Structural Insights Into the Assembly of Paramyxoviruses

CAPSIDS, MATRICES AND VESICLES – STRUCTURAL INSIGHTS INTO THE ASSEMBLY OF PARAMYXOVIRUSES

Lassi J. P. Liljeroos

Institute of Biotechnology and Department of Biosciences, Division of Genetics Faculty of Biological and Environmental Sciences University of Helsinki

and

Viikki Doctoral Programme in Molecular Biosciences University of Helsinki

ACADEMIC DISSERTATION

To be presented for public examination with the permission of the Faculty of Biological and Environmental Sciences of the University of Helsinki, in the lecture hall 2402 of Biocenter 3, Viikinkaari 1, on October 11th 2013 at 12 o’clock noon.

HELSINKI 2013

Supervisor

Research Director, Docent Sarah J. Butcher Institute of Biotechnology University of Helsinki

Thesis committee

Professor Adrian Goldman Professor Markku Kulomaa Department of Biosciences Institute of Biomedical Technology Faculty of Biological and University of Tampere Environmental Sciences University of Helsinki

Reviewers

Docent Tero Ahola Dr. Peter B. Rosenthal Department of Food and Division of Physical Biochemistry Environmental Sciences MRC National Institute for Faculty of Agriculture and Forestry Medical Research University of Helsinki London, UK

Opponent Custos

Professor Kay Grünewald Professor Liisa Holm Oxford Particle Imaging Centre Institute of Biotechnology and Division of Structural Biology Department of Biosciences Wellcome Trust Centre for Human Genetics Faculty of Biological and University of Oxford, UK Environmental Sciences University of Helsinki

ISBN 978-952-10-9272-5 (paperback) ISBN 978-952-10-9273-2 (PDF; http://ethesis.helsinki.fi) ISSN 1799-7372

Unigrafia Helsinki 2013

Teresalle ja Islalle

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-Douglas Adams, Dirk Gently’s Holistic Detective Agency

TABLE OF CONTENTS

Original publications ...... i Abbreviations ...... ii Summary ...... iii A. INTRODUCTION ...... 1

1. Virus structure ...... 2

1.2. Structures of negative-sense RNA viruses ...... 3

1.2.1. Structures of paramyxoviruses ...... 8

2. Assembly of negative-sense RNA viruses ...... 10

2.1. Role of matrix protein in assembly ...... 11 2.2. Assembly of measles virus ...... 13 2.3. Assembly of human respiratory syncytial virus ...... 17

3. Entry of negative-sense RNA viruses ...... 19 4. Electron cryotomography as a tool in studying virus structure ...... 21

4.1. Sample preparation for electron cryotomography ...... 23 4.2. Image formation in the electron microscope ...... 24 4.3. Electron cryotomography ...... 26 4.4 Subvolume averaging ...... 28

B. AIMS OF THE PRESENT STUDY ...... 31 C. MATERIALS AND METHODS ...... 32 D. RESULTS AND DISCUSSION ...... 33

1. The N0-P complex ...... 33 2. Budding of MV and HRSV and structures of the released virions ...... 35

E. CONCLUSIONS AND PERSPECTIVES ...... 42 F. ACKNOWLEDGEMENTS...... 43 G. REFERENCES ...... 45

ORIGINAL PUBLICATIONS

This thesis is based on the following articles, which are referred to in the text by their Roman numerals.

I Liljeroos, L., Huiskonen, J. T., Ora, A., Susi, P. and Butcher, S.J. (2011) Electron cryotomography of measles virus reveals how matrix protein coats the ribonucleocapsid within intact virions. Proc Natl Acad Sci U S A. 108(44):18085-90

II Liljeroos, L., Krzyzaniak, M. A., Helenius, A. and Butcher, S.J. (2013) Architecture of respiratory syncytial virus revealed by electron cryotomography. Proc Natl Acad Sci USA. 110(27):11133-8

III Liljeroos, L., Butcher, S.J. Expression, purification and characterization of measles virus N0-P complex. Manuscript.

Additional unpublished data will be presented.

ABBREVIATIONS

3D-cryoEM three-dimensional electron cryomicroscopy ARE apical recycling endosome ART algebraic reconstruction technique CCD charge-coupled device cryo-EM electron cryomicroscopy cryo-ET electron cryotomography DQE detective quantum efficiency EBOV Ebola virus ER endoplasmic reticulum ESCRT endosomal sorting complex required for transport F fusion protein FEG field emission gun G G glycoprotein H hemagglutinin HA influenza virus hemagglutinin HIV human immunodeficiency virus HN hemagglutinin-neuraminidase HRSV human respiratory syncytial virus HSV herpes simplex virus L-domain late domain MARV Marburg virus MCNC matrix-covered nucleocapsid MTF modulation transfer function MuV mumps virus MV measles virus MVB multivesicular body MVE multivesicular endosome N nucleoprotein NA neuraminidase NDV Newcastle disease virus NMR nuclear magnetic resonance PFU plaque-forming unit PIV parainfluenza virus PNT phosphoprotein N-terminal end RABV rabies virus RNP ribonucleoprotein SeV Sendai virus SH small hydrophobic protein SIRT simultaneous iterative reconstruction technique SNR signal-to-noise ratio -ssRNA negative single strand RNA TEM transmission electron microscope VLP virus-like particle VSV vesicular stomatitis virus WBP weighted back-projection WT wild-type

SUMMARY

Paramyxoviridae constitute a family of pleomorphic, enveloped viruses including several human pathogens. Understanding of the structure and assembly of paramyxoviruses has been hindered by the lack of whole-virion three- dimensional structures. In this work, measles and human respiratory syncytial viruses were studied with three-dimensional electron microscopy and biochemical analysis of recombinant proteins. The analysis revealed significant differences in the structure and assembly of the two viruses. The differences were most notable in the way the matrix protein, the main factor driving budding from host cell, was organized inside the virions. In measles virions, the matrix was found to cover the genome-containing ribonucleocapsid, whereas in human respiratory syncytial virus the matrix was lining the inner surface of the membrane vesicle. These differences have implications on models of how each ribonucleoprotein complex assembles and how the viruses bud from the host cell. The early control of measles ribonucleoprotein assembly was subsequently in- vestigated to further reveal the details of the precise manner in which the intricate molecular ballet of viral assembly is orchestrated inside the host cell. The results presented in this thesis expand the understanding of enveloped virus structure and assembly, which is important in rational approaches to fight the pathogenic members in the group.

iii

A. INTRODUCTION

Viruses are obligatory parasites that bidity are greatest in the developing rely on the host cell for replication. countries, but also significant in the They are like letters of genetic infor- industrialized countries. Economi- mation that, in order to be duplicat- cally, prevention and preparation for ed, require a copy machine. The copy possible viral threats, like pandemic machine is a bacterial, archaeal or influenza, causes considerable costs. eukaryotic cell. Most viruses carry In the 2009 H1N1 influenza pandem- components of this machine inside ic, the costs were mainly incurred by the viral particle in the form of struc- vaccination programs, where many tural proteins encoded by genes in countries decided to purchase the the viral genome that can be made of vaccine for the whole population. In either RNA or DNA. Relying on the the absence of a general viral antibi- host cell for existence, viruses have otic, vaccines are the most efficient evolved to thrive in as myriad condi- arms to fight pathogenic viruses. tions as living organisms. They can Successful vaccination, however, re- be found in places intuitively adverse quires anticipation as normally it is to life, such as hot, acidic springs too late to vaccinate when disease (Bize et al., 2008). It has been esti- symptoms arise. With the difficulty mated that there are over 1030 viruses in predicting the vastness and pace of in the biosphere (Hendrix 2002) and epidemics, preventive measures can in most environments they outnum- easily be either under- or overesti- ber the host cells by an order of mated. Also, having to develop a new magnitude (Wommack et al., 2000). vaccine for every season for highly The vast majority of these viruses are variant viruses, such as influenza, bacteriophages, viruses of the bacte- results in high costs for society. De- ria. Eukaryotic viruses, although spite the efficacy of vaccines against more modest in abundance, also many viruses, some have dodged all come in a great variety. There are attempts to develop one. Examples approximately 200 different viruses include the human immunodeficien- known to cause disease in humans of cy (HIV) and human respiratory syn- which more than one in four is con- cytial (HRSV) viruses. sidered an emerging or re-emerging The difficulty of developing a pathogen (Woolhouse et al., 2005). general anti-viral drug arises mainly Of all the known human pathogens, from the parasitic and highly variable viruses constitute approximately 15 nature of viruses. They rely on the %, the rest being mainly bacteria, host cell for many of their functions, fungi, helminths and protozoans in like replication, transport, exit and decreasing order of prevalence. Vi- entry, and therefore blocking these ruses are overrepresented in the functions is difficult without affect- emerging and re-emerging patho- ing uninfected cells and causing con- gens, RNA viruses comprising 37 % sequent toxicity to the whole organ- of the total 177 pathogens considered ism. Because viruses do not have to belong to this group (Woolhouse similar unifying features as bacteria et al., 2005). do (e.g. a peptidoglycan cell wall), it Viral diseases cause great socio- is difficult, if not impossible to devel- economic burden throughout the op an inhibitory molecule that would world. Virus related death and mor- have as broad a spectrum as some of

the bacterial antibiotics do. One electron cryomicroscopy (cryo-EM) group of antivirals, effective for sev- and light microscopy. Enveloped eral different viruses, is the nucleo- pleomorphic (from the Greek: many- side analogues that mimic the natu- formed) viruses, like paramyxovirus- ral nucleosides used in DNA synthe- es, are especially amenable to a spe- sis. Acycloguanosine, for example, is cial application of cryo-EM called effective against several members of electron cryotomography (cryo-ET) the Herpesviridae family and is the for three dimensional studies of the main treatment for herpes simplex whole virion. virus (HSV) infections. The mecha- Paramyxoviruses are envel- nism of action of such compounds oped, nonsegmented, negative-sense relies on the mimicry of cellular nu- RNA viruses that include a number cleosides, and they are preferentially of human pathogens, like HRSV, incorporated into the viral DNA re- measles (MV), mumps (MuV) and sulting in termination of DNA syn- parainfluenza (PIV) viruses. For MV thesis. and MuV an effective vaccine has In the fight against viral diseas- been available for several decades es, it is instrumental to investigate and they have been largely eradicat- the molecular details of the viruses ed from countries with well- and the interplay between the virus coordinated vaccination programs, and the cell. Zooming in to the but remain a major problem in many smallest atomic-level detail is re- developing countries. Due to increas- quired in order to be able to rational- ing reluctance to be vaccinated and ly engineer molecules that can be the consequent decrease in herd im- used to prevent or treat infection. munity, there have been outbreaks of This level of detail for a virus in the these viruses recently in countries cellular context can only be achieved like U.S.A., Germany, France, U.K. with a combination of imaging tech- and Finland (Kay et al., 2011, Muscat niques, such as x-ray crystallography, 2011). Human vaccines are yet to be nuclear magnetic resonance (NMR), developed for PIV and HRSV.

1. VIRUS STRUCTURE

Viruses are generally in the range of viruses, three-dimensional electron 20 - 1000 nm in diameter. Most cryomicroscopy (3D-cryoEM) cou- known viruses have an icosahedral pled with image averaging, as well as capsid. Icosahedral viruses include a x-ray crystallography have been the vast number of different virus fami- methods of choice for virus studies. lies infecting all domains of life. The Consequently, especially icosahedral genomes of icosahedral viruses are viruses are overrepresented in the varied, being of DNA or RNA, single- pool of three-dimensionally charac- or double-stranded, linear or circu- terized virus structures (> 300 icosa- lar. Helical viruses are also common, hedral virus structures and < 50 ple- especially within viruses infecting omorphic virus structures are availa- plants, the archetype being tobacco ble in the electron microscopy data mosaic virus (Stubbs et al., 2012). bank, accession date 09.07.2013). Due to the highly ordered, symmet- An icosahedron is composed of rical shape of icosahedral and helical 20 facets, thus having six five-fold,

10 three-fold and 15 two-fold sym- ical viruses can be utilized in struc- metry axes. Viral icosahedral capsids ture determination and consequently are 60-fold symmetric and the sim- helical virus structures have been plest viruses with the so-called T = 1 solved to atomic resolution by x-ray symmetry have 60 copies of the cap- fiber diffraction (Namba et al., 1989) sid protein in the shell. One triangu- and to near-atomic resolution by 3D- lar facet of the icosahedron is there- cryoEM (Clare et al., 2010, Sachse et fore composed of three copies of the al., 2007). capsid protein in an identical envi- Outside the majority of icosa- ronment. Most viruses have more hedral and helical viruses, most vi- than one copy of the protein(s) per ruses are enveloped and pleomorphic asymmetric unit, which increases the although some like the Alphaviridae size of the capsid, thus allowing more and Flaviviridae are enveloped and space for larger genomes. In these icosahedral. The Herpesviridae, capsids the proteins are not in exact- some Togaviridae and Cystoviridae ly equivalent relationship to the are exceptional in that they are en- neighboring ones and their positions veloped and pleomorphic, but con- in the capsid are thus referred to as tain an icosahedral capsid inside the quasi-equivalent (Caspar et al., envelope. Some members of the 1962). Although the majority of the Bunyaviridae family produce both icosahedral virus structures solved to icosahedral and pleomorphic parti- atomic resolution have been solved cles (Överby et al., 2008). Enveloped by X-ray crystallography, some have viruses have a host-derived lipid been solved by 3D-cryoEM, some by membrane that protects the genome both such as that of human adenovi- and associated proteins and also rus (Liu et al., 2010, Reddy et al., provides means for cell entry via 2010, Zhang et al., 2013a, Zhang et membrane fusion. The vast majority al., 2010). of known enveloped pleomorphic Helical viruses can be found viruses are eukaryotic, although within plant viruses and bacterio- some have also been found in bacte- phages. Most of them have a single riophages and archaeal viruses capsid protein that forms a helix to- (Ogawa et al., 1985, Pietilä et al., gether with nucleic acid, in most cas- 2012). For pleomorphic viruses, av- es ssRNA [for a review, see (Stubbs eraging methods like x-ray crystal- et al., 2012)]. The helix essentially lography, NMR or single particle 3D- comprises the whole virus. Helical cryoEM are not suitable for whole- nucleocapsids can also be found in virus structure determination. Thus, many other viruses, like filoviruses, only after the relatively recent wide- rhabdoviruses and paramyxoviruses, scale application of cryo-ET, 3D but they are different in that the heli- structures of these viruses have also cal nucleocapsid comprises only part begun to be revealed (Subramaniam of the whole virion. The symmetrical, et al., 2007). often highly ordered structure of hel-

1.2. STRUCTURES OF NEGATIVE-SENSE RNA VIRUSES

Negative strand RNA (-ssRNA) vi- human pathogens. The group com- ruses comprise a large group of vi- prises eight virus families: Bor- ruses, many of which are significant naviridae, Filoviridae, Paramyxo-

viridae and Rhabdoviridae that have allow acidification of the virion inte- a nonsegmented genome; and Are- rior during the endocytotic entry. M2 naviridae, Bunyaviridae, Ophioviri- has also been shown to have a role in dae and Orthomyxoviridae that have the final pinching off from the host a segmented genome. Pathogens like cell (Rossman et al., 2010). Some of influenza (family Orthomyxoviri- the paramyxoviruses have, in addi- dae), rabies (RABV, Rhabdoviridae), tion to the attachment and fusion Ebola (EBOV, Filoviridae) viruses, proteins, a third small hydrophobic MV and HRSV are members of the (SH) membrane protein. The role of group. All animal –ssRNA viruses this protein is unclear, but functions are enveloped and they are pleo- in host cell TNF-α signaling inhibi- morphic, except some of the viruses tion and as a cation selective, oligo- in the Rhabdo- and Bunyavirus fam- meric ion channel, have been sug- ilies (Figure 1). Rhabdoviruses are gested (Carter et al., 2010, Fuentes et bullet-shaped (Ge et al., 2010, al., 2007, Gan et al., 2008, He et al., Guichard et al., 2011) and some of 2001, Li et al., 2011, Wilson et al., the bunyaviruses have icosahedral 2006). symmetry (Freiberg et al., 2008, 3D structures of several Överby et al., 2008). –ssRNA viruses using 3D-cryoEM The majority of –ssRNA viruses have been described (Bharat et al., have a helically-arranged ribonucle- 2012, Ge et al., 2010, Harris et al., ocapsid (RNP) that contains the ge- 2006, Loney et al., 2009, Överby et nome wrapped either inside (Green al., 2008). Influenza virion structure et al., 2006) or outside (Tawar et al., has now been described for both a 2009) a nucleoprotein (N) helix. In mainly spherical X31 strain (Harris addition to the RNP, the host- et al., 2006, Wasilewski et al., 2012) derived lipid membrane encloses the and a mainly filamentous RNA-dependent RNA polymerase(s) A/Udorn/72 strain (Calder et al., and the matrix proteins. In all mem- 2010, Vijayakrishnan et al., 2013). In bers of the group there are one or the Udorn strain the M1 matrix pro- more viral membrane-spanning gly- tein was assembled in a regular, fil- coproteins that are required for host amentous array directly under the cell attachment, entry and in the case membrane bilayer and could be dis- of influenza and some paramyxovi- rupted with low pH treatment ruses, also detachment. In some vi- (Calder et al., 2010). In the X31 ruses, like influenza, filo- and strain, M1 lined the inner side of the rhabdoviruses, the attachment and membrane but was not tubularly or- fusion functionalities reside in the ganized (Harris et al., 2006). In same glycoprotein (hemagglutinin, Marburg virus (MARV), a member of HA for influenza, G for rhabdo- and the Filoviridae family, similar regu- filoviruses), whereas in others these larity to influenza Udorn, was detect- roles have been divided into two sep- ed in the VP40 matrix layer (Bharat arate proteins (e.g. hemagglutinin, H et al., 2011). In vesicular stomatitis and fusion, F in MV). Influenza also virus (VSV), the M matrix protein has two glycoproteins, but the sec- formed a left-handed helical struc- ond, the neuraminidase (NA) helps ture in register with the RNP (Ge et in virus escape from the cell surface al., 2010) and was in close proximity by cleaving off sialidase to which the to both the membrane and the RNP. HA attaches. Influenza has, in addi- For Sendai virus (SeV), a member of tion, a proton channel forming pro- the Paramyxoviridae, patches with tein M2 the main role of which is to thickened membrane possibly

Figure 1. Typical morphologies of different animal –ssRNA virus families. The spike glycoproteins are drawn as two different spikes for virus families that have separate genes for two glycoproteins. RNP and the matrix protein are depicted in blue and orange, respectively harboring M, were reported (Loney et al., 2006, Green et al., 2006) , in et al., 2009). A cylinder-like struc- HRSV seven (Tawar et al., 2009) and ture, possibly composed of M, was in SeV six (Calain et al., 1993). In also found, but filamentous virions most cases, the RNA is protected by were not reported. N and is RNAse resistant. The tight Structures of RNP-like assem- packing and protection of RNA by N blies have been studied by x-ray crys- raises the question of how the poly- tallography and 3D-cryoEM for some merase can access the RNA. It is like- of the –ssRNA viruses (Albertini et ly that local disassembly of the RNP al., 2006, Bhella et al., 2004, Green is required, but direct evidence is still et al., 2006, Schoehn et al., 2004, lacking. In influenza RNPs, however, Tawar et al., 2009). All, except the RNA is partially exposed and HRSV, were reported to have left- susceptible to RNAse degradation handed RNPs (Tawar et al., 2009). (Moeller et al., 2012). The influenza For HRSV, the handedness was not, RNPs are assembled in an unusual however, determined experimentally. manner in that they fold on them- The diameters of the RNPs vary be- selves to form an antiparallel double tween 15 nm in HRSV (Bhella et al., helix leaving a short loop between 2002) and ~70 nm in VSV (Ge et al., the intertwined helices, the free ends 2010) and they can be up to 1 µm in interacting with the polymerase length in the paramyxoviruses. The (Arranz et al., 2012, Moeller et al., number of RNA bases per N protein 2012). varies so that in RABV and VSV nine For many of the –ssRNA virus- nucleotides bind to one N (Albertini es, crystal structures of the spike pro-

teins have been solved (Hashiguchi proteolytic cleavage by cellular pro- et al., 2007, Lee et al., 2008, teases in order to be active. Unlike McLellan et al., 2011, Roche et al., other –ssRNA viruses, HRSV re- 2007, Varghese et al., 1983, Wilson quires two cleavages in order to be et al., 1981, Yin et al., 2005). All, ex- active for fusion (Gonzalez-Reyes et cept bunya- and rhabdoviruses, have al., 2001). class I fusion proteins of which SeV F Conversely to most of the was the first described (Homma et –ssRNA virus fusion proteins that al., 1973). Bunyaviruses have class II share the same general features, the fusion proteins and spike assemblies attachment functionalities are highly on virions that are quite different variable. This is mainly due to the from those of other –ssRNA viruses very different receptors of the virus- (Bowden et al., 2013, Huiskonen et es. The majority, including influenza al., 2010, Huiskonen et al., 2009). and most of the paramyxoviruses, Rhabdoviruses have class III fusion bind to sugar moieties on the cell proteins that combine features from surface, yet some (e.g. MV, Hendra both class I and class II fusion pro- and Nipah viruses) bind to specific teins (Roche et al., 2006, Roche et protein receptors. Although crystal al., 2007). Class I fusion proteins are structures of isolated ectodomains of expressed in the host cell in a many –ssRNA spike proteins have pretriggered, metastable, spring- been solved, the in situ structures on loaded conformation and they un- virions have only been reported for dergo dramatic refolding upon trig- influenza (Harris et al., 2013), Tula, gering by either low pH or a more Rift valley fever and Bunyamwera specific signal from binding to the viruses (Bowden et al., 2013, host cell surface (Harrison 2008). Huiskonen et al., 2010, Huiskonen et Class I and II fusion protein undergo al., 2009) from the Bunyaviridae irreversible conformational changes and PIV3 (Ludwig et al., 2008) from upon triggering, whereas for class III the Paramyxoviridae. In PIV3, F, fusion proteins the pH-related where discernible, was reported to be changes are reversible. Other than in the postfusion conformation low pH, the triggering signals are not (Ludwig et al., 2008). In the absence known, but they are thought to be of other F in situ structures, it is not relayed by the attachment protein known if that is a common feature in after binding to the receptor (Chang the family. For MV and HRSV the et al., 2012, Navaratnarajah et al., two glycoproteins have also been 2011). For HRSV, a receptor for the suggested to form a complex on the fusion protein has also been identi- cell surface, with the association oc- fied (Tayyari et al., 2011). Thus, it is curring already in the endoplasmic possible that in the case of HRSV the reticulum (ER) for MV. It has yet to attachment protein only indirectly be determined if they exist as such enhances the perhaps lower affinity on the virion (Low et al., 2008, interaction between the fusion pro- Plemper et al., 2001). tein and its receptor nucleolin. It has Matrix proteins are the main vi- also been noted that HRSV lacking ral factors responsible for budding the attachment glycoprotein G is in- (Liljeroos et al., 2013). They general- fectious, albeit at a lower level than ly reside in close contact with the wild-type (WT) (Techaarpornkul et inner side of the viral membrane. al., 2001). All of the class I fusion Although all –ssRNA matrix proteins proteins are trimeric type II trans- share many functions, like bringing membrane proteins and require a the RNP and glycoproteins together

at the budding sites, their structures Influenza M1 is completely different are highly varied. Paramyxoviral, in structure to the others, being filoviral and bornaviral matrix pro- completely α-helical (Sha et al., tein structures share similar mainly 1997). Arena and bunyaviruses do β-sheet folds (Figure 2) (Dessen et not have a matrix protein per se, but al., 2000, Money et al., 2009, arenaviruses have a small protein Z Neumann et al., 2009), although that has similar functions to the ma- bornaviral M is composed of only trix proteins (Neuman et al., 2005, one of the two domains found in Strecker et al., 2003). Many of the paramyxoviral and filoviral matrix matrix proteins have a propensity to proteins. Rhabdoviral M has a simi- self-oligomerize into sheets or heli- lar secondary structure composition ces, which is important for their but is unrelated in topology (Gaudier budding functionality. et al., 2002, Graham et al., 2008).

Figure 2. Comparison of matrix protein structures of –ssRNA viruses. Ribbon diagram of the atomic models from (A) HRSV M (PDB ID 2VQP) (Money et al., 2009), (B) EBOV VP40 (PDB ID 1ES6) (Dessen et al., 2000), (C) Borna disease virus M (PDB ID 3F1J) (Neumann et al., 2009), (D) VSV M (PDB ID 2W2R) (Gaudier et al., 2002), (E) influenza A M1 (PDB ID 1EA3) (Arzt et al., 2001) and (F) Lassa virus Z (PDB ID 2KO5) (Volpon et al., 2010). For Lassa virus Z the structured core domain between amino acids 26-79, is shown. The full length protein is 99 amino acids long and the termini are unstructured. Reprinted from (Liljeroos et al., 2013) with permission from the publisher.

1.2.1. Structures of paramyxoviruses

The family Paramyxoviridae con- Paramyxoviruses expose the ec- tains two subfamilies: Paramyxovir- todomains of two membrane- inae and Pneumovirinae. The Para- spanning glycoproteins, F and the myxovirinae subfamily has seven attachment protein on the mem- genera: Aquaparamyxovirus, Avula- brane surface. The HN attachment virus, Ferlavirus, Henipavirus, protein of respiro-, rubula- and Morbillivirus, Respirovirus and avulaviruses binds to sialic acids and Rubulavirus. The Pneumovirinae contains hemagglutination and neu- subfamily is smaller having only two raminidase activities. Morbillivirus- genera: Metapneumovirus and es, like MV, have protein receptors Pneumovirus. Of the human patho- and have lost the neuraminidase ac- gens, HRSV and human metapneu- tivity of their attachment protein H movirus belong to the Pneumoviri- (Muhlebach et al., 2011, Noyce et al., nae, all others to the Paramyxoviri- 2011, Tatsuo et al., 2000). Pneumo- nae. All paramyxoviruses are envel- and henipaviruses have an attach- oped pleomorphic viruses that are ment protein G that lacks both he- highly variable in size and shape. magglutination and neuraminidase Although the vast majority of these activites. The crystal structures of viruses are thought to be spherical or some paramyxoviral attachment pro- close to spherical in shape, filamen- teins have been solved alone and tous forms have also been reported with their receptors (Hashiguchi et (Bächi et al., 1973, Compans et al., al., 2011, Santiago et al., 2010, Xu et 1966, Yao et al., 2000). Although the al., 2008, Zhang et al., 2013b). They family includes several human path- all share a similar β-propeller fold in ogens, there was only one whole viri- their head domain despite the vari- on 3D paramyxovirus structure re- ous binding partners they have ported on SeV when the current evolved to bind. The oligomerization study was initiated (Loney et al., state on the virions in most cases is 2009). SeV was reported to vary be- tetrameric (Brindley et al., 2010, tween 110 and 540 nm in diameter, Yuan et al., 2008). was densely covered by the surface Crystal structures of the F pro- glycoproteins and contained multiple tein ectodomain in a prefusion state copies of the helical RNP. The RNP have been solved for PIV5 and HRSV appeared similar to what was earlier and in the postfusion state for PIV3, reported for purified RNPs (Bhella et NDV, and HRSV (McLellan et al., al., 2002). Indications of the pres- 2013, McLellan et al., 2011, Swanson ence of M under the membrane were et al., 2010, Welch et al., 2012, Yin et derived from the thickness meas- al., 2005, Yin et al., 2006). The pre- urements of the membrane, where fusion structures resembled each some regions had a thickness up to other in the overall shape, but were 12 nm. In the absence of other stud- different in certain antigenic sites ies, two of the open questions in the and location of the fusion peptide field were firstly, how widely these that inserts into the target cell mem- observations apply to other para- brane (McLellan et al., 2013). The myxoviruses, and secondly, what postfusion conformation was found causes some of the paramyxoviruses to be highly similar in all the struc- to adopt a filamentous shape? tures solved, having the canonical six-helix bundle in the stalk and an 8

overall golf-tee-like morphology. a left-handed helix. In general, crys- Surprisingly, in the only analysis so tallizing the nucleoprotein of the far of the glycoproteins on virions, F paramyxoviruses is exceedingly chal- was found in the postfusion confor- lenging due to the fact that regard- mation in PIV3 (Ludwig et al., less of the expression system used, 2008). Whether this was due to they bind to cellular RNA and oli- sample preparation, the fact that on- gomerize into a heterogenous mix- ly regions on virions with distinct ture of RNP-like helical structures. spikes, were used for analysis, or that These helices cannot easily be disas- F naturally mainly is in the postfu- sembled as the protein-RNA binding sion conformation on virions, has yet is tight and the RNA is ribonuclease to be confirmed. Since all of the protected. Hence the early stages of paramyxoviruses undergo virus-cell assembly of the RNP have been fusion at neutral pH, the fusion trig- tricky to access structurally. gering mechanism is more complex The M protein of paramyxovi- than that for pH-dependent fusion ruses is generally thought to line the proteins. For MV it has been shown internal side of the lipid bilayer. It that the fusion triggering signal is has a tendency to oligomerize as hel- initiated by H binding to the main ices (McPhee et al., 2011) and has receptor CD150 and that a conforma- been suggested to form a tubular lay- tional shift in the central stalk region er also on the RNP in disrupted vi- of the H tetramer is required rus-infected cells (Almeida et al., (Brindley et al., 2012). 1963, Brown et al., 1987). A crystal Paramyxoviruses have a non- structure of HRSV M has been pub- segmented genome typically approx- lished and it consists of two predom- imately 15 kb in length. Consequent- inantly β-sheet containing domains ly, being approximately 15 – 20 nm that are connected by an unstruc- in diameter, the RNPs can reach tured linking region (Money et al., lengths of up to 1 µm. RNPs from 2009). One side of the protein has an different viruses have varying helical extensive positively-charged surface parameters and slightly different ap- that extends to both domains. This pearance, but all have a general her- surface was suggested to interact ringbone-like structure (Bhella et al., with the negatively-charged phos- 2002). Recombinant RNPs from MV pholipids in the virion thus placing and HRSV have been reconstructed this side towards the membrane. In from electron micrographs and flexi- the absence of the whole virus 3D bility was detected even within a sin- structure, it is currently not known gle nucleocapsid (Bhella et al., 2004, how the M is organized in HRSV vi- Schoehn et al., 2004, Tawar et al., rions although indications of helical 2009). A crystal structure of an RNP- assemblies in the filamentous virions like decameric ring of HRSV has have been reported (Bächi et al., been solved and in this structure the 1973). Additionally, it is unclear RNA was wrapped around the ring whether the M-covered RNPs found on the outside in a groove on N in preparations with disrupted MV (Tawar et al., 2009). This structure (Almeida et al., 1963) are an authen- was then modeled into a 3D electron tic feature of the virion, or merely an microscopy helical reconstruction of experimental artifact. the RNP from electron microscopy as

2. ASSEMBLY OF NEGATIVE-SENSE RNA VIRUSES

Assembly and exit of enveloped vi- enriched in detergent insoluble cho- ruses is a complicated process, where lesterol (Scheiffele et al., 1999). all essential viral components have to Some viral proteins, like MV F, when be at the correct time at the place of expressed alone, have a tendency to assembly in suitable numbers. They localize into lipid rafts (Pohl et al., rely on the host cell as a supply of 2007). It is enough for one of the lipids for encapsidation of their ge- viral proteins to have this tendency if netic material during the journey the viral protein interaction network from one host cell to the next. They covers all the other components that exit the host cell via an event gener- are consequently also pulled to lipid ally called “budding” that initiates by rafts for budding. Many –ssRNA vi- gathering the viral components to ruses, like MARV, MV and HRSV the cell membranes. Next, they begin also show preference for budding to extrude from the cell enclosing the from one side of polarized epithelial viral genome and viral structural cells (Blau et al., 1995, Roberts et al., proteins inside the nascent extru- 1995, Sänger et al., 2001). Interest- sion. After a final pinching off from ingly, the MARV glycoprotein GP, the cell, the viral components have when expressed alone, is transported become enclosed into a lipid bilayer to the apical side whereas viral bud- hijacked from the host cell. During ding happens exclusively from the the process, the glycoproteins are basolateral side (Sänger et al., 2001). incorporated into the virion mem- Bunyaviruses, conversely to other brane via protein-lipid and protein- –ssRNA viruses, bud from the Golgi protein interactions with the mem- apparatus (Kuismanen et al., 1982, brane and the internal viral proteins. Novoa et al., 2005). The main role of the glycoproteins is Replication of most animal to act as docking and entry apparat- –ssRNA viruses takes place entirely uses to the next target cell, but they in the cytoplasm, influenza and Bor- are also important in inducing mem- na disease viruses being the only ex- brane curvature, interacting with the ceptions replicating in the nucleus. internal viral components and in di- Most of the enzymatic activities re- recting the budding to correct sites in quired for virus multiplication are the membrane. included in the viral proteins. Yet, Many of the –ssRNA viruses the viruses often rely on the host bud from specific microdomains on cell’s machinery for many aspects, the plasma membrane, called lipid especially for transport due to the rafts (Manie et al., 2000, Panchal et poor diffusion of the viral compo- al., 2003, Scheiffele et al., 1999). nents in the crowded cytoplasm. The These regions are enriched in sphin- requirement for transport includes golipids, cholesterol and proteins not only the glycoproteins that go (Lingwood et al., 2010). The budding through the normal ER – Golgi ex- preference from the lipid rafts has port route of transmembrane plasma been established mainly by detecting membrane proteins, but also colocalization of viral proteins and transport of the RNP and matrix pro- lipid raft markers in cells and the teins to the assembly sites. Addition- lipid composition of viruses has been ally, host components have roles in found to resemble that of lipid rafts, regulating viral proteins by post-

translational modifications and in Of the viral components, the the final pinching off of the virus matrix proteins are vital in assembly from the host cell. The host compo- and budding. They act in a glue-like nents include the endosomal com- manner between the viral compo- plexes required for transport nents and the membrane, bringing (ESCRT)(Bieniasz 2006), actin all the components together at the (Bohn et al., 1986, Carlson et al., budding sites. The role of matrix pro- 2010, Cudmore et al., 1995), micro- teins and their crucial interactions tubules (Penfold et al., 1994), and with other viral and cellular proteins COP-coated vesicles (Yamayoshi et are discussed next. al., 2008).

2.1. ROLE OF MATRIX PROTEIN IN ASSEMBLY

The important role of matrix pro- ruses, expression of the matrix proteins in virus budding was first tein alone is enough to cause release shown with temperature sensitive of VLPs (Jasenosky et al., 2001, mutants of SeV and VSV (Knipe et Justice et al., 1995, Pohl et al., 2007). al., 1977, Yoshida et al., 1979). Bud- For some viruses, however, coex- ding of both viruses was inefficient at pression of N with one of the surface elevated temperatures and altered glycoproteins is required or enhances the morphology of VSV (Schnitzer et VLP release (Li et al., 2009, Licata et al., 1979). Complementation with al., 2004, Noda et al., 2002, Schmitt separately expressed M restored the et al., 2002). The paramyxoviral budding ability (Lyles et al., 1996). Newcastle disease virus (NDV) ma- Also, deletion of the whole M gene trix has even been shown to be able from RABV resulted in 5 x 105 fold to deform and bud into phospholipid reduced release from cells bilayer vesicles upon in vitro incuba- (Mebatsion et al., 1999). Mutations tion of purified components in the M gene have also been linked (Shnyrova et al., 2007). For influen- to altered pathogenesis. In the sub- za virus, the role of M1 in budding is acute sclerosing panechephalitis not as clear as it is for other –ssRNA (SSPE) strains of MV, the M gene has viruses as conflicting reports on M1’s undergone hypermutation of U to C ability to drive VLP budding have residues, which results in impaired been published (Chen et al., 2007a, budding and consequently more cell- Gomez-Puertas et al., 2000). It is associated virus (Patterson et al., likely, however, that M1 defines the 2001). SSPE is a lethal neurogenera- virion shape as in the filamentous tive disorder with an incidence of particles it is assembled as a helix approximately 4-11 cases per and in the spherical particles as a 100 000 measles infections (WHO sphere (Calder et al., 2010). Yet, in 2006). the absence of demonstrated mem- The role of different viral pro- brane and glycoprotein free M1 helix, teins in budding has widely been as- it is uncertain whether the shape de- sessed by expressing individual pro- termining factor is M1 or perhaps a teins in mammalian cells and analyz- helical array of surface glycoproteins. ing whether vesicles with those par- In addition to the role in viral ticular proteins are released as virus- particle release, the matrix proteins like particles (VLP). With many vi- interact with multiple other viral

components to be included in the tivesicular endosomes (MVE)] and in budding virion. Interactions so far the final membrane scission step at described include the surface glyco- the end of cytokinesis. Topologically, protein tails (Ali et al., 2000a, Ali et these processes are similar to virus al., 2000b, Ghildyal et al., 2005b) budding from the plasma membrane. and the RNP (Ge et al., 2010, Iwasaki Thus, it is not surprising that viruses et al., 2009, Noton et al., 2007). For have evolved to hijack components of MV the interacting region on N was VPS to their advantage and redirect mapped to two C-terminal leucine them to the plasma membrane (Fig- residues, mutations in which result- ure 3). Interestingly, for EBOV a ed in impaired virus growth (Iwasaki role for COPI and COPII vesicles in et al., 2009). In most cases, the in- VP40 transport and budding has teraction of M and N is thought to be been demonstrated (Yamayoshi et direct, but for HRSV a mediator role al., 2010, Yamayoshi et al., 2008). for another viral protein, M2-1, has COPI and COPII are normally in- been described (Li et al., 2008). volved in ER-Golgi transport and are Many viruses make use of the not present in the proximity of the cellular vacuolar protein sorting plasma membrane. EBOV thus hi- (VPS) pathway in budding. Viral ma- jacks the machinery and redirects it trix proteins contain late domains (L- for transport to the plasma mem- domains) that interact with compo- brane. nents of this pathway. L-domains, so Despite the well-established termed because mutations in them role of matrix proteins in –ssRNA cause defects at a late stage in bud- virus assembly and budding, it is not ding, were first found in retroviral clear what provides the driving force gag matrix protein (Göttlinger et al., for deforming the cell membrane. 1991, Parent et al., 1995, Wills et al., Evidence from the study of isolated 1994), but have since been found in NDV M budding into phospholipid many other viruses including filo- vesicles suggested that multimeriza- rhabdo- paramyxo- and arenaviruses tion of M provides the energy, but (Harty et al., 1999, Li et al., 2009, cellular proteins could also have a Licata et al., 2003, Urata et al., role in this. For MARV it has been 2006). Influenza, instead, is not de- shown that actin is incorporated into pendent on VPS for budding and M1 virions and disrupting F-actin fila- does not contain L-domains (Bruce ments by cytochalasin D treatment et al., 2009, Watanabe et al., 2010). significantly reduced VP40 VLP The dependency on VPS for budding budding (Kolesnikova et al., 2007). is generally judged based on whether In a proteomic analysis of HRSV, dominant negative expression of actin was also detected in purified VPS4, a protein functioning in the virus preparations (Radhakrishnan pinching-off stage of VPS, impairs et al., 2010), although it was not budding. ESCRT complexes 0, I, II studied whether it was in the globu- and III are components of VPS and lar or filamentous form. In MV, fila- are normally involved in sorting pro- mentous actin has been detected proteins to late endosomes [also termed truding into the viral buds with the multivesicular bodies (MVB) or mul- barbed ends (Bohn et al., 1986).

Figure 3. ESCRTs have a role in several topologically similar membrane scission events. HIV is able to hijack the ESCRTs normally functioning in vesicle budding into late endosomes and in cytokinesis via its gag matrix protein. Reprinted by permission from Macmillan Publishers Ltd: Nature (Raiborg et al., 2009), © (2009).

2.2. ASSEMBLY OF MEASLES VIRUS

MV is one of the most infectious hu- (SLAM, CD150), which is the main man pathogens known and therefore receptor for MV (Tatsuo et al., requires high coverage of vaccination 2000). Recently, nectin-4 was identi- in the population for herd immunity. fied as an MV receptor on epithelial It is uncommon in industrialized cells not expressing CD150 countries, but poses a significant (Muhlebach et al., 2011, Noyce et al., health risk in the developing coun- 2011). The laboratory-adapted vac- tries especially to children under the cine strains, but not the WT strains age of five. In 2008, it caused the can also utilize the ubiquitous CD46 death of over 160 000 people world- as their receptor. The immunosup- wide (WHO 2009). The main reason pression caused by the virus is mani- for the mortality is the immunosup- fold, and includes CD150-mediated pression that follows from the infec- lymphopenia, prolonged cytokine tion. In countries with poor hygiene imbalance and silencing of peripher- and healthcare consequent second- al blood lymphocytes (Avota et al., ary infections can lead to serious ill- 2010). ness or death. The virus replicates in MV genome codes for eight pro- the cytoplasm of lymphocytes and teins: N, P, C, V, M, F, H, and L in 3’ dendritic cells expressing the signal- - 5’ order (Figure 4). The C and V ing lymphocyte activating molecule proteins are expressed from the P

gene. C is expressed from an alterna- V are nonstructural proteins and tive initiation codon and V has the N- their main roles have been shown to terminus of P, but the C-termini of lie in inhibiting host innate immuni- the two proteins are different. This is ty response by interfering with inter- accomplished with mRNA editing via feron signaling (Andrejeva et al., addition of one or more untemplated 2004, Palosaari et al., 2003, Shaffer G residues, which results in frame et al., 2003, Takeuchi et al., 2003). shifting (Cattaneo et al., 1989). C and

Figure 4. Gene organization of the MV genome. Gene order is drawn in the 3’ to 5’ direction from left to right. Adapted by permission from Macmillan Publishers Ltd: Nature Reviews Microbiology (Moss et al., 2006), © (2006).

MV assembly in the infected lular RNA (Bhella et al., 2002). The cell cytoplasm begins with the syn- N interacting regions on P have been thesis of a new copy of the RNA ge- mapped to the XD domain and to the nome. All viral RNA synthesis is car- very N-terminal end (PNT) (Harty et ried out by the RNA-dependent RNA al., 1995). PNT has been reported to polymerase that is composed of the be unstructured, but partial folding large (L) protein and its accessory could be induced with trifluoroetha- phosphoprotein (P). All enzymatic nol (Karlin et al., 2002). In the crys- activities reside on L whereas P func- tal structure of VSV N0-P the PNT tions as an adaptor between L and folded as an α-helix, occupied a the template RNP. It is not known groove in a hinge region on N that how the RNP template is read by the was adjacent to the RNA-binding polymerase but a model has been groove (Figure 5) (Green et al., suggested, where the L-P complex 2006, Leyrat et al., 2011). N0-PNT cartwheels around the helical RNP, interactions were mainly hydropho- with the tails of the tetrameric P con- bic, whereas N interactions with stantly associating and disassociating RNA were ionic. For SeV, the PNT from N (Longhi 2009). P is a largely amino acids responsible for N0 bind- intrinsically unstructured protein ing have been mapped to residues with a central helical tetramerization 33-41 (Curran et al., 1995). In the domain and a C-terminal XD domain absence of a paramyxoviral N0-P that binds to the C-terminal part of N crystal structure, it is not known (N(TAIL)) in transcription and repli- what the details of the N0-P interac- cation. In addition to the mediator tion are. Knowing the precise region role between N and L, P serves a of PNT required to keep N in its chaperone role for N until assembled RNA-free N0 form would allow for onto the growing RNP (Curran et al., the design of peptide inhibitors as 1995). This is required as free N (de- has already been shown for RABV noted N0 in the RNA-free form), hav- (Castel et al., 2009). ing a high affinity for RNA, would otherwise bind unspecifically to cel-

Figure 5. Structure of VSV N in complex with PNT (A) and RNA (B). PNT prevents N from binding to RNA by occupying a groove in a hinge region of N that is adjacent to and partly overlapping with the RNA-binding site. Adapted from (Leyrat et al., 2011).

After replication and assembly 1994). In normal infection N co- of the RNP, the next step is to have localized with M to the plasma mem- the RNP, polymerase, M and the gly- brane, but formed small dots in the coproteins transported to the bud- perinuclear area when the interac- ding sites. How RNP is transported, tion was abrogated. In electron mi- is largely unknown, but the polymer- croscopy of plastic-embedded sec- ase is thought to be transported as a tions from infected cells, two mor- complex with RNP. Actin and tubulin phologies for the RNP were detected, have both been reported to be im- thinner “smooth filaments” and wid- portant in MV infection, but their er “granular filaments” (Dubois- precise roles are unclear. Actin has Dalcq et al., 1973, Oyanagi et al., been suggested to have a role in re- 1971). Another study reported “fuzzy lease of the virus from the cell and nucleocapsids” in the cytoplasm of tubulin in replication (Bohn et al., infected cells and these were shown 1986, Moyer et al., 1990, Stallcup et to contain M (Brown et al., 1987). al., 1983). For the closely related SeV Thus, it is possible that M covers the it was shown that RNPs are trans- RNP in infected cells and RNP is ported along microtubules in associ- transported to the lipid rafts for ation with intracellular vesicles budding as cargo, employing the (Chambers et al., 2010). Conversely same cellular machinery that to a number of other –ssRNA virus- transport M to the plasma mem- es, MV assembly and budding is brane when expressed alone. If M ESCRT independent (Salditt et al., covers the RNP in a tubular assem- 2010). bly, a transition from this assembly The viral M protein has been to the membrane would be required, shown to have a crucial role in the as according to the present model, in transport of RNP to the budding sites the released virions M forms a layer on the plasma membrane, but also in next to the inner leaflet of the mem- transcription inhibition (Iwasaki et brane (Figure 6) (Moss et al., al., 2009, Suryanarayana et al., 2006). Another possibility is that M

stays bound to the RNP inside the ized in membrane rafts, but raft lo- virion and does not form a layer di- calization is enhanced in the pres- rectly under the membrane. To shed ence of F (Pohl et al., 2007). F is lo- light into this uncertainty, structural calized in lipid rafts when expressed studies of the released virion are re- alone whereas H localization to lipid quired. rafts requires the presence of F (Vincent et al., 2000). So, even though M is the main coordinator of assembly, F has an important role in taking all the components to the lipid rafts, where the budding is thought to occur (Vincent et al., 2000). The requirement of F for H translocation to lipid rafts also indicates that they have an interaction, which has also been shown by fluorescence microscopy and pulse-chase analysis (Plemper et al., 2001). In polarized epithelial cells MV shows preference for budding from the apical side (Blau et al., 1995). The glycoproteins require expression of M in order to be transported to the apical side (Maisner et al., 1998, Naim et al., 2000). Thus, M and F have a mutual relationship in that M helps in trans- porting F to the correct side of the cell for budding and once at the Figure 6. A textbook model for the MV virion. membrane, F helps M to translocate In the model the RNP is randomly arranged into lipid rafts. The RNP and H seem inside the virion and M is bound to the inner to be freeloaders in the process. leaflet of the membrane. Adapted by permis- What provides the final pinch- sion from Macmillan Publishers Ltd: Nature ing off functionality for the budding Reviews Microbiology (Moss et al., 2006), © (2006). virion, is not known. This functionality could be a feature of M as purified M from the closely related NDV can M, when expressed alone, bud into phospholipid vesicles drives the release of VLPs with a sim- (Shnyrova et al., 2007). Also, MV ilar efficiency to that of viral budding budding is ESCRT independent (Pohl et al., 2007). Thus, like many (Salditt et al., 2010) and it does not other matrix proteins, it has an in- have a protein, like the M2 of influ- herent ability to direct vesicle release enza that would be capable of mem- from cells. In the absence of the gly- brane scission (Rossman et al., coproteins, some M is found local- 2010).

2.3. ASSEMBLY OF HUMAN RESPIRATORY SYNCYTIAL VIRUS

HRSV is a common virus that in purified virus preparations both healthy adults causes cold-like symp- spherical and filamentous particles toms, but can be severe in young are present (Gower et al., 2005). children and the elderly (Falsey et Currently it is not known which al., 2005, Nair et al., 2010). It is re- morphology dominates in clinical sponsible for over 20 % of acute low- infection, but both appear to be in- er respiratory infections world-wide fectious in vitro (Gower et al., 2005). and has been estimated to cause the Similarly to MV and other death of 100 000 children under the paramyxoviruses, the N protein has age of five (Nair et al., 2010). Unlike an inherent RNA-binding activity for MV, an effective vaccine has and it readily forms RNP-like struc- proven difficult to develop. Hurdles tures when expressed in insect or for vaccine development include the bacterial cells (Meric et al., 1994, early age of infection, innate immun- Murphy et al., 2003). The N- ity evasion by the virus, adaptive terminal part of N was shown to be immunity incapable of preventing responsible for the helical assembly subsequent infections and lack of a and a deletion mutant of N that con- suitable animal model (Graham tained only the first 92 N-terminal 2011). Treatment is limited to passive amino acids of the total 391 could immune-prophylaxis for neonates assemble into helices (Murphy et al., under high risk of severe infection 2003). Similarly to other paramyxo- (Shadman et al., 2011) and after on- viruses, a role for P in preventing set of the disease the purine analog unspecific binding to cellular RNA, ribavirin is sometimes used. has been described for HRSV P In addition to the proteins (Castagne et al., 2004). The regions common for all paramyxoviruses, on P responsible for this function HRSV has genes encoding for SH, have not been characterized. secondary matrix M2-1 and M2-2, In polarized epithelial cells, and two nonstructural proteins, NS1 HRSV buds from the apical side. F, and NS2. M2-1 and M2-2 are ex- when expressed alone, localizes to pressed from the same gene, M2-2 the apical side. F is not, however, being expressed from an internal required for sorting of the internal overlapping coding frame. M2-1 has components to the apical side as been shown to be a transcription an- VLPs lacking both glycoproteins still ti-terminator, enhancing transcrip- bud from the apical side (Batonick et tional chain elongation and read- al., 2008). RSV, similarly to MV, is through at gene junctions (Collins et thought to bud from lipid raft micro- al., 1996, Hardy et al., 1998). It is domains in the plasma membrane also a structural component of the (McCurdy et al., 2003). HRSV M, virion and has a role in transport of when expressed alone, binds to cellu- RNP to the budding sites (Li et al., lar membranes (Henderson et al., 2008). M2-2 has been ascribed a role 2002). In viral infection it is found in the transcription-to-replication partly in lipid rafts, but lipid raft transition (Bermingham et al., 1999). association is dependent on F Characteristic for HRSV are (Henderson et al., 2002). As with the filamentous filopodium-like pro- most of the –ssRNA viruses HRSV M trusions seen at the cell surface of is associated with the RNP during infected cells (Mitra et al., 2012). In infection and also interacts with the 17

cytoplasmic tails of the glycoproteins port was found to be controlled by (Ghildyal et al., 2005b, Ghildyal et ubiquitinylation of M and depleting al., 2002, Shaikh et al., 2012a). The free cellular ubiquitin resulted in in- M2-1 protein has also been shown to hibition of budding (Wang et al., have a role in transport of the RNP 2010). from the replication sites to the A number of cellular compo- plasma membrane. It was suggested nents have also been indicated to be to act as a mediator between the RNP important for HRSV assembly. These and M (Li et al., 2008). The interac- include actin and Rab11 family inter- tion between M and F has been acting protein 2 (FIP2), a major pro- shown to be crucial for the formation tein associated with the apical recy- of viral filaments on the cell surface cling endosome (ARE) involved in and this interaction was shown to be protein trafficking in polarized cells disrupted by a single phenylalanine (Burke et al., 1998). Importantly, like mutation in the cytoplasmic tail of F MV, RSV budding has been shown to (Oomens et al., 2006, Shaikh et al., be ESCRT-independent (Utley et al., 2012a). The M and F proteins appear 2008). Similarly to MV, it is not to coordinate the budding so that F known how, in the absence of first initiates a bud at a lipid raft, ESCRTs, the final pinching off from which cannot elongate into a fila- the host cell occurs. FIP2 however ment before M is recruited to the site has been shown to control budding (Mitra et al., 2012). The recruitment as a defective FIP2 mutant caused a of M occurs presumably via the in- failure in the final stage of HRSV teraction between the F cytoplasmic budding (Utley et al., 2008). Fila- tail and M and further interactions mentous actin has been observed at with M and RNP, mediated by M2-1, the base of viral filamentous protru- guarantee the incorporation of all sions (Jeffree et al., 2007). Yet, it internal components. G and SH are was shown that viral filaments are dispensable for budding and quite formed independently of host cyto- surprisingly for the whole infection skeleton and related proteins, and in vitro, although G does elevate in- actin was suggested to be important fectivity (Techaarpornkul et al., not in assembly of the filament but in 2001). G interacts specifically with M anchoring the viral filaments (Shaikh with its cytoplasmic tail, but has also et al., 2012b). HRSV activates Ras been reported to form a complex gene family member A (RhoA), a with F on the cell surface (Ghildyal et small GTPase known to regulate the al., 2005b, Low et al., 2008). actin cytoskeleton, and inhibition of RSV M has been found to un- RhoA compromises filament assem- dergo nuclear-cytoplasmic shuttling. bly. Inhibition resulted in blunted It is imported via importin β1 and filaments and a shift towards spheri- exported at a late stage of infection cal particle production (Gower et al., by exportin 1 (Crm1) (Ghildyal et al., 2005). Interestingly, a matrix-less 2003, Ghildyal et al., 2009, Ghildyal HRSV mutant also showed blunted et al., 2005a). Like MV M, it inhibits filament formation, which might in- transcription and this function was dicate a requirement for ARE in postulated to be regulated by con- proper M transport and assembly. trolled export from the nuclear res- How the matrix, when present at the ervoir of M (Ghildyal et al., 2009). filament assembly sites, causes elon- Nuclear-cytoplasmic shuttling has gation of the filaments, is not known. also been shown to occur in Nipah virus infection. In that case the ex-

3. ENTRY OF NEGATIVE-SENSE RNA VIRUSES

The first step in any infection process the receptor binding functionality is is attachment to a host cell. Viruses often a part of the fusion protein. have evolved to use various types of Structurally the fusion protein func- molecules on the cell surface as re- tionality dominates in these proteins, ceptors for attachment. The main as it requires much more complex types of molecules used as viral re- mechanics than the simple binding ceptors are proteins, carbohydrate to the receptor. Interestingly, para- moieties on glycoproteins and lipids myxoviruses, which have separate (Morizono et al., 2011). The type and proteins for both functionalities, of- number of receptors used by differ- ten have the neuramidase activity on ent viruses vary greatly even within the attachment protein. That activity viral families and receptors are the on influenza is on the separate NA most important factors in defining protein. Thus, from an evolutionary tropism. Protein receptors tend to be perspective, the receptor-binding the most specific as they can be ex- activity on influenza HA might have pressed in very limited cell types and initially resided on the NA. thus restrict the viral infection to After attachment to the target specific cells. Carbohydrates, like cell, a virus needs to penetrate at sialic acids used by many viruses, are least one lipid membrane. For envel- present widely in different types of oped viruses, two membranes, that of animal cells and result in more wide- the host cell and that of the virus, spread infection of different cell have to be fused. Enveloped viruses types. Yet, within sialic acid binding take two particular means to achieve viruses, tropism can be conferred by this: endocytosis followed by fusion preferential binding to certain types with the endocytotic vesicle or direct of sialic acid. Human influenza, for fusion with the plasma membrane. example, binds specifically to um- Of the –ssRNA viruses influenza, brella-shaped α2-6 linked sialic acids filo- rhabdo-, bunya-, arena- and that are present mainly in the epithe- some paramyxoviruses use the for- lial cells of the respiratory tract, mer route, whereas most of the whereas avian influenza binds to paramyxoviruses use the latter. The cone-shaped α2-6 and α2-3 linked vast majority of viruses utilizing the sialic acids (Chandrasekaran et al., host endocytotic machinery enters 2008). Mutations in HA that allow the cell via clathrin-mediated endo- binding to human-type sialic acids cytosis and is dependent on acidifica- are thought to have a major role in tion of the endosome. The lowered influenza crossing the species barri- pH is required for triggering of the er. fusion protein, which results in fu- The ligands for the receptors, sion of the viral and endosome the viral attachment proteins, are as membranes. The degree of acid sen- varied as their receptors. In icosahe- sitivity of the fusion protein deter- dral viruses, the receptor binding mines the stage of endosome matu- protein is part of the capsid and in ration, where the virus penetration some viruses like the African horse- occurs. VSV, for example fuses with sickness virus it is a prominent large early endosomes at around pH 6, feature on the capsid (Manole et al., whereas influenza and Uukuniemi 2012). Within the –ssRNA viruses, virus (a member of the Bunyaviridae

family) fuse with late endosomes at a proteins do not form a complex in- pH closer to 5 (Lozach et al., 2010, side the cell and are thought to inter- White et al., 1981). act only after attachment protein Some of the –ssRNA viruses en- binding to the target cell (Paterson et ter via macropinocytosis, a ligand- al., 1997). induced clathrin-independent pro- It is well established that the cess normally used for uptake of fluid stalk region of the attachment pro- and solutes (Mercer et al., 2012). tein plays a key role in triggering F Most of the viruses dependent on for fusion (Ader et al., 2012, Apte- macropinocytotis for entry are also Sengupta et al., 2013, Bose et al., dependent on acidification of the 2011, Brindley et al., 2013, Maar et macropinosome. However, HRSV al., 2012, Melanson et al., 2004). and possibly others rely on proteolyt- Based on two different crystal forms ic activation by cellular proteases in observed for MV H in complex with the macropinosome and do not re- the receptor CD150 , it was suggested quire acidic conditions (Krzyzaniak that a sliding movement between the et al., 2013). The filoviruses are ex- two dimers of the tetramer results in ceptional in that they require both a conformational change of the stalk low pH and proteolytic cleavage by that then causes triggering of F host pH-dependent proteases for (Hashiguchi et al., 2011, Nakashima fusion (Chandran et al., 2005). et al., 2013). The two states of the H Viruses that are independent of tetramer have also been detected in endocytosis and acidic pH for entry native polyacrylamide gel electro- fuse directly with the cell membrane phoresis of free and receptor-bound and have evolved alternative ways for H (Brindley et al., 2012). For para- ensuring suitable time and place for myxoviruses with HN as the attach- fusion triggering. These include the ment protein an additional mecha- requirement for binding to a co- nism for control has been observed. receptor in addition to the primary In crystal structures of HN the large receptor for HIV and relay of recep- head domains were found in two dis- tor binding signal from the attach- tinct orientations; bent down to ment protein to the fusion protein by block any possible interaction be- altered lateral contacts for paramyx- tween F and the HN stalk, or bent up oviruses. For most paramyxoviruses exposing the trunk (Welch et al., the signaling is thought to occur by 2013, Yuan et al., 2011). It was sug- either dissociation or formation of a gested that HN binding to the recep- complex between the attachment and tor would shift the heads up and al- fusion protein (Chang et al., 2012). low F to interact with the trunk, a In most cases the interaction is virus prerequisite for F triggering (Welch specific as substituting the attach- et al., 2013). Similar large conforma- ment protein to that of another virus tional shifts have not been observed from the family does not produce for paramyxoviruses with H or G as infectious virions (Das et al., 2000, the attachment protein. As in most of Hu et al., 1992). MV H and F form a these viruses F is thought to form a complex already in ER of the host complex with the attachment protein cell before budding and are likely to already before target cell attachment, remain as a complex until F trigger- they are unlikely to employ such a ing for fusion (Plemper et al., 2001). mechanism. With most paramyxoviruses (ones The actual mechanism of fusion with HN as the attachment protein), is incompletely understood although however, the attachment and fusion multiple structures of the fusion pro-

tein ectodomains in both pre- and unfavorable process to which the re- postfusion conformations have been folding of F is thought to provide the solved (Bullough et al., 1994, energy. Despite the wealth of infor- McLellan et al., 2011, Roche et al., mation already obtained, the lack of 2006, Roche et al., 2007, Swanson et intermediate state structures leaves al., 2010, Welch et al., 2012, Wilson great gaps to the model. The struc- et al., 1981, Yin et al., 2005, Yin et ture of the fusion pore, for example, al., 2006). In the current model for has not been described and it is not Class 1 fusion protein mediated fu- known how many copies of the F sion (Harrison 2008, Plemper 2011), trimer are required to construct a triggering of F first results in expo- fusion pore. sure of the hydrophobic fusion pep- After fusion of the viral and tides that get casted out in a spring- host membranes, uncoating of the like manner. The fusion peptides RNP from the matrix protein is re- then insert into the target membrane quired before another round of infec- connecting the two membranes. tion can begin. How and where this Next, F refolds back into a compact occurs, is poorly understood for most structure bringing the two mem- –ssRNA viruses. For influenza it is branes in close apposition. The re- known that a drop in pH results in folding is driven by the formation of disassembly of the M1 from the viral a highly stable six-helix bundle by membrane possibly preceded by a the so-called heptad repeats present conformational change and thinning in both ends of each F monomer of of the M1 layer (Calder et al., 2010, the trimer. Finally, the two mem- Fontana et al., 2012). M1 then disso- branes fuse opening a fusion pore, ciates from the RNP prior to RNP probably via a hemifusion interme- transport to the nucleus where repli- diate, and the virion cargo gets deliv- cation takes place (Martin et al., ered to the cytoplasm. Fusion of the 1991). two membranes is an energetically

4. ELECTRON CRYOTOMOGRAPHY AS A TOOL IN STUDYING VIRUS STRUCTURE

Eukaryotic cells are typically over 10 stead, electrons at voltages of 200 – µm in size, smallest bacteria approx- 300 kV used in modern electron mi- imately 1 µm and viruses in the order croscopes have a wavelength of ~2 of tens to hundreds of nanometers in pm which is more than adequate for diameter. Eukaryotic and to some virus studies even at atomic resolu- extent also bacterial cells can be tion. X-ray crystallography can studied using visible light microsco- sometimes be used, but by definition, py. Resolution in any imaging tech- only for samples that can be crystal- nique is limited by the wavelength of lized and crystallization of even large the radiation used to inspect the regular viruses is often difficult. specimen, which for visible light Thus, electron microscopy is usually ranges between 400 and 700 nm. the method of choice. Therefore, in order to be able to vis- When the electrons hit the ualize details within viruses, visible sample inside the vacuum of a light microscopy cannot be used. In- transmission electron microscope

(TEM), the vast majority of the elec- indeed is a copy of the other is often trons pass through the sample unaf- unreliable or impossible by eye and fected. Those that interact with the therefore computational methods are sample scatter either elastically or normally used. inelastically. In elastic scattering, the For 3D studies of biological ob- energy of the incident electrons does jects in transmission electron mi- not change, only the direction. This croscopy, multiple images of the change in electron direction corre- same object in different orientations sponds to a change in the phase of have to be obtained. This is done by the diffracted electron wave and can one of two means: by collecting mul- be used in phase contrast imaging. In tiple images of an object present in inelastic scattering, some of the en- multiple copies in different orienta- ergy is transferred to the specimen, tions in the sample (e.g. a suspension which results not only in damage to of an icosahedral virus) or by tilting the sample by ionization, chemical the sample inside the microscope bond rearrangements or free radical and collecting images of the same formation, but also results in noise in object from multiple orientations. the image (Orlova et al., 2011). For The former is called single particle heavy metal stained specimens the reconstruction and the latter tomog- changes are tolerable and such sam- raphy. Of the two, tomography is ples can be imaged with a bright more versatile as it can be used to electron beam resulting in high con- image unique objects unlike single trast. Also the high scattering cross particle reconstruction. Tomography sections of the heavy metal atoms is therefore ideal for 3D imaging of provide higher contrast than the light pleomorphic viruses and is currently atoms in biological samples. In order the method of choice not only for to preserve the sample in its un- pleomorphic virus studies but also stained native state, cryo-fixation is for structural studies of the cell (Gan used. In the absence of heavy-metal et al., 2012, Guerrero-Ferreira et al., stain the radiation damage becomes 2013, Subramaniam et al., 2007). a significant hurdle. It is indeed the Using single particle methods com- most significant factor preventing bined with the powerful utilization of straightforward imaging of biological icosahedral symmetry of some virus- samples in atomic detail. In order to es, atomic resolution reconstructions avoid excessive damage to the sam- have been obtained (Liu et al., 2010, ple, dim electron beam and short Zhang et al., 2010). Resolutions from exposure of the sample have to be electron cryotomographic studies are used. Due to the low number of elec- currently more modest (typically 3 - trons passing through the sample 4 nm), but no fundamental re- and contributing to the collected im- strictions exist for obtaining atomic age, the images are noisy and poor in resolution from tomography when contrast. To overcome this issue, av- combined with averaging of repeat- eraging of multiple low-contrast im- ing structures within the tomograms. ages of an object present in multiple Challenges in averaging of tomo- copies can be used to enhance the graphic data are discussed in section contrast. Verifying that one object 4.4.

4.1. SAMPLE PREPARATION FOR ELECTRON CRYOTOMOGRAPHY

All biological reactions take place in aqueous solutions. Thus, in order to view biological molecules in native or close-to-native state they should be imaged in such conditions. Electrons have a rest mass which results in poor penetration in air and therefore a high vacuum must be maintained in the electron microscope column. To prevent dissipation of the water from the sample, the sample can be fixed by cooling it to low enough temperatures for the water to solidi- fy. Ice at atmospheric temperatures is crystalline and composed of a hexagonal lattice of water molecules. Formation of water crystals destroys the intricate biological structures and thus fixation by ordinary freezing is not useful for electron cryomicroscopy. However, water, when cooled rapidly to temperatures below -140 °C, forms a metastable, amorphous state called vitrified water (Figure 7). Fast enough cooling can be achieved with rapid immersion into liquid ethane cooled by liquid nitrogen (Adrian et al., 1984, Dubochet et Figure 7. Three forms of solid water viewed al., 1988) normally using a gravity- with an electron microscope. Corresponding driven guillotine. Warming up the diffractograms are shown in the middle. In A) sample causes an irreversible phase hexagonal ice, in B) cubic ice, and in C) vitre- transformation to cubically or hex- ous water are shown. In C) the absence of intensity peaks in the diffractogram indicates agonally crystallized water, which that the water molecules are not ordered, destroys the biological sample (Fig- whereas in A) and B) the intensity peaks or ure 7). Thus, the sample has to be rings indicate an ordered state. Reprinted from maintained at all times under -150 °C (Dubochet et al., 1988) with permission from the publisher. normally with the help of liquid nitrogen, the boiling point of which is - 196 °C at atmospheric pressure. such as mammalian cells, high pres- The cooling rate close to the sure can be used to prevent crystalli- surface of the specimen is fast zation of water. In this high-pressure enough for vitrification, but quickly freezing the sample is frozen at pres- slows down towards the interior. sures up to 200 MPa, and this way Therefore, only thin samples of up to samples as thick as 200 µm can be maximally a few micrometers in vitrified (Vanhecke et al., 2008). The thickness can be vitrified using method is based on freezing point plunge-freezing. For thicker samples, depression, reduction in the rate of

ice crystal formation and reduction bution of the sample on the grid. of ice crystal growth at high pressure. Next, the excess sample is removed However, due to the poor penetra- from the grid by blotting with a piece tion of the electron beam through the of filter paper to leave only a thin sample, the optimal sample thickness layer of sample on the grid. Immedi- for TEM is less than 200 nm. Thus, ately after blotting, the grid is high-pressure frozen samples have to dropped in the tip of tweezers to liq- be processed into sections, preferably uid ethane using a guillotine. The under 200 nm thick that can then be vitrified samples can then be stored viewed with the microscope. Cryo- in liquid nitrogen for years until use. electron microscopy of vitrified sec- To preserve the vitrified state, the tions (CEMOVIS) is a rising tech- sample has to be cooled also during nique that currently requires skilled transfer to the microscope and dur- hands to be successful (Al-Amoudi et ing imaging. For this, a special liquid al., 2004). nitrogen cooled sample holder is For viruses in suspension, used. For samples to be used for elec- plunge-freezing is normally used. In tron tomography, colloidal gold practice, ~3 µl of sample is pipetted beads are normally added to the onto a holey carbon film supported sample before plunge-freezing. by a copper mesh grid. The carbon Those provide high contrast features layer is normally first ionized with a in the images that are useful in glow-discharger or a plasma cleaner alignment of the tomographic tilt to make the carbon layer charged series. and thus hydrophilic for even distri-

4.2. IMAGE FORMATION IN THE ELECTRON MICROSCOPE

Similarly to a conventional light mi- ochromatic than the electrons from croscope, a TEM is composed of a thermionic guns and are used in all source of electrons, a lens system to modern TEMs for high resolution condense, focus and project the analysis. beam, and a viewing screen, film or a In the FEG the electrons are ex- camera to view and record the tracted from the emitter surface of a formed image. As electrons are nega- Zr02 coated sharp tungsten crystal tively charged particles, electromag- tip by a voltage gradient and subse- netic lenses instead of refractive quently accelerated with a potential glass lenses are used to focus the of typically 200 – 300 kV (Orlova et beam. The electron source has typi- al., 2011). Throughout the TEM col- cally been a thermionic heated tung- umn the beam is guided by electro- sten filament or a lanthanium hexa- magnetic beam deflectors, parallel- boride (LaB6) crystal. Of these two, ized or focused by electromagnetic LaB6 as an electron source provides a lenses and restricted by metallic ap- more coherent, brighter beam. Yet, ertures with small holes in them. the most coherent and brightest First the divergent electron beam is beam is provided by field emission parallelized and focused by a con- gun (FEG) electron sources. The FEG denser lens system with apertures. electrons also are also closer to mon- Next, the beam passes through the

sample, mostly unaffected, but some based on their energy (Angert et al., of the electrons undergo elastic or 2000, Schroder 1992). This energy- inelastic scattering events. The image filtering can remove inelastically and initial magnification is then scattered electrons deflected with formed by the objective lens which is low angles that have passed through the most critical part for high resolu- the objective aperture. Energy filters tion imaging and where most image are routinely used in modern cryo- aberrations originate in. After the TEMs to enhance contrast. objective lens, the beam passes Contrast in TEM is formed by through an aperture, which is used to two different principles. Amplitude prevent inelastically scattered elec- contrast results from absorption of trons with high deflection angles electrons to the sample via inelastic from contributing noise to the image. interactions and in essence means The formed image is further magni- that some parts of the sample do not fied by a magnifying lens system let electrons pass through as well as which is adjusted to obtain different other parts and therefore show as final magnifications. Finally, the darker areas in the image. Amplitude formed image is viewed from a phos- contrast for unstained biological phorus screen or recorded on film or specimens with mainly light ele- a camera. The camera has commonly ments, such as hydrogen, carbon, been a charge-coupled device (CCD) nitrogen and oxygen, only contrib- connected to an electron scintillator, utes approximately 7 % of the total but direct electron detectors (DED) contrast (Toyoshima et al., 1988). without a scintillator, are becoming The main fraction of the contrast more popular due to their high detec- comes from the interference between tive quantum efficiencies (DQE) and the unaffected electron wave gone the resulting low level of noise through the sample and the diffract- (Bammes et al., 2012). To reduce the ed wave with elastically scattered noise in the formed image, it is also electrons. Transfer of contrast in possible to filter the electrons after TEM is described by the contrast they have passed through the sample transfer function (Baker et al., 1999):

( ) ( ) ( ) ( ( )) ( ( ))

where ( ) ( the electron source. Thus an FEG

) , v is the spatial fre- provides much more subtle attenua- quency per Å, Famp is the fraction of tion of high frequencies than thermi- amplitude contrast, λ is the electron onic guns. The phase contrast com- wavelength in Ångströms, Δf is the ponent of the CTF is a periodic sine underfocus in micrometers and Cs is function that is affected by defocus the spherical aberration of the mi- and the spherical aberration of the croscope objective lens in millime- objective lens. Thus, the inherent ters. CTF is a periodic function that spherical aberration has a phase is modulated by components of both shifting effect on the function and amplitude and phase contrast and is the effect varies with the defocus of attenuated at high spatial frequen- the lens. Different defoci result in cies by the envelope component high signal at different spatial fre-

( ) (where δ describes beam co- quencies depending on the positions herence) of the CTF originating from of the minima and maxima of the the instabilities and incoherence of CTF. Focusing slightly under the ex-

act focus enhances low frequency can be accurately estimated from the components and is normally used for positions of the so-called Thon rings imaging. Overfocusing would in visible in the power spectrum of the principle do the same, but in overfo- image that correspond to CTF zeros. cus the amplitude and phase contrast Since there are zeros in the CTF, in components of CTF have opposite order to cover the full spatial fre- signs at low frequencies, which re- quency range, multiple images at sults in destructive interference and different defoci are required. Cor- consequently low signal. recting for the effects of CTF are al- As the phase contrast compo- ways used in high-resolution cryo- nent of CTF is a sine function, its EM, but are beginning to be used values vary between 1 and -1. In or- also in cryo-ET. Previously the data der to have human interpretable da- were often lowpass filtered to the ta, the negative regions of the func- first CTF zero to avoid artefacts from tion need to be phase flipped. The the negative CTF values. This howev- positions of CTF zeros can be calcu- er often limits the obtainable resolu- lated from the function if the spheri- tion to ~3 – 4 nm and is to be avoid- cal aberration coefficient and defocus ed if high resolutions are strived for. are known. The defocus of an image

4.3. ELECTRON CRYOTOMOGRAPHY

Cryo-ET has been widely used for magnification. Low magnification is ultrastructural studies of pleo- not only useful for easy location of morphic viruses (Briggs et al., 2006, the positions to be imaged, but it also Cyrklaff et al., 2005, Grünewald et minimizes the electron dose on the al., 2006, Harris et al., 2006, Pietilä sample before the actual data collec- et al., 2012), cellular structures (Al- tion. Once the microscope is aligned, Amoudi et al., 2007, Brandt et al., the region of interest in the sample is 2010, Maimon et al., 2012, Psencik et positioned to the center of the field, al., 2009) and virus-cell interactions the beam brightness is set, and a tilt (Carlson et al., 2010, Ibiricu et al., series is collected. Typically the tilt 2011). Samples for cryo-ET are pre- series is collected from -60° to +60° pared either by plunge vitrification and an image recorded every two or cryo-sectioning. Typically, colloi- degrees. Thus in total approximately dal gold of 5 - 10 nm in diameter is 60 images of the same object are rec- included in the sample suspension orded. For optimally thin regions before vitrification. For cryo-sections (~100 nm in thickness) with an ener- of cells, this cannot however be done, gy-filter equipped TEM, tilting the but quantum dots in organic solvents sample up to ±70 ° can yield useful can be added directly on the vitrified data. The reason for not tilting to sections and used in a similar man- higher tilts is that because the sam- ner for alignment of the tilt series ple has slab geometry, the more it is (Masich et al., 2006). tilted the longer distance the elec- After having successfully vitri- trons have to travel in the ice layer. fied the sample and inserted it into a The thicker the ice is the higher is the TEM, regions of interest and of thin probability that the electrons under- enough ice are searched with low go inelastic scattering or scatter

more than once which leads to noise clearly visible even in the high tilt in the image. The mean free path images, their trajectories through the describing the mean distance of two tilt series can be used in alignment. consecutive scattering events for 200 Alternatively, a cross correlation kV accelerated electrons is approxi- based fiducialless alignment can be mately 270 nm in vitreous water performed, but it tends to be less ro- (Diebolder et al., 2012). bust and accurate than that using The thickness of the sample fiducial markers. Once the positional varies with 1 / cos α, where α is the and angular relationship of each im- tilt angle. What follows is that at 60° age to the next in the tilt series is de- the relative sample thickness is twice termined, a 3D reconstruction can be and at 70° almost three times that at calculated. This is most commonly 0°. To obtain approximately even done using weighted back-projection contrast in the images at all tilts, the (WBP), but also alternative methods beam intensity is often varied ac- like simultaneous iterative recon- cordingly throughout the tilt series. struction technique (SIRT) or alge- Like in all cryo-EM, the electron dose braic reconstruction technique (ART) that the sample can tolerate without are sometimes used. The principle of destruction of fine details is a major electron cryotomography is summa- limiting factor. For reasons poorly rized in Figure 8. understood, it appears that when the According to the central section electron dose is divided to multiple theorem, for weak phase objects such separate exposures, higher total dose as thin biological samples, the 2D can be used. In single particle cryo- Fourier transform of a projection of a EM the dose per image is normally 3D object corresponds to a central limited to ~10-20 e / Å2 depending section in the 3D Fourier transform on the coherence and the resolution of the object (Bracewell 1956, aimed for, whereas in cryo-ET total Crowther et al., 1970, De Rosier et doses of ~100 e / Å2 are commonly al., 1968). The thickness of the sec- used. In order to limit the total dose tion is inversely proportional to the in cryo-ET, focusing and tracking of thickness of the sample. According to the position are done in a region that Crowther et al., for an ideal object is a few micrometers off the actual the maximal resolution of the recon- tilt series collection site. struction is determined by the num- Once the tilt series is collected, ber of projections and the diameter a 3D tomogram can be calculated. of the object by the following formula Multiple program suites are available (Crowther et al., 1970): for this, but they generally rely on the same principles (Heymann et al.,

2008, Korinek et al., 2011, Kremer et al., 1996). First, due to the inaccura- cies in the microscope stage and go- where dx is the resolution, D is the niometer, the precise tilt angle and object diameter and N is the number position of the individual images are of projections. In order to reproduce determined in a process called a completely faithful reconstruction alignment. Here, the gold beads can of the 3D object, the entire Fourier be used as fiducial markers. As they space should be evenly sampled. The have high density and are therefore limited tilting range in electron

Figure 8. Principle of electron cryotomography. (A) Vitrified specimen is tilted inside the microscope to collect 2D projection images of the object of interest in different orientations. The tilting range typically varies from ±60° to ±70° with increments of 1.5° to 3°. The images are recorded with a CCD or a DED. As a result, a series of projections of the specimen is obtained (B). After alignment of the projection images, a 3D model of the original specimen can be reconstructed with WBP (C), SIRT or ART. Reprinted from Biophysical Chemistry, 100/1-3, Grünewald et al., Prospects of electron cryotomography to visualize macromolecular complexes inside cellular compartments: implications of crowding, Pages 577-591, © (2003), with permission from Elsevier. tomography results in a missing tion along the beam. The missing wedge-shaped region of information region of information can be reduced in the Fourier space, commonly re- from a wedge to a cone by collecting ferred to in the literature as the two orthogonal tilt series of the same “missing wedge”. The missing wedge region of interest. This is however causes anisotropic resolution in the rarely done in cryo-ET due to the reconstruction, which is poorest limited acceptable electron dose. along the beam direction and best in the directions perpendicular to the beam. Consequently, the tomograms are poorly interpretable in the direc-

4.4 SUBVOLUME AVERAGING

To enhance contrast and alleviate results in enhanced contrast because missing wedge artifacts, multiple the random noise in the reconstruc- copies of a particular object in differ- tions in proportion to the nonran- ent orientations in the tomograms dom signal from biological features can be averaged together. Averaging decreases with an increasing number

of averaged volumes. Before averag- et al., 2003), glycoprotein spike and ing, the subvolumes are extracted gag of HIV (Briggs et al., 2009, and aligned together by methods White et al., 2010), influenza HA adopted from the single particle (Harris et al., 2013), Chlamydomo- field. The main difference is that in nas flagellum microtubule doublets subvolume averaging, all procedures (Pigino et al., 2011), nuclear pore are carried out on 3D volumes in- complexes (Beck et al., 2007, stead of the 2D images in single par- Maimon et al., 2012) and COPI coat- ticle reconstruction. Subvolume av- ed vesicles (Faini et al., 2012). A erages of an increasing number of basic workflow for subvolume aver- cellular and viral structures have aging is illustrated in Figure 9. been published and include the herpes simplex virus capsid (Grünewald

Figure 9. A simplified workflow for subvolume averaging.

The most commonly used reconstruction, multiple runs with method for alignment is a rotational different initial templates can be per- and translational search of a sub- formed and they all should converge volume against a template using to the same final average. In sub- cross correlation as a similarity volume alignment, the missing measure. To avoid template bias in wedge is problematic because the the final reconstruction, the initial subvolumes have a tendency to align template should be an object lacking to their missing wedges. To avoid fine features. Typically, a low-pass this, many of the programs only in- filtered random average of the exclude regions with information in tracted subvolumes is used as an ini- Fourier space in the analysis, or re- tial template. To validate the final scale the similarity measure accord- 29

ing to the fraction of overlap of the 2009, Zanetti et al., 2009). Due to missing wedge (Bartesaghi et al., the low DQE of the CCD cameras 2008, Forster et al., 2008, Heumann used, the modulation transfer func- et al., 2011, Schmid et al., 2008). For tion (MTF) of the camera dampens objects with large protruding fea- the signal at high resolutions. Thus, tures, alignment to the missing often careful boosting of the high wedge can be prevented well, but it frequency signal with an inverse of tends to be more difficult for objects the MTF is required to bring out de- with fine features. tail (Xiong et al., 2009). DEDs To obtain a faithful reconstruc- should reduce this need as their tion of the original object by simple MTFs fall off at higher spatial fre- averaging, the subvolumes would quencies than those of CCD cameras. have to evenly cover the orientation In any averaging method it is space. Because a perfectly even dis- important to average together only tribution of subvolumes in all orien- homogenous particles. In cryo- tations is practically impossible to tomograms SNR is higher than in 2D obtain, the average volume is never a micrographs and the three- perfect estimate of the true object. dimensionality helps in identifying The imbalances in the data distribu- identical particles by eye, but often a tion can, however be compensated computational classification method for by dividing the average with the is required for objectivity. Classifica- average of the missing wedges of all tion is widely used in single particle subvolumes included in the average. processing, but has only recently Another approach used for sub- been adapted to subvolume analysis. volume averaging that does not rely Similarly to alignment of the sub- on cross correlation is based on max- volumes, the missing wedge causes imum-likelihood refinement and is some difficulties in classification as also widely used in the field (Scheres well and easily leads to classification 2012, Sorzano et al., 2004). to groups differing in the orientation For high resolution subvolume of the missing wedge. Ways to cir- averages, correction for the CTF is cumvent this problem have however required. Two main factors compli- been described (Forster et al., 2008, cate the correction in comparison to Frank et al., 2012, Heumann et al., the CTF correction in single particle 2011, Stolken et al., 2011, Winkler et processing. First, as the sample is al., 2009, Xu et al., 2012, Yu et al., tilted in most of the images, a gradi- 2011) and these methods have been ent of defoci is present in each image. applied to sort out heterogeneity in Second, the individual images have structures like influenza spikes extremely low signal-to-noise ratios (Fontana et al., 2012), HIV spikes (SNR) as a total dose of ~100 e / Å2 (Liu et al., 2008), and axonemes has to be distributed across the whole (Heuser et al., 2012). tilt series. These are, however not fundamental unsolvable issues and methods have been developed that can relatively well correct the CTF effects in the images (Xiong et al.,

B. AIMS OF THE PRESENT STUDY

At the start of this study in 2008, no 3D whole virus structures of paramyxoviruses had been published. We chose two members of the family for cryo-ET analysis. The viruses were selected based on their significance as human pathogens and the potential benefit of the results for helping to fight the viruses. The specific aims were:

1) To characterize the ultrastructure of measles virus, determine how the RNP is organized and solve the controversy of how the matrix protein is assembled in released virions (Study I).

2) To characterize the ultrastructure of human respiratory syncytial virus, investigate how the matrix protein is assembled in spherical and filamentous viruses, and how the glycoproteins and the RNP are organized in released virions. To combine the obtained structural data with previous biochemical knowledge for an assembly model (Study II).

3) To express, purify and characterize a measles virus RNA-free N-P complex. To develop a method for producing the complex in pure, large enough quantities for crystallization (Study III).

C. MATERIALS AND METHODS

The main method in the studies was grams repeating features of interest cryo-ET combined with subvolume were extracted, iteratively aligned averaging (Study I and II). This and averaged to obtain the final av- method was chosen as currently it is erage structure. In study I Jsubtomo the only method suitable for studies (Huiskonen et al., 2010) and in study on detailed 3D-stuctures of envel- II PEET (Nicastro et al., 2006) pro- oped, pleomorphic viruses. All imag- gram packages were used for sub- ing for cryo-ET was done on plunge- volume processing. The rationale for vitrified virus samples using an FEI using different program packages in F20 electron microscope. Typically, study I and study II was that of the ±60° tilt series were collected with 2° two packages only PEET offered a steps at a nominal magnification of classification option required for 39400x resulting in a sampling of sorting out the spike heterogeneity in 0.38 nm / pixel on a Gatan Ultrascan study II. For classification the prin- 4000 CCD camera. Tomograms were cipal component decomposition and calculated in IMOD (Kremer et al., clustering features in PEET were 1996) using WBP and typically used. The imaging and image pro- binned to have a final sampling of cessing details are described in the 0.77 nm / pixel. From the tomo- methods section of each study.

Table 1 Methods used in the study Method Used in study virus culture and purification I, II molecular cloning I, III recombinant protein expression I, III electron cryotomography I, II subvolume averaging I, II 3D classification II electron microscopy of negatively stained samples I, III electron microscopy of plastic embedded stained sections II immunosorbent electron microscopy I fluorescence microscopy II size exclusion chromatography and multi-angle light scat- III tering SDS-polyacrylamide gel electrophoresis III blue native polyacrylamide gel electrophoresis III limited proteolysis and mass spectrometry III

D. RESULTS AND DISCUSSION

One of the main aims in this study assembly intermediate of the RNP by was to shed light on the way para- recombinant protein production and myxoviruses assemble in the host cell in vitro biochemical analysis (study and how the viral components are III). The results are summarized in a organized in the virion released from model for budding in Figure 10 and the cell (studies I and II). We also will be discussed in the text in the looked at an early stage of assembly order in which the events are thought for MV by characterizing a pre- to occur during virus assembly.

1. THE N0-P COMPLEX

To address the very first step in MV where deletion of the first 10 N- budding, the encapsidation of the terminal amino acids of P led to over genomic RNA, we set out to produce a 90 % decrease in signal in a mam- an intermediate RNA-free N0-P malian two hybrid system (Harty et complex in E. coli (Study III). The al., 1995). Also, for SeV and VSV the two proteins were co-expressed and critical regions for N0-P interactions purified as a complex using an N- have been mapped to amino acids terminal hexahistidine tag on P. The 33-41 and 11-30, respectively (Chen complex was verified to be RNA-free et al., 2007b, Curran et al., 1995). and did not form helical RNPs which The C-terminal domain of N is confirmed that co-expression of the known to be unstructured in the ab- two proteins leads to formation of a sence of a binding partner (Longhi et soluble RNA-free N0. Due to the al., 2003) and thus might prevent highly protease-sensitive nature of P, crystallization of the protein. In and the fact that major parts of the search of a solid structured con- protein are intrinsically unstructured struct, guided by limited proteolysis (Bourhis et al., 2006), we construct- and mass spectrometry of the cleav- ed a number of C-terminally truncat- age products, we cloned an N con- ed versions of P that were co- struct lacking the protease sensitive expressed with N. This was done for C-terminus. This construct was com- two reasons: to map the region on P posed of the 408 N-terminal amino that is required to keep N in the N0 acids on N. We co-expressed this form and to obtain a stable N0-P con- construct together with GST- struct amenable for biochemical hexahistidine-P1-49 and purified the characterization and crystallization. complex with Ni-NTA resin followed We expressed some of the shortest P by on-beads enterokinase digestion constructs as GST fusion proteins as and size-exclusion chromatography they could not be expressed without (SEC). In SEC-multi-angle light scat- a bulky tag and determined that the tering (SEC-MALS) analysis, the pu- first 49 N-terminal amino acids of P rified complex was determined to be were enough to bind N and prevent a 52±2 kDa 1:1 heterodimer, which is N assembly on RNA. The results similar to the crystallized VSV N in were consistent with an earlier report complex with VSV P1-60. In

Figure 10. Assembly of MV and HRSV. Initially, the newly synthesized genomic RNA is encapsi- dated with N into a helical RNP (green box). Before assembly onto the helix, N is prevented from binding to cellular RNA by P. For MV the first 49 N-terminal amino acids of P are enough for this chaperone function. This N0-P complex is then thought to be dissociated by the polymerase (blue) to assemble N on the RNP. In MV the assembled RNP is then covered by M to form a matrix-covered nucleocapsid (MCNC), which is required for transport of the RNP to the budding sites. At the budding site, the MCNC are organized as bundles that are incorporated into the nascent bud on the membrane (blue box). After the final pinching off from the membrane, the RNP remains covered by M and has become enveloped in membrane that also carries the viral glycoproteins on the outside. The spikes are transported to the budding sites independently of the MCNC via the ER-Golgi route. Unlike in MV, HRSV RNPs are not covered by M, although the transport of RNP to the budding sites requires M. The interaction between RNP and M is mediated by M2-1. For poorly understood reasons, HRSV M undergoes nuclear-cytoplasmic shuttling before being transported to the budding site with M2-1 and RNP. At the budding site HRSV budding is initiated by F, after which M assembly into a tubular structure elongates the bud into a filament (yellow box). The RNP is incorporated into the virion via the interaction with M, mediated by M2-1. After pinching off from the cell, in some of the particles F converts into the postfusion conformation and M disassembles from the membrane causing the particle to adopt a spherical form.

addition to SEC-MALS we studied likely that the PNT in MV binds to N0 the negatively-stained complex in in a similar manner to that in VSV. electron microscopy and the sample This could be confirmed by crystalliz- appeared to mainly contain a mono- ing and solving the structure of the disperse complex clearly different N1-408-P1-49 complex. Knowing the from the helical assembly observed precise nature of the interaction in the Ni-NTA resin flow-through. would lead to a better understanding This further confirmed that we had of how N assembly on RNA is con- produced an N0-P complex (Study trolled and how it could potentially III). be prevented. Also, being able to To characterize the main mode break the complex by mild detergent of interaction in the MV N1-408-P1-49 treatment provides a means to obtain complex, we screened for release of pure N0, a form of the protein long N1-408 from GST-hexahistidine-P1-49 sought for RNP assembly studies bound on Ni-NTA beads in different (Bourhis et al., 2006). conditions. We found the interaction to be mainly hydrophobic as Triton- X 100 caused release of N1-408 from the complex, but neither high NaCl nor KCl concentrations (up to 2 M) resulted in N1-408 release. Thus, it is

2. BUDDING OF MV AND HRSV AND STRUCTURES OF THE RELEASED VIRIONS

The association of the matrix protein served had an additional layer of with the RNP in transport of the RNP density on the outside and the RNPs to the budding sites has been well were found in large aggregates. On established for both MV and HRSV anti-N grids, only 0.8 % of RNPs had (Ghildyal et al., 2002, Iwasaki et al., a similar covering, and the RNPs 2009, Li et al., 2008). Yet, to what were not aggregated. Thus, the addi- extent M is present in the transport- tional layer observed was composed ed complex, had not been well de- of M and it resulted in aggregation of scribed. For MV, early electron mi- the RNPs. The appearance of these croscopic studies indicated that M is matrix-covered nucleocapsids present on the RNP in significant (MCNC) was similar to the RNPs enough numbers to form a “fuzzy” with an additional covering detected layer on the RNP (Brown et al., earlier in MV-infected cells (Almeida 1987), contrary to the commonly et al., 1963). held view reported in many review Having verified that the tubular articles/text books. To confirm the structure covering MV RNPs was presence of M on the surface of RNPs composed of M, it was of interest to in infected cells and for a more de- see whether this assembly was re- tailed picture of the complex, we car- tained in the budded virus, or wheth- ried out immunosorbent electron er M was relocated to the inner sur- microscopy on MV infected cell ly- face of the membrane, or perhaps sates (Study I). On MV anti-M grids, both. Because both HRSV and SeV M approximately 30 % of the RNPs ob- had been reported to form tubular

structures (Heggeness et al., 1982, cal, but HRSV also formed filamen- McPhee et al., 2011) it was essential tous virions although 95 % were to compare HRSV with MV and SeV close-to-spherical. In the filamentous (Study I and II). In a cryo-ET study HRSV, two layers of density were of 11 released SeV virions MCNCs observed under the membrane, were not reported (Loney et al., which was not the case in MV. The 2009). As in MV only ~20 % of the one closest to the membrane was particles contained MCNC, it is pos- most likely formed by M as it is sible that analysis of a larger number known to be capable of forming heli- of SeV virions could reveal these cal structures on membranes and is structures in SeV too. In general in required for filamentous virion elon- tomography, getting a comprehen- gation in infected cells (McPhee et sive description of the sample is dif- al., 2011, Mitra et al., 2012). The ficult as in diluted samples such as identity of the innermost layer was many viral preparations one tilt se- less clear, but it was likely to be com- ries might have only one virion in it. posed of the M2-1 protein as it is In the future automated data collec- known to mediate the M-RNP inter- tion and processing should help in action and is a structural component overcoming this issue. of the virion (Li et al., 2008). We We produced MV (Helsinki subjected the M and RNPs of both strain, isolated from a patient in viruses to subvolume alignment and 1964 and the Edmonston vaccine averaging to analyze more closely the strain) and HRSV (A2 strain) in cell nature of the M and RNP assemblies. culture and purified the viruses from As the MCNC were readily visi- the cell culture supernatants. Initial- ble in the tomograms of MV, we ly, only supernatant viruses were manually defined the initial positions used to allow analysis of the released of the structures for subvolume anal- fraction of virions. Later, for HRSV, ysis. Next, partially overlapping seg- we compared the morphology of the ments of the tubular structures were released fraction and a fraction re- extracted and averaged and this av- leased from cells by freeze-thawing erage used as a refinement template. and found it to be essentially similar. In iterative refinement of the average The titers of the preparations from structure, we noticed that the M lay- supernatants were on the order of er outside the RNP did not refine to 109 and 108 plaque-forming units any distinct structure, but remained (PFU) / ml for MV Helsinki strain blurry as if the subvolumes were (study I) and HRSV (study II), re- randomly aligned. The RNP, however spectively. We considered these titers rapidly started to acquire a left- high as generally both are thought to handed single-start helical structure be viruses difficult to produce in high similar to that which had earlier been titers. Yet, the preparations were di- published for recombinant RNP-like lute for cryo-ET as often only one or helices (Bhella et al., 2004, Schoehn a few virions could be included in a et al., 2004). Fortunately, excluding tilt series. the inner helix from the refinement In cryo-ET of the released viri- by masking resulted in rapid refine- ons, the most striking difference be- ment of the M-layer to a left-handed tween the MV and HRSV was that in five-start helix with a ridge-to-ridge MV virions the RNP was indeed distance of 7.2 nm. Thus, the RNP mainly covered by M, whereas in and M both form left-handed helices HRSV this was not the case. MV viri- but have different symmetry. Form- ons were spherical or close-to spheri- ing such a complex requires flexibil-

ity in the interactions between M and of the missing wedge in the tomo- N. Such flexibility could be conferred grams, they were likely to be linked. by the intrinsically unstructured C- Subsequently, in a tomogram of an terminus of N in which the interac- aggregate of material released from tion with M is known to occur infected cells, we detected a connec- (Iwasaki et al., 2009). tion between two MCNC tubes that After having determined that M based on its thickness appeared to be forms a helix around the RNP in MV naked RNA (Figure 11). Therefore we studied how the MCNC are orga- the linkages between the MCNC in nized in the virions. This was done by the virions could be formed of naked template matching using the sub- RNA too thin to detect in the crowd- volume averages of the individual ed environment of the virion interior. helices as templates. The MCNC To summarize, our results suggest were found to form bundles where that in MV the matrix coats the RNP the M-layers of adjacent helices were already inside the host cell and that in close contact. The adjacent RNPs state is maintained in the released were often anti-parallel whereas the virion. Thus deforming the host M-helices did not appear to have di- membrane in MV budding is unlikely rectionality at the resolution of the to be a result of a sheet-like assembly reconstruction (4.4 nm). The pres- of M under the plasma membrane ence of a dyad axis in the M helix was that would cause curvature to the shown by the almost perfect correla- membrane. However, the MCNC as- tion of the reconstruction with its sembly does not exclude a possible 180° rotated copy (rotation around role for M in shaping the membrane the axis perpendicular to the helical as the edges of the MCNC bundles axis). As the individual helix seg- were often in close proximity to the ments in a bundle were on average membrane. It is also possible that only approximately 100 nm in not all of the M is in MCNC and some length, multiple segments would be of the protein could be bound to the required to accommodate a full ge- membrane in small patches not read- nome. Thus, even though we could ily visible in the tomograms. not detect the connections, possibly due to thin connections or the effects

Figure 11. MCNC are possibly connected by naked RNA. In a slice from a tomogram of a broken MV virion, thin and long linkers can be seen. The appearance and width of the linker suggests that it is composed of free RNA. Alternatively it could be RNA decorated with N that has been unwound from the typically helical RNP. In the inset a piece of the structure where RNP has unwound from inside of the M helix is shown.

We generated radial profiles by be composed of a seven-start helix aligning and averaging subvolumes with a ridge-to-ridge distance of 7.8 extracted from the surfaces of spher- nm. The repeat distance corresponds ical MV and HRSV as well as fila- to that observed in the Fourier trans- mentous HRSV particles. In the av- forms of the filament surface. It is erages of spherical particle sub- therefore possible that a similar alt- volumes no other layers than the hough inverted assembly of M was membrane were discernible. In the present in both. average of filamentous particles the In a study published while our M-layer was at 6.1 nm from the work was in progress, the ultrastruc- membrane and the putative M2-1 ture of NDV by cryo-ET was de- layer at 6.9 nm from the M layer. scribed (Battisti et al., 2012). Mor- Although it was possible that M phologically, most NDV particles formed a helical assembly under the were spherical without an apparent filamentous HRSV membrane, we matrix layer under the membrane, could not confirm this most likely but also elongated particles were ob- due to the fact that most of the fila- served. In the elongated particles mentous virions were flattened in the part of the inner side of the mem- thin ice required for high resolution brane had an M-covering similar in tomograms. However, a repeating appearance to the one we detected in feature at approximately 8 nm fre- HRSV filaments. In the same article quency could be detected in the lay- also a crystal structure of M was er. Similar observations have been solved and it could be fitted into a reported for influenza M1 and MARV subvolume average of the M-layer. M VP40 (Bharat et al., 2011, Calder et appeared to form a sheet with 7 nm al., 2010). In a number of filamen- repeat distance in orthogonal direc- tous virions, the M-layer continued tions, which is approximately similar as a curved sheet when it had de- to what we saw in HRSV. The ultra- tached from the membrane. This in- structures of MV, HRSV, SeV and dicated that M had an inherent abil- NDV are shown for comparison in ity to form tubular structures and Figure 12. that these structures were stable in The RNPs in HRSV and regions the absence of a membrane. Thus, it of MV RNP without M-layer were is likely that the filamentous shape in more curved and randomly oriented HRSV is determined by the assembly than the MCNC. This was indicative of M into tubes. This is supported by of M’s role in making the MV RNPs the fact that the presence of M is re- straight and rigid. In HRSV the viri- quired for formation of long fila- ons were packed to a varying extent ments at the cell surface (Mitra et al., with the RNP material and in fila- 2012). The filamentous regions on mentous virions long stretches of virions varied in diameter between RNP could often be seen aligned 70 nm and 190 nm, which indicated along the longitudinal axis. In NDV, that M assembly into tubular struc- the RNPs in elongated virions were tures could accommodate multiple aligned similarly to HRSV and were different symmetries. The flexibility in close contact with the M-layer extended to even more extreme cases (Battisti et al., 2012). As HRSV viri- as exemplified by a virion that con- ons were more tightly packed with tained an inverted M-tube with the the RNPs, it was difficult to judge membrane and spikes inside the whether the RNPs were specifically tube. We reconstructed this tube in bound to the M/M2-1-layer or subvolume averaging and found it to whether the adjacent positioning was

Figure 12. Tomographic slices of paramyxoviruses. In MV the RNP is covered by M, whereas in HRSV and NDV the RNP is not covered, but M forms a layer next to the internal side of the membrane. In SeV M does not cover the RNP nor form a continuous layer under the membrane. MCNC are shown with white arrowheads and RNPs without M with black arrowheads. Star denotes an area of the NDV membrane free of glycoprotein spikes and M. Adapted from study I; study II; Loney et al. 2009; Battisti et al. 2012 with permission from the publishers. due to the crowded interior of the the RNP was reported to be right- virion. handed based on experimenting with We analyzed the structure of different helical averaging of the the HRSV RNP in essentially a simi- tomographic structure and estimat- lar manner to that used for MV RNP ing cross-correlations with the origi- and found the helix to be left- nal structure. The data were, howev- handed. This was in direct contradic- er, not shown (Battisti et al., 2012). tion with a previously published Helical averaging per se does not model (Tawar et al., 2009). To verify give the absolute hand, but use of a that the tomogram calculation re- control tomographic sample would tained the correct absolute handed- have done. ness, we collected tilt series and cal- The spike glycoproteins on MV culated tomograms of left-handed and SeV virions formed a densely nanogold decorated DNA-origami packed layer from which it was diffi- structures (Kuzyk et al., 2012). While cult to discern individual proteins our article was in press, the authors (Loney et al., 2009). In NDV, the of the conflicting article published a spikes were packed to similar densi- second article correcting their origi- ties as in MV and SeV, but showed nal mistake in the handedness preferential lateral location to the (Bakker et al., 2013). Their mistake regions with M-layer under the arose from not experimentally de- membrane (Battisti et al., 2012). In termining the handedness, but decid- HRSV the individual spikes, alt- ing on it based on modeling the hough densely packed in the mem- RNP-like ring X-ray structure as a brane could be discerned and ana- helix (Tawar et al., 2009). Correction lyzed by subvolume averaging. We of the handedness is important in attempted subvolume averaging also this case because it affects the direc- for MV, but were unable to classify tion of the RNA in the model. That in the subvolumes for each of the two turn has implications on models of spikes. In HRSV spherical virions the how the polymerase could access the spikes appeared to be of one single RNA in the structure (Bakker et al., type with a golf-tee like shape re- 2013). It has also been suggested that sembling the HRSV F in a postfusion all the –ssRNA RNPs are left handed conformation (PDB ID 3RRT) (Bharat et al., 2011). Yet, for NDV, (McLellan et al., 2011). The same

type of spike could also be detected most likely represented F. As the on the filamentous regions, but there golf-tee shaped class was reminiscent also another smaller spike was pre- of the HRSV postfusion F crystal sent. Of all the virions, only those structure, and the small spike re- with the small spike appeared to sembled in shape the prefusion have some local order in the spike structure of PIV5, we attempted to fit layer. The small spikes were ar- the crystal structures into the aver- ranged roughly in lines on the sur- ages. Initially, we fitted the prefusion face and the lines were often approx- F from PIV5 (PDB ID 4GIP) (Welch imately perpendicular to the longitu- et al., 2012) into the small spike den- dinal axis of the filament. sity (Study II). Subsequently, a crys- Because HRSV has two glyco- tal structure of the prefusion ecto- protein spikes, we found ourselves domain of HRSV F was published facing a problem of sorting which (PDB ID 4JHW) (McLellan et al., type of spike was G, which F, and 2013). As this structure was biologi- whether or not F was perhaps pre- cally more relevant to our study, we sent in more than one conformation. fitted that into the small spike densi- To begin with, we cultured and puri- ty instead of the PIV5 F (Figure fied a mutant of HRSV that lacked 13A). We also fitted the postfusion SH and G on the surface. This HRSV F into the golf-tee spike densi- rgΔGΔSH strain has been shown to ty (PDB ID 3RRT) (McLellan et al., be infectious and in general F on the 2011). The dimensions of the sub- surface is enough for a productive volume average and the crystal struc- HRSV infection in vitro ture were similar and the crystal (Techaarpornkul et al., 2001). On the structure appeared to fit well into the mutant virions, the majority of the subvolume average density (Figure spikes appeared to be of the golf-tee 13B). Although the limited resolu- type thus identifying it as F. Initially, tion of the subvolume averages pre- we inferred that the other smaller vented reliable numerical assessment spike should then be G. To be objec- of goodness of the fits, it was clear tive, we did computational classifica- that the small spike corresponded to tion analysis on the spike sub- the prefusion and the golf-tee spike volumes. The classification resulted to the postfusion F. From our analy- in two well-defined classes one of sis, the structure and presence of G which was similar to F and the other was left unresolved. It is possible similar to the small spike. Surpris- that G was similar enough in shape ingly, some of the subvolumes classi- to one of the F conformations that it fied into the small spike class origi- did not separate into its own class or nated in the rgΔGΔSH tomograms. it was not prominent enough or pre- Additionally, an average calculated sent in too low numbers to be includ- from only the rgΔGΔSH subvolumes ed in the analysis. Using G-specific in the small spike class clearly re- antibodies for labeling the virus prior sembled more the small spike than to analysis could help in clearing this the golf-tee spike. Thus, both classes uncertainty.

Figure 13. Crystal structures of HRSV F in prefusion (A) and postfusion (B) states fitted into the subvolume average densities. The isosur- faces in A and B were rendered at 2 σ and at 5 σ from the mean, respectively.

HRSV F is exceptional in that it following scenario is likely: Initially, requires two cleavages by a furin-like most of the virions were filamentous. protease to be fusion active (Zimmer With time, during preparation or et al., 2001). It has recently been storage, the matrix layer disassem- shown that HRSV entry involves bled from some virions’ membrane macropinocytosis and that the sec- making the particles spherical in ond cleavage occurs inside the target shape. In the same process, F con- cell after internalization of the virion verted into the postfusion confor- (Krzyzaniak et al., 2013). Thus, also mation rendering the virions inactive in the virus preparation used in our (Figure 10). The conversion of F study, F was uncleaved at the second and disassembly of M are correlated site and regardless of this the majori- because prefusion F was not present ty had converted into the postfusion in spherical particles, but appear not state. Currently it is unclear why the to be strictly temporally connected as conversion should happen before postfusion F was also present in fil- attaching to the target cell, but it is amentous virions. Accordingly, it is possible that the virions with the rather the conversion of F than dis- converted F are inactive as the parti- assembly of M that begins the trans- cle-to-infectivity ratio of HRSV prep- formation process. Thus, it is possi- arations have been reported to be ble that during entry, triggering of F high, which was also the case in our for fusion leads to disassembly of M. preparations (50 ± 10:1). Additional- Disassembly of M is likely to be rely, we noticed that the fraction of fil- quired before the next round of repli- amentous virions in the preparation cation can begin. went down on freezing and incuba- tion at 37 °C (5.2 %, 1.1 % and 0.9 % for fresh, -80 °C frozen and 37 ° C incubated virus, respectively) and this correlated with a drop in infectivity (90 % drop in -80 °C frozen compared to fresh virus). Although not causally proven in our study, the

E. CONCLUSIONS AND PERSPECTIVES

In this study we have conducted at least as many new questions as cryo-ET studies on two important they were able to answer. The most pathogens from the Paramyxoviri- intriguing ones regarding the RNP dae family. We also produced and assembly are: How does P of para- characterized a complex of MV N and myxoviruses prevent N from binding P that is important in the early stages to RNA and at least equally im- of virus assembly. The results indi- portantly, how is P then released cated clear differences in the virion from N to bind RNA when it is re- structure between MV, HRSV and quired? Phosphorylation of P could other paramyxoviruses. The differ- possibly have a role in this. It would ences were most noticeable in the be of great interest to study if the way the matrix protein was assem- RNP could be assembled in vitro and bled and how it defined the shape of whether the sequence or structure of the whole virion. For MV we were RNA has a role in efficient encapsi- able to rectify a commonly adopted dation. Could the N-P interaction be model for the virus architecture by prevented with PNT mimicking pep- showing that M did not line the in- tides or drug compounds? Trials with ternal side of the membrane but was RABV demonstrate that this might wrapped as a helix around the RNP. be possible (Castel et al., 2009). In HRSV we analyzed in detail all the The greatest gaps in knowledge main components of the virion. This on enveloped virus assembly lie in analysis showed that M and F were understanding the virus-host inter- responsible for the filamentous mor- actions required for assembly and phology observed for a fraction of the release of the virion and spread to virions. We also corrected a mistake adjacent cells. What are the compo- in the earlier literature regarding the nents required from the cell for handedness of the RNP and showed transport of viral proteins, shaping that F is present in both pre- and the cellular membrane and final scis- postfusion conformations in the viri- sion of the virion off the membrane? ons. Addressing these topics will require Although we mainly studied vi- careful analysis of all aspects com- ruses isolated from the cell, a fair bining structural biology, cell biology amount of information on the as- and biochemistry of the isolated sembly of the viruses could also be complexes. Undoubtedly, future derived. As structure is most useful technological development combined when it can be interpreted in terms with the true medical need will evi- of biological functionality, we com- dently lead to answers to these cur- bined our structural models with da- rently unresolved questions and ta from biochemical and cell biologi- open paths to new currently un- cal literature into models for virus mapped territories. assembly. As is typical for any scientific study, the studies reported here raise

F. ACKNOWLEDGEMENTS

The work for this thesis was carried moved on. It has been a real pleasure out between 2008 and 2013 at the to work in an environment with so Finnish Centre of Excellence in Virus many colorful personalities. Research (2006-2011), at the Insti- I want to acknowledge Ari O. tute of Biotechnology at the Universi- for the unbelievable enthusiasm and ty of Helsinki under the supervision energy that you spread to the people of Docent Sarah Butcher. The work around you. Thanks for teaching me was funded by Finnish Academy, so many of your practical “old tricks” Viikki Doctoral Programme in Mo- in the lab to help the everyday work. lecular Biosciences (VGSB) and Eu- Thanks for the interesting discus- ropean Molecular Biology Organiza- sions of various kinds during these tion (EMBO). years. You are a walking encyclope- I want to express my warmest dia of everything. Furthermore, I am thanks to Sarah. You have guided me extremely grateful for the initial virus through this project. This project preparations you made for the mea- that sometimes led me to misty dark sles project. Without those this pro- mires of slow progress, and even ject would have ended before it even there I often managed to hit a wall. really begun. You have taught me to find light Thanks to Shabih for being where I could barely notice a spark. such a great and friendly officemate. You have not held my hand, but al- You have always found the time to ways been available and offered a answer my questions and help with signpost at a crossing or a ladder things you master and I don’t. over a wall when I have needed help. Thanks to Violeta for keeping I especially would like to me aware of the superiority of gar- acknowledge you for seeing your stu- dening over work. It has been a real dents and employees not only as pleasure to get to know you. I have working force, but humans who you really enjoyed all the discussions on treat with respect. various topics unrelated to work dur- I want to express my gratitude ing the years. to our collaborators Dr Juha Huis- Thanks to Pasi for teaching me konen, Docent Petri Susi, Dr Magda- how to do electron microscopy and to lena Krzyzaniak and Professor Ari Eevakaisa for a lot of help in data Helenius. Without your input, this collection for my projects. work would not have been possible. I want to thank Professors Thanks to Juha for teaching me Adrian Goldman and Markku Kulo- the principles and more of electron maa for the advice you have given tomography. Thank you for making during the thesis committee meet- me feel welcome to visit both Mar- ings. Thanks also to Tero Ahola and tinsried and Oxford. Also, thanks for Peter Rosenthal for reviewing this your never-ending patience in sitting thesis. Your comments were very with me through several nights at the helpful. microscope during my visit to Mar- I want to thank my mom and tinsried. dad for the great support throughout I would like to thank all the my studies and life in general. Raija I present members of the Butcher lab want to thank for providing the calm as well as you who have already

environment of Nokian mökki where well. You have also kept in check that I wrote a major part of this thesis. my work-life balance has all the time My deepest gratitude goes to been on a sound basis. my beloved wife Teresa. You have Finally, I want to thank our been the best support imaginable. wonderful baby daughter Isla for the You have helped me stand up when I joy and happiness you have brought have felt like giving up. You have pa- into our lives in these past few tiently read through almost every months. You have helped me com- text and presentation I have pro- plete this work much faster than I duced during these years. You have would have done would I not have always shown interest in what I have expected such a nice change in life. been doing, or at least pretended

Lassi Liljeroos Helsinki, September 2013

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