirus DIGE difference in-gel electrophoresis DNA Desoxyribonukleinsäure dsDNA Doppelstrang DNA dsRNA Doppelstrang RNA E early EBV Epstein-Barr-Virus EHV Equines Herpesvirus eIF Eukaryotischer Translations-Initiationsfaktor EIHV1 Endotheliotropes Elefantenherpesvirus ER Endoplasmatisches Retikulum ESI Elektrospray-Ionisation F Fusionsproteinsprotein FTICR Fouriertransformation-Ionenzyklotronresonanz-Massenspektrometrie g Glykoprotein γ late Gag Gruppenspezifisches Antigen GaHV Gallid herpesvirus H Hämagglutinin H5N1 Hämagglutinin 5 -Neuraminidase 1 HA Hämagglutinin HAUSP herpesvirus-associated ubiquitin-specific protease HCMV Humanes Zytomegalievirus HeLa Henrietta Lacks HEp-2 Humane Epithelzellen 106 HHV Humanes Herpesvirus HIV-1 Humanes Immundefizienz-Virus Typ-1 HN Hämagglutinin-Neuraminidaseprotein hnRNP Heterogenes Nukleäre Ribonukleoprotein HP hoch pathogen Hsp Hitzeschockprotein HSV Herpes Simplex Virus Huh7 Humane Hepatoma Zellen Hve herpesvirus entry mediators ICAT isotope-coded affinity tag IcHV1 Channel catfish virus ICP infected cell protein ICPI intrazerebralen Pathogenitätsindex IE immediate-early ILTV Infektiöse Laryngotracheitis IRS internal repeat iTRAQ isobaric tag for relative and absolute quantitation JNK c-Jun N-terminale Kinasen kb Kilobasen kbp Kilobasenpaare kDa kiloDalton KSHV Kaposi-Sarkom-Herpesvirus L late L RNA-abhängigen-RNA Polymerase LC Flüssigkeitschromatographie LLT large latency transcript LP niedrig pathogen M Matrixprotein MALDI matrix assisted laser desorption/ionisation MAP Mitogen-aktiviertes Protein McHV1 Macacine herpesvirus 1 MCMV Murines Zytomegalievirus MDBK Madin Darby bovine kidney cells MDV Marek´sche Krankheit MHC major histocompatibility complex mRNA Boten-RNA MS Massenspektrometrie MS/MS Tandem-Massenspektrometrie MuHV1 Murid herpesvirus 1 MusHV1 Dachs Herpesvirus N Neuraminidase NA Neuraminidase NDV Newcastle Disease Virus nm Nanometer NP Nucleoprotein ori Replikationsursprung OsHV1 Austern Herpesvirus p Protein P Phosphoprotein pH potentia hydrogenii PI3K Phosphoinositid-3-Kinase PMF peptide mass fingerprint Procaspase Pro Cystein-Aspartat spezifische Protease PrV Pseudorabiesvirus PTM Posttranslationale Modifikation Q Quadruopol RaHV Ranid herpesvirus 107 RGG Arginin-Glycin-Glycin RING really interesting new gene RNA Ribonukleinsäure RNase A Ribonuklease A RNP Ribonukleoproteinkomplex SAP145 spliceosome-associated protein 145 SARS schweres akutes respiratorisches Syndrom SDS Natriumdodecylsulfat SELDI surface-enhanced laser desorption/ionization SILAC stable isotope labeling by amino acids in cell culture snRNP small nuclear ribonucleoproteins SuHV1 Suid herpesvirus 1 TOF time-of-flight TRS terminal repeat UL unique long US unique short USP7 ubiquitin-specific-processing protease 7 vhs virion-host-shutoff VP Virus Protein VSV Vesicular Stomatitis Virus VZV Varizella-Zoster Virus z.B. zum Beispiel

108

X.2 Publikationen X.2.1 Veröffentlichungen

Quantitative whole-cell proteome analysis of pseudorabies virus-infected cells.

Skiba M., Mettenleiter T.C., Karger A.

J Virol. 2008 Oct;82(19):9689-99.

Gene expression profiling of Pseudorabies virus (PrV) infected bovine cells by combination of transcript analysis and quantitative proteomic techniques.

Skiba M., Glowinski F., Koczan D., Mettenleiter T.C., Karger A.

Vet Microbiol. 2010 Jun 16;143(1):14-20.

Influence of Insertion Site of Avian Influenza Virus Hemagglutinin (HA) Gene Within the Newcastle Disease Virus Genome on HA Expression.

Ramp K., Skiba M., Karger A., Mettenleiter T.C. and Römer-Oberdörfer A.

J. Gen. Virol. Im Druck, doi:10.1099/vir.0.027268-0

A comprehensive proteome map of bovine cerebrospinal fluid.

Brenn A, Karger A, Skiba M, Ziegler U, Groschup MH.

Proteomics. 2009 Nov;9(22):5199-205.

X.2.2 Tagungsbeiträge:

Quantitative whole-cell proteome analysis of pseudorabies virus-infected cells.

Skiba M., Mettenleiter T.C., Karger A.

3rd Mini-Herpesvirusworkshop, 19. September 2008, Berlin, oral presentation.

Gene expression profiling of Pseudorabies virus (PrV) infected bovine cells by combination of transcript analysis and quantitative proteomic techniques.

Skiba M., Glowinski F., Koczan D., Mettenleiter T.C., Karger A.

3rd European Society for Veterinary Virology - Veterinary Herpesvirus Symposium, 22.-24. April 2009, Greifswald, oral presentation

109

X.3 Eidesstattliche Erklärung Hiermit erkläre ich, dass diese Arbeit bisher von mir weder an der Mathematisch- Naturwissenschaftlichen Fakultät der Ernst-Moritz-Arndt-Universität Greifswald noch einer anderen wissenschaftlichen Einrichtung zum Zwecke der Promotion eingereicht wurde.

Ferner erkläre ich, dass ich diese Arbeit selbständig verfasst und keine anderen als die darin angegebenen Hilfsmittel benutzt habe.

Martin Skiba

110

X.4 Lebenslauf Persönliche Daten

Name: Martin Skiba

Geburtsdatum: 11.12.1980

Geburtsort: Bochum

Familienstand: ledig

Ausbildung

1987 - 1991 Grundschule Bochum-Dahlhausen

1991 - 2000 Gymnasium Theodor-Körner-Schule Bochum-Linden

Abschluss: Abitur

2000 - 2001 Zivildienst im Martin-Luther-Krankenhaus Bochum- Wattenscheid

2001 - 2007 Studium der Biochemie an der Ernst-Moritz-Arndt-Universität Greifswald

Abschluss: Diplom-Biochemiker

Diplomarbeit am Friedrich-Loeffler-Institut, Bundesforschungsinstitut für Tiergesundheit auf der Insel Riems:

„Quantitative Proteomanaylse von Herpesvirus infizierten Zellen mittels stabiler Isotopenmarkierung: Entwicklung von Strategien zur Vorfraktionierung.“

2007 - 2010 Dissertation am Friedrich-Loeffler-Institut auf der Insel Riems:

„Analyse des Wirtszellproteoms und der Expression viraler Proteine in virusinfizierten Gewebekulturen durch quantitative Massenspektrometrie mit metabolisch eingeführten stabilen Isotopen.“ 111 X.5 Danksagung Die vorliegende Arbeit wurde am Friedrich-Loeffler-Institut, Insel Riems, im Institut für

Molekularbiologie angefertigt. An dieser Stelle möchte ich allen danken, die durch ihre Hilfsbereitschaft zur Fertigstellung dieser Arbeit beigetragen haben.

Mein besonderer Dank gilt

Herrn Prof. Dr. Dr. h.c. Thomas C. Mettenleiter für die Überlassung des Themas, die stete Bereitschaft zur wissenschaftlichen Diskussion, die wertvollen Anregungen und die Unterstützung zur Veröffentlichung der erhaltenen Ergebnisse.

Herrn Dr. Axel Karger für die exzellente Betreuung in allen theoretischen und praktischen Aspekten dieser Arbeit und die Bereitschaft seine langjährige Laborerfahrung zu teilen.

Frau Dr. Angela Römer-Oberdörfer für die stetige Unterstützung und für die Einblicke in das Newcastle Disease Virus auf molekularer Ebene.

Frau Dr. Barbara Klupp für die freundliche Überlassung der PrV-US3 Deletionsmutante und Herrn Dr. Günther Keil für die Hilfe bei der Erstellung der Mikroarrayanalysen.

Allen Labormitgliedern und Freunden für die angenehme Zusammenarbeit und für die moralische Unterstützung zum Fortgang dieser Arbeit.

Mein herzlichster Dank gilt meinen Eltern für ihre andauernde und vorbehaltlose

Unterstützung.

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