To my family
POPULÄRVETENSKAPLIG SAMMANFATTNING
Virus är väldigt små partiklar. Så små att de, med endast ett fåtal undantag, inte kan ses med mikroskop. De kan orsaka infektioner hos människor, djur, växter och allt annat som lever. Det finns många olika sorter, vilka har skilda egenskaper och infekterar olika värdar och typer av celler. De virus som behandlas i den här avhandlingen tillhör arten Enterovirus. De är mycket små även jämfört med andra virus. De har en arvsmassa, också kallat “genom”, bestående av en RNA-sträng som skyddas av ett 20-hörningt proteinhölje. Till de mer välkända enterovirusen hör poliovirus som kan infektera och bryta ned nerver samt rhinovirus, som orsakar majoriteten av alla förkylningar. Det virala RNAt “kodar” för ett antal proteiner. Några av dessa utgör byggstenarna för det skyddande höljet, medan andra omorganiserar den infekterade cellen och möjliggör kopiering av arvsmassan. När ett virus ska infektera en cell binder den först till strukturer på cellens yta. Arvsmassan förs sedan in i cellen där värdens egna proteiner börjar syntetisera virala proteiner med arvsmassan som mall. Därefter kopieras det virala RNAt, även detta sker med genomet som mall, och proteinerna som utgör det skyddande höljet, strukturproteinerna, sätts samman och innesluter de nyproducerade RNA- molekylerna. När människor och andra liknande organismer fortplantar sig får avkomman hälften av sin arvsmassa från vardera av sina föräldrar. Detta bidrar till att arten kan fortsätta evolvera och anpassa sig. För Enterovirus går inte detta. Istället sker artens anpassning till nya omständigheter genom hög mutationsfrekvens, d v s att det blir fel i kopieringen av arvsmassan. Dessutom kan det bildas virus med en arvsmassa som är en kombination av två olika virus genom att proteinet som sätter ihop den nya RNA-kopian hoppar över till en annan ”mall”, detta kallas för rekombination. Upptäckterna som är beskrivna i denna avhandling är uppdelade i fem arbeten där olika delar av virusens livscykel har studerats. Detta har gjorts genom omvänd genetik, vilket innebär att ändringar görs i virusets arvsmassa, varefter skillnader i virusets beteende studeras. Virusets ändrade arvsmassa, utan proteinhöljet, förs då in i cellen där den börjar kopieras, precis som att det hade förts in på naturlig väg. I det första arbetet visade vi att det gick att kombinera de icke- strukturella delarna av genomet med strukturella delar från andra virustyper inom samma. I arbete nummer två upptäckte vi att viruset kunde återhämta sig efter att arvsmassan ändrats så att den inte längre fungerade som en mall för protein-syntes. I det tredje arbetet lyckades vi öka effektiviteten med vilken viruset började kopieras efter att arvsmassan förts in i cellen. Detta kan i framtiden komma att användas för effektivare och säkrare virusbehandling mot cancertumörer (viral onkolys). För att studera virus är det viktigt att få dem att föröka sig i cellkultur, d v s celler som har anpassats till att leva i cellodlingskärl. Ingen har än så länge fått Rhinovirus-C att växa i cellkultur och i det fjärde arbetet kunde vi konstatera att skälet till det antagligen ligger i att viruset inte kan binda till cellen, alternativt att arvsmassan inte naturligt kan föras in i cellen. Det finns två strategier för virus att ta sig ut ur infekterade celler, antingen utnyttjar de värdens transport ut ur cellen (persisterande virus) eller så har de sönder cellen (lytiskt virus). I det sista arbetet tittade vi på olikheter i förändring hos celler som infekterats med persisterande respektive lytiskt virus och kunde konstatera att det lytiska viruset har nästan 10 gånger så stor påverkan på cellen som det persisterande. Som helhet har de projekt som ingår i denna avhandling bidragit till en djupare förståelse för olika stadier av livscykeln hos Enterovirus. Upptäckterna som gjorts kommer att underlätta framtida utveckling av vacciner, antivirala medel och viral onkolys.
LIST OF PUBLICATIONS
This thesis is based on the following articles, which are referred to in the text by Roman numerals. All published papers are reproduced with permission from the respective publisher.
I. Efficient replication of recombinant Enterovirus B types, carrying different P1 genes in the coxsackievirus B5 replicative backbone N. Jonsson*, A. Sävneby*, M. Gullberg K. Evertsson, K. Klingel and A.M. Lindberg. Virus Genes, Published online February 8, 2015
II. Virus derived from an Enterovirus B construct efficiently reverts from a frameshift mutation immediately beyond the translation initiation site A. Sävneby, N. Jonsson, P.A. Svensson, S. Hafenstein and A.M. Lindberg. Submitted.
III. Improved replication efficiency of echovirus 5 after transfection of colon cancer cells using an authentic 5' RNA genome end methodology S. Israelsson, A. Sävneby, J-O. Ekström, N. Jonsson, K. Edman and A.M. Lindberg. Investigational New Drugs. 32(6):1063-70, 2014.
IV. Restricted replication of the rhinovirus C34 prototype in standard cell lines A. Sävneby, F. Lysholm, N. Jonsson, T. Allander, A.M. Lindberg and B. Andersson. Manuscript.
V. Gene expression in rhabdomyosarcoma cells infected with cytolytic and non-cytolytic variants of coxsackievirus B2 Ohio A. Sävneby, J. Luthman, F. Nordenskjöld, B. Andersson and A.M. Lindberg. Manuscript.
* These authors contributed equally LIST OF ABBREVIATIONS
A adenine CPE cytopathic effect CRE cis-acting replication elements CV coxsackievirus dsRNA double-stranded RNA E echovirus ER endoplasmic reticulum EV enterovirus FMDV foot-and-mouth disease virus GMK green monkey kidney IF immunofluorescence staining IRES internal ribosomal entry site NGS next generation sequencing nt nucleotides ORF open reading frame PCR polymerase chain reaction PFU plaque-forming units PV poliovirus qRT-PCR quantitative reverse transcription polymerase chain reaction RD rhabdomyosarcoma RV rhinovirus TCID50 tissue culture infectious dose 50 % UTR untranslated region VP viral protein
TABLE OF CONTENTS
Introduction ...... 11 What are viruses and why are they interesting? ...... 11 Dead or alive? ...... 11 Deaths caused by viruses ...... 12 Historical perspective ...... 12 The discovery of viruses ...... 12 The development of vaccines ...... 12 Aim of thesis ...... 13 Taxonomy and pathogenesis ...... 14 Taxonomy ...... 14 Pathogenicity ...... 14 Picornaviridae ...... 15 The Enterovirus genus ...... 17 History of enteroviruses ...... 18 Enteroviruses in society ...... 18 The enteroviral genome and proteins ...... 20 The 5’UTR ...... 20 IRES ...... 21 The polyprotein ...... 21 The P1 region proteins ...... 22 The P2 region proteins ...... 23 2A ...... 23 2B ...... 23 2C ...... 23 The P3 region proteins ...... 24 3A ...... 24 3B ...... 24 3C ...... 25 3D ...... 25 The 3’UTR ...... 25
Main differences between enteroviruses and other picornaviruses ...... 26 The capsid ...... 27 Elucidating the capsid structure ...... 27 Capsid functions ...... 27 The capsid structure ...... 28 The capsid surface ...... 29 The infectious cycle ...... 30 Attachment ...... 30 Entry ...... 32 Translation ...... 33 RNA replication ...... 34 Assembly ...... 34 Release ...... 35 Evolution of enteroviruses ...... 36 Mutations ...... 36 Recombination ...... 36 Selective pressure ...... 37 Working with enteroviruses ...... 38 Cell culturing ...... 38 Immortal cell lines ...... 38 Propagating the virus ...... 39 Passaging of virus ...... 40 Transfection ...... 40 Reverse genetics ...... 40 Quantifying viruses and determining their presence ...... 41 Plaque assay ...... 41 Tissue culture infectious dose 50 % ...... 42 qPCR ...... 42 Immunofluorescence staining ...... 43 Western Blot analysis ...... 44 Aims and objectives ...... 45 Results and discussion ...... 46 Efficient replication of Enterovirus B recombinant ...... 46 Independent evolution of genomic regions ...... 46 The replication competent backbone ...... 46 Confirmation of replication ...... 47 Conclusion ...... 48 Initiation of enterovirus translation ...... 48 Measuring infectivity ...... 48 Sequence analysis ...... 48 Alternative means of translation initiation ...... 49 Conclusion ...... 49 Increased efficiency of RNA replication activation ...... 50 Lytic ability of E5N in colon cancer cell lines ...... 50
Oncolytic virotherapy with infectious nucleic acid ...... 50 The importance of an authentic 5’end ...... 51 Transfection of HT29 cells with infections RNA ...... 51 Conclusion ...... 52 Towards the propagation of Rhinovirus C in standard cell culture ...... 52 Detection of the RV-C34 type and construction of a cDNA clone ...... 52 Transfections of immortal cells and attempts to passage pico58 ...... 53 Viral translation and RNA replication ...... 53 Conclusion ...... 53 Effects of viral replication on the host cell ...... 54 Experimental set-up ...... 54 Differences in intracellular viral load ...... 54 Differentially expressed genes ...... 55 Annotated genes ...... 55 Conclusion ...... 55 Concluding remarks ...... 56 Future perspectives ...... 57 Acknowledgements ...... 58 Cited literature ...... 60
INTRODUCTION
What are viruses and why are they interesting? The word ‘virus’ is originally a Latin word meaning ‘poison’ or ‘toxin’. Viruses co-exist with life wherever it occurs. They are so small that, with few exceptions, they cannot be seen using a regular light microscope. That something so small can affect us, and the lives around us, in such an all- encompassing way is so very fascinating to me, and the reason why I wanted to study viruses. Dead or alive? A question that is commonly asked is whether viruses are dead or alive. For as long as I have known anything about viruses my answer has been “both” and, though not everyone agrees with me, I am not alone in this view [1]. When a virus has been transcribed, translated, processed, assembled and released as a fully formed virus particle, or virion, it has all the potential to infect a new host. It is a birth of sorts and yet; ironically, this is when the virus is perceived as dead. There is no clear consensus of the definition of life [2], but most agree that it includes the ability to metabolize and reproduce. After the virus has been released from the host cell it has no such ability. Viruses are obligate parasites, which mean that they cannot complete their lifecycle without infecting a host cell that provides the necessary resources. When a virus enters into a cell it loses its shell, envelopes are left behind and the capsids are transformed to release the nucleic acid. In short, the virus particle is broken into pieces, and this is when the virus becomes alive. Of course, not all viruses look and act the same; in fact there is a huge variety of viruses with different sizes, shapes, hosts, genomic material and outer barriers; but what they all have in common is that they use the resources of a host cell to fulfill their lifecycles. Some viruses integrate their genome into the host genome, whereas others shut down the translation of the host cell proteins. There are a variety of tricks and tactics but they all have the same purpose: to facilitate an ideal
11 environment to replicate and form new virions. After a complete virus particle is formed and released from the host, the virus is ‘dead’, and the cycle starts over again. Deaths caused by viruses When the World Health Organization (WHO) compiled their list of the top ten causes of deaths in 2012 worldwide, the human immunodeficiency virus (HIV), causing acquired immunodeficiency syndrome (AIDS), was in sixth place. Lower respiratory tract infections and diarrheal disease (number four and seven on the list, respectively) are also in large parts caused by viral infections. In low-income countries, infectious diseases are the dominant cause of death. Approximately one third of deaths in such countries are caused by lower respiratory tract infections, HIV/AIDS, diarrheal diseases, malaria (parasite) and tuberculosis (bacteria) [3].
Historical perspective The discovery of viruses In 1892 Dimitrii Ivanowsky showed that filtered extracts of a plant with tobacco mosaic disease were still infectious. The implication was that something smaller than all known bacteria was the cause of this infectious disease [4]. Six years later Martinus Beijerinck presented a similar study. He also concluded that it was a replicating agent and not a toxin. Toxins can be diluted, but replicating entities will continually increase their number of copies. Beijerinck called this infectious agent ‘virus’ and described it as a fluid [5]. This started a debate that would go on for 25 years regarding whether viruses were liquids or particles. In 1917 the first plaque assay was performed and in 1939 the first electron micrograph of a virus was produced, laying all theories of an infectious liquid to rest. Viruses were confirmed to be infectious particles different from bacteria and other pathogens [6]. The development of vaccines At the very end of the 18th century, E. Jenner noticed that the milkmaids that were exposed to cowpox seemed to evade smallpox. He formulated the hypothesis that the lesions the milkmaids got on their hands after contact with animals with cowpox somehow protected them from the similar but more severe smallpox infections. He tested the hypothesis by taking puss from a cowpox lesion and inoculating a boy with it. The boy experienced some initial symptoms of infection, but they soon subsided. Investigations into whether this inoculation had given immunity, carried out by exposing the boy to smallpox, indicated that the boy now was protected. Jenner did some further
12 studies and coined the term vaccine, after the Latin word vacca, meaning cow. Because of vaccination campaigns smallpox was declared eradicated in 1980 [7]. Numerous other vaccines have been developed, drastically reducing cases of potentially fatal diseases such as measles and poliomyelitis [8].
Aim of thesis The viruses studied in this thesis belong to the Picornaviridae family. The most well known virus in the family, belonging to the Enterovirus (EV) genus, is the poliovirus (PV), the cause of poliomyelitis. Other well recognized members are the viruses belonging to the Rhinovirus (RVs) species, and the Foot-and-mouth disease virus (FMDV) species. The former are the cause of 30-50 % of all common cold cases [9] and the latter causes foot-and-mouth disease in cloven-hoofed animals, which is one of the most contagious diseases affecting mammals [10]. The aim of this thesis is to provide a deeper understanding of the different aspects of enteroviral replication. This has been achieved through reverse genetic studies of species EV-B and RV-C.
13 TAXONOMY AND PATHOGENESIS
Taxonomy Taxonomy is the division of organisms into subgroups. Seven orders of viruses exist, and these are sub-divided into 27 families. In addition to these, there are many virus families that have not been assigned to an order. The Picornaviridae family belongs to the order Picornavirales and has 29 genera: Aphthovirus, Aquamavirus, Avihepatovirus, Avisivirus, Cardiovirus, Cosavirus, Dicipivirus, Enterovirus, Erbovirus, Gallivirus, Hepatovirus, Hunnivirus, Kobuvirus, Kunsagivirus, Megrivirus, Mischivirus, Mosavirus, Oscivirus, Parechovirus, Pasivirus, Passerivirus, Rosavirus, Sakobuvirus, Salivirus, Sapelovirus, Senecavirus, Sicinivirus, Teschovirus and Tremovirus [11] (Table I). Picornaviridae is by far the largest family and out of the 29 genera, 3 are new members included in the latest Virus Taxonomy update in February 2015 [12]. Traditionally, the organization of viruses into genera and species was based on physical and serological properties as well as the pathogenicity of the virus in natural infections or in animal models. With the possibility of nucleic acid sequencing, and better knowledge of viral genomes, taxonomic classification has become increasingly sequence-based. Today, picornaviruses are divided into genera, species and types by the sequence of the first coding section of the viral genome (P1).
Pathogenicity Pathogenesis describes the ability of a pathogen, like bacteria, viruses or parasites, to cause disease. Many picornaviruses cause infections without triggering any apparent symptoms, so called non-pathogenic infections, although this is not the case for all of them. Picornaviruses can cause an astounding number of different diseases in humans and other animals including: poliomyelitis, encephalitis, gastroenteritis, aseptic meningitis,
14 myocarditis, foot-and-mouth disease, hepatitis and respiratory diseases ranging from severe infections to the common cold [13]. There have also been indications of members of the Picornaviridae family being an underlying cause of type 1 diabetes [14].
Picornaviridae Many picornaviruses are associated with diarrheal disease; among them are the members of the genera Avisivirus, Cosavirus, Gallivirus, Passerivirus and Salivirus [15-19]. The Oscivirus genus contains a single species, Oscivirus A, which has one of the largest picornavirus genomes with over 9243 nucleotides (nt) [20]. Hepatovirus A constitutes the only member of the Hepatovirus genus and is most often contracted from contaminated food or drinking water [13]. Tremovirus, the genus of avian encephalomyelitis virus, is most closely related to Hepatovirus [21]. Duck hepatitis A virus constitutes the Avihepatovirus genus, which causes hepatitis in ducklings [22]. Another picornavirus that causes hepatitis in birds is Megrivirus [23]. Hunnivirus and Teschovirus are closely related and both contain only one species [24]. Kobuvirus includes three species, one of which can infect humans; Aichivirus A [12], associated with gastroenteritis; this was recently reported to circulate in Sweden [25]. Aquamavirus, Mischivirus, Mosavirus, Pasivirus, and Rosavirus are all recently discovered viruses and are not known to be associated with a disease [26-30]. Cadicivirus is the sole member of the Dicipivirus genus and is the only picornavirus with two open reading frames (ORFs). This is facilitated by a second internal ribosome entry site (IRES) element (described in more detail in the following chapter) in the middle of the first ORF [31]. Cardiovirus is divided into Encephalomyocarditis virus and Theilovirus and has been isolated in over 30 different host species [13]. Sapelovirus contains three virus species infecting ducks, monkeys and pigs [13, 32, 33]. The Seneca Valley virus, of the Senecavirus genus, was discovered as a contaminant in a lab cell culture [34]. It can infect humans but is non-pathogenic and is being tested as an oncolytic virus in phase II trials [35, 36]. The Parechovirus genus includes Human parechovirus and Ljungan virus. The latter is found in rodents while the former is known to cause gastrointestinal, respiratory and neurological disease in humans [37]. Erbovirus causes respiratory disease in s [38], as does Equine rhinitis A virus of the Aphthovirus genus. Aphthovirus has four species, three of which cause respiratory infections and the fourth is the FMDV. FMDV is extremely infectious in cloven-hoofed animals. Apart from the suffering of infected animals, the disease has great economic impact worldwide [39, 40]. Recently, three new genera were included in the Picornaviridae family; Kunsagivirus, Sakobuvirus, and Sicinivirus [41-43].
15 Table I. The 26 genera and 46 species of the Picornaviridae family [12]. For each species examples of associated disease(s) and the natural host(s) are given. The 3 new genera in the Picornavirus family are indicated with an asterisk (*). Genera Species Associated disease(s) Host(s) Ref Aphthovirus Bovine rhinitis A virus Respiratory disease Bovine [11] Bovine rhinitis B virus Respiratory disease Bovine [11] Equine rhinitis A virus Respiratory disease Equine [38] Foot-and-mouth disease virus Foot-and mouth disease Ungulates [44] Aquamavirus Aquamavirus A No known disease associated Seal [26] Avihepatovirus Duck hepatitis A virus Hepatitis Avian [22] Avisivirus Avisivirus A Enteritis Avian [15] Cardiovirus Encephalomyocarditis Encephalitis, myocarditis Rodents [45] Theilovirus Encephalomyelitis, Gastroenteritis Rodents, Human [19] Cosavirus Cosavirus A Gastroenteritis Human [16] Dicipivirus Cadicivirus A No known disease associated Canine [31] Enterovirus Enterovirus A Hand-foot-and-mouth disease Human, simian [46] Enterovirus B Respiratory disease, myocarditis Human, simian [46] Enterovirus C Poliomyelitis, meningitis Human [46] Enterovirus D Respiratory disease Human, simian [47] Enterovirus E Respiratory disease, enteritis Bovine [48] Enterovirus F Respiratory disease, enteritis Bovine [48] Enterovirus G Polioencephalomyelitis, enteritis Porcine, Ovine [49] Enterovirus H Diarrheal disease Simian [50] Enterovirus J Diarrheal disease Simian [50] Rhinovirus A Respiratory disease Human [51] Rhinovirus B Respiratory disease Human [51] Rhinovirus C Respiratory disease Human [51] Erbovirus Equine rhinitis B virus Respiratory disease Equine [38] Gallivirus Gallivirus A Enteritis, growth depression Avian [17] Hepatovirus Hepatitis A virus Hepatitis Human [52] Hunnivirus Hunnivirus A No known disease associated Bovine, Ovine [53] Kobuvirus Aichivirus A Gastroenteritis Human [54] Aichivirus B No known disease associated Bovine [55] Aichivirus C No known disease associated Porcine [56] Kunsagivirus* Kunsagivirus A No known disease associated Avian [41] Megrivirus Melegrivirus A Hepatitis Avian [23] Mischivirus Mischivirus A No known disease associated Bats [27] Mosavirus Mosavirus A No known disease associated Rodent, Avian [28] Oscivirus Oscivirus A No known disease associated Avian [20, 57] Parechovirus Human parechovirus Encephalitis, meningitis Human [37] Ljungan virus Encephalitis, myocarditis Rodent [58] Pasivirus Pasivirus A No known disease associated Porcine [29] Passerivirus Passerivirus A Gastroenteritis Human [18] Rosavirus Rosavirus A No known disease associated Rodents [30] Sakobuvirus* Sakobuvirus A Feline [42] Salivirus Salivirus A Gastroenteritis Human [19] Sapelovirus Porcine sapelovirus Enteritis Porcine [32] Simian sapelovirus No known disease associated Simian [13] Avian sapelovirus Hepatitis Avian [33] Senecavirus Seneca Valley virus No known disease associated Human [59] Sicinivirus* Sicinivirus A No known disease associated Avian [43] Teschovirus Porcine teschovirus Encephalomyelitis Porcine [60] Tremovirus Avian encephalomyelitis virus Encephalomyelitis Avian [61]
16 The Enterovirus genus The papers included in this thesis are focused exclusively on the EV-B (Papers I, II, III and V) and RV-C (Paper IV) species of the Enterovirus genus. The Enterovirus genus is the largest in the Picornaviridae family, consisting of 12 species; Enterovirus A-H and J as well as Rhinovirus A-C [12]. The phylogenetic relationship of these viruses is shown in Figure 1.
Figure 1. Maximum likelihood amino acid tree of the P1 Capsid demonstrating the relationships between enteroviruses. Avian sapelovirus was used as an out-group and the trees were built with 1000 bootstrap replicates. The scale bar indicates amino acid substitutions per site. Courtesy of Michelle Wille. The species marked with a dot are the ones studied in the papers included in this thesis. A complete phylogenetic tree of the Picornaviridae family can be found in [24].
17 History of enteroviruses In all likelihood, viruses have evolved alongside humans for as long as we have existed. There is a stele (Figure 2) dated to the 18th Egyptian dynasty (1403 - 1365 BC) depicting a man with a drop foot, a characteristic injury after a poliovirus infection [62]. Poliovirus belongs to the EV-C species and has played an important part in our history. Poliovirus infections are usually mild, but in 1-2 % of cases the virus will enter the central nervous system and cause poliomyelitis, which is a severe paralytic disease [63]. It became a significant health issue in the 20th century in both Europe and North America, where the number of infections Figure 2. Ancient stele depicting an Egyptian man with a potential peaked in the 1940s and 50s. This prompted a poliovirus injury [62]. search for polio-like viruses, which resulted in the discovery of other enteroviruses such as coxsackieviruses (CVs) A and B and a number of enteric viruses isolated from asymptomatic patients that were named echoviruses (E) after enteric (where they were found) cytopathic (killed cells in culture) human (found in human) orphan (not associated with disease). However, it has since become clear that echoviruses do cause a number of human diseases [64].
Enteroviruses in society Enteroviruses infect mainly by the fecal-oral route and usually give rise to mild infections. However, exceptions do exist; for instance coxsackieviruses can cause severe infections of the respiratory system, heart, nervous system and pancreas [64]. Some enteroviruses have also been indicated as a cause of type I diabetes [14]. The Rhinovirus species has recently been included in the Enterovirus genus. There are more than 150 types in the three species of rhinoviruses [11, 65]. Rhinoviruses have been proposed to be a causative agent of asthma and 30-50 % [66] of all cases of the common cold, two of the most economically costly diseases in our society [67]. In the last decades, two enteroviruses have started to cause alarm. Enterovirus A71 (EVA71) and more recently enterovirus D68 (EVD68) are now associated with more severe infections than previously. EVA71 normally causes hand-foot-and-mouth disease, but lately it has also been associated with severe neurological complications and fatality [68]. EVD68 was
18 discovered in 1962 but was rarely associated with disease. This has changed in the last three years, with EVD68 being reported in clustered outbreaks causing severe and, in a few cases, even fatal respiratory infections in America, Asia and Europe [69]. Last year (2014), the United States was struck with a large outbreak of severe respiratory infections caused by EVD68. At the same time, the number of children with polio-like symptoms increased and EVD68 has been isolated from some of these patients [70]. Enteroviruses are very common and frequently cause infections both in humans and in other animals. Consequently, they have a large impact on our society, both medically and economically.
19 THE ENTEROVIRAL GENOME AND PROTEINS
The enterovirus genome is approximately 7500 nucleotides long and consists of single stranded RNA with positive polarity. It is very similar to an mRNA, and therefore the viral genome is translated by the host cell ribosomes upon host cell entry. The genome is made up of five regions (Figure 3); the 5’ untranslated region (UTR), the protein-coding region divided into P1, P2 and P3 and the 3’UTR. The 5’UTR contains structures that help facilitate translation and replication of the genome. The P1 region codes for the structural proteins that make up the viral capsid while the P2 and P3 regions code for the non-structural proteins that are involved in genome replication and protein processing. The 3’UTR has a Poly(A)tail and is involved in the replication of the viral genome [71]. Within the genome there are three cis- acting replication elements (CREs), found in the 5’UTR, 3’UTR and in varying locations of the protein-coding region [72].
5’UTR P1 P2 P3 3’UTR VPg Poly(A)
Figure 3. Schematic illustration of the enterovirus genome. The protein-coding region has a single reading frame that codes for the enteroviral polyprotein. This protein is divided into three regions; P1, which is made up of the capsid proteins, as well as P2 and P3, which are processed into seven non-structural proteins. On each side of the open reading frame there are untranslated regions, the 5’ and 3’ UTR.
The 5’UTR The 5’UTR of the enteroviral RNA genome is approximately 620-750 nt long and is arranged into elaborate structures with pseudoknots and stem-loops. The sequence begins with UU that binds the VPg (viral protein, genome- linked) protein (which will be described later in this chapter) and then
20 continues with the oriL (origin of replication at the left) which is one of the three CREs [72]. In enteroviruses, the oriL has a characteristic structure and is often referred to as the ‘cloverleaf’ (Figure 4), a structure essential for initiation of replication of the RNA genome [73]. IRES The majority of the 5’UTR is made up of the internal ribosomal entry site (IRES), which consists of well-ordered, intricate secondary and tertiary structures. At the functional domains of these structures the host cell proteins and viral proteins assemble to form a translation complex that can facilitate cap-independent translation. To date, four different types (I-IV) of IRES have been identified and members of the Enterovirus genus have type I (Figure 4). These are highly conserved regions; hence there is very little sequence variation within an IRES type [71]. When the translation complex has formed, it starts scanning the RNA to find a start codon where translation begins. The distance from the IRES to the translation initiation site is 18-40 nt in the Rhinovirus species [71], but up to 150 nt for the Enterovirus species. The initiation site is in a Kozak context [74] (RNNAUGG, where R=purine, N=any nucleotide and the initiation codon is AUG), which is a conserved sequence in mRNAs, surrounding the start codon. [75].
The polyprotein The protein-coding region is translated as one ORF resulting in a polyprotein of approximately 2200 amino acids. During translation the polyprotein is auto-cleaved in-between regions P1 and P2 (Figure 5). A viral protease then separates the P2 and P3 regions and continues with most of the processing of
Figure 4. Structural features of the enteroviral 5’UTR. The cloverleaf can be seen to the left in the model. The remaining stem-loop structures comprise the picornavirus type I IRES element. Adapted from Lin et al. [76].
21 the protein precursors. Altogether there are 11 separate enteroviral proteins: viral protein (VP)4, VP2, VP3, VP1, 2A, 2B, 3C, 3A, 3B (also known as VPg), 3C and 3D. The proteins of the P1 precursor are the structural proteins that make up the viral capsid, while all other proteins are non-structural. It is a very small genome and many of the proteins and their precursors have multiple functions [77].
Structural proteins Non-structural proteins
Polyprotein
P1 P2 P3
VP0 VP3 VP1 2A 2BC 3AB 3CD
VP4 VP2 VP3 VP1 2A 2B 2C 3A 3B 3C 3D
Figure 5. Schematic illustration of the protein processing. As the open reading frame of the enteroviral genome is translated the newly forming polyprotein is rapidly processed into 11 separate proteins by viral proteases.
The P1 region proteins The P1 protein is further cleaved into VP0, VP3 and VP1. These are the building blocks that make up the viral capsid, the protective shell surrounding the viral genome. When the capsid is formed and the genomic RNA encapsidated, VP0 is cleaved into VP4 and VP2 [71]. The P1 coding region is much more diverse than the rest of the genome. Comparisons of this protein-coding region are an important part of determining what species a virus belongs to [78]. Furthermore, the VP1 coding region is used to divide the species into types [79]. The structural proteins are used for attachment to the cells and their diversity is part of what allows the virus to adapt to the environment. In Paper I, the P1-coding region of CVB5 Dalldorf (CVB5D) was replaced with the P1 region of other viruses of the same type, to study the viability of such viruses. There have also been reports of interactions of the P1 proteins with proteins inside the host cell; for example VP2 of CVB3 interacts with
22 proapoptotic protein Siva [80] and enteroviral VP3 interacts with viral protein 2C [81]. The impact of a mutation in the VP1-coding sequence on the gene expression of infected cells was investigated in Paper V.
The P2 region proteins There are three proteins included in the P2 region, which is the second region to be synthesized during translation. These proteins have diverse functions such as genome processing, RNA replication and host membrane reorganization. 2A The enteroviral 2A protein is a chymotrypsin-like protease with multiple functions. The initial cleavage of the polyprotein in the junction of the P1 and P2 regions is due to the autocatalysis of the N-terminus of the 2A protease (2Apro) [71, 82]. In some cases 2Apro also causes host cell translation shut-off by cleaving the cellular protein eIF4G, which is necessary for cap-dependent translation [83, 84]. 2Apro has been shown to cause DNA fragmentation in both PV and EVA71 and to catalyze cleaving of a number of important cellular proteins, thus causing enteroviral induced apoptosis [85-87]. In addition, 2A has been reported to reduce, and even stop, the splicing of certain cellular pre-mRNA [88, 89]. The C-terminal amino acids of the enteroviral 2Apro also play a role in viral RNA-synthesis, the mechanism by which this occurs is still unknown [90]. 2B The 2B protein is hydrophobic and associates with membranes of the host cell, such as those found in the endoplasmic reticulum (ER) and the Golgi apparatus [91]. It is sometimes referred to as a viroporin, due to its ability to form membrane pores. During infection the formation of pores increases the influx of extracellular Ca2+, leading to the release of Ca2+ from the ER and Golgi into the cytoplasm [91, 92]. This disrupts the traffic of cellular proteins through the Golgi and hinders the secretion of cellular proteins [92, 93]. Ca2+ is an important intracellular signaling substance, and this disrupted homeostasis affects organelle regulation, for example through suppressed caspase activation and prevention of apoptotic cell death [94]. 2C The precursors 2BC and C are also involved in membrane rearrangement, producing replication vesicles [95]. Moreover, 2C has ATPase activity [96]
23 and an RNA helicase motif [97]. This motif interacts with the 3’UTR of the negative stranded RNA genome, suggesting it might be involved in positive stranded RNA synthesis [98]. Furthermore, it has been shown that 2C is required for initiation of negative stranded RNA synthesis [99], and is associated with virion assembly [81]. The crucial role of 2C in combination with a fairly conserved amino acid sequence makes it a good target for antiviral agents [100-102]; a well- known example is guanidine hydrochloride [103].
The P3 region proteins The proteins of the P3 region are non-structural, with a wide range of functions. The four proteins 3A-D have functions such as cleaving proteins and disturbing the host cell translation, to interfering with protein secretion, and assisting in various steps in the viral genome replication cycle. 3A Like 2B, 3A is a hydrophobic membrane protein that interferes with the protein transport from ER to Golgi [104]. In addition, 3A has a role in evasion of the host immune response, by inhibiting the expression of the major histocompatibility complex 1 (MHC-1) and thereby preventing antigen presentation [105]. The precursor 3AB also binds to intracellular membranes and anchors the replication complex to the membrane vesicles [106]. When bound, 3AB stimulates self-cleavage of another precursor, namely 3CD, into 3C protease (3Cpro) and polymerase (3Dpol) [107] and activates the polymerase activity of 3Dpol [108]. 3B The smallest of the enteroviral proteins is 3B, which is also called VPg [71]. VPg binds covalently to the 5’end of either the positively or negatively stranded RNA genome and primes replication of the genome. The coupling of VPg to the genome occurs through an uridylylation of VPg, facilitated by interactions of the CRE oriI (origin of replication internally) and the 3CD precursor protein [82]. The oriI is located in different parts of the genome depending on the virus, for example it can be found in the 2C coding region of the PV [109] and CVB3 [110], 2A of RV-A [111], VP1 of RV-B [112] and VP2 of RV-C [113].
24 3C 3C is a cysteine protease (3Cpro) and, together with its precursors, it is responsible for most of the polyprotein cleavage, starting with cleavage at the junction between P2 and P3. 3Cpro and 3CDpro are responsible for all the processing of the polyprotein apart from the cleavage between P1 and P2 and the maturation cleavage of VP0 [71]. Just like 2Apro, these proteins also target host cell proteins. This causes disruption of a number of host cell functions such as mRNA transcription [114] and cap-dependent translation [115]. 3D The protein at the C-terminal portion of the viral polyprotein is an RNA- dependent RNA polymerase (3Dpol). This polymerase specifically copies polyadenylated RNA when it has a uracil-bound primer. During an infection, that primer is the VPg protein, which also activates the 3Dpol by stimulating the cleavage of the precursor 3CD into 3Cpro and 3Dpol [108]. The structure of 3Dpol is similar to that of other polymerases; a ‘cupped hand’ with a ‘thumb and a finger’ that moves along the RNA strand. It can replicate both the positive and negative strand, and, when the negative strand is used as a template, multiple polymerases work simultaneously to synthesize RNA strands of positive polarity [116].
The 3’UTR The 3’UTR is important for virus translation and initiation of replication of negative stranded RNA. It has also been associated with determination of tissue tropism [117, 118]. It is much shorter than the 5’UTR and the sequence is more divergent between picornaviruses. Just like the 5’UTR, the 3’UTR is folded into secondary and tertiary structures, forming a CRE, here called oriR, (origin of replication at the right). The purpose of the oriR is not entirely clear. It has been suggested to be important for the initiation of minus-stranded RNA, but the underlying mechanism is not known [118]. The number of stem-loops in the domain varies between one and three, for example RVB14 has one, PV has two and CVB4 has three. Surprisingly, it has been demonstrated that interchanging the stem-loop region of these viruses does not affect viability. It can even be removed completely for PV and CVB4 without diminished viability in HeLa cells [117-120]. It has been established that those proteins important for PV RNA replication, namely 3AB and 3CDpro, specifically interact with the 3’UTR [121]. The poly(A)tail is on average 50 adenines (As) in length [122] and starts approximately 40-100 nucleotides downstream of the stop-codon terminating the synthesis of the polyprotein [71, 118]. The length of the
25 poly(A)tail has implications for viability; whereby if the tail is shortened (to less than 20 A) the virus loses viability [123].
Main differences between enteroviruses and other picornaviruses The family of picornaviruses is large and there is a fair amount of diversity among the member viruses. For example they cause different symptoms, have different genome sizes and host tropisms. One of the most striking differences is that some of the members of the Picornaviridae family; namely Aphthovirus, Cardiovirus, Erbovirus, Gallivirus, Hunnivirus, Kobuvirus, Mischivirus, Mosavirus, Oscivirus, Passerivirus, Salivirus, Sapelovirus, Senecavirus and Teschovirus; have an additional protein, the Leader (L) protein that is coded at the 5’ end of the ORF [11]. The L protein is highly diverse between genera, both in sequence and in function. The L protein of some genera, like the Aphthovirus, has a function quite similar to the enteroviral 2A protein [124], while the L protein of kobuviruses are involved in RNA synthesis and encapsidation [125]. Unlike the other non-structural proteins that are present in all picornaviruses, the 2A protein exists in multiple structurally and functionally different variants. Apart from the variant found in enteroviruses, there are other 2A proteins with proteolytic functions, but the 2A protein can have entirely different roles, for example involvement in regulation of cell proliferation and virion assembly [71]. In most picornaviruses the VP0 protein is a precursor that is cleaved into VP4 and VP2, but in some picornaviruses, like Parechovirus, this never happens and the virus capsid is formed with three proteins: VP0, VP1 and VP3 [11]. The Enterovirus genus is the only member of the Picornavirus family that has IRES type I, the rest have types II-IV. Enteroviruses are also the only member with a cloverleaf formation at the 5’end of the genome. Other picornaviruses have the oriL in the same location but in an unbranched stem formation [71].
26 THE CAPSID
Elucidating the capsid structure In the 1950s, a poliovirus epidemic plagued the western world and extensive research was focused on learning more about the virus and how to combat it. The new method of propagating viruses in cell cultures greatly simplified purification, and, combined with methods such as ultracentrifugation and electron microscopy (Figure 6), several chemical properties of the poliovirus were determined, for instance its uniform spherical structure of approximately 27 µm [126]. In 1955 the first images of crystallized enterovirus particles, poliovirus MEF1, were published, significantly advancing our understanding of the structure of the virus capsid [127]. However, it was not until 1985 that the first high-resolution virion structure of an enterovirus was published; first poliovirus [128], followed by RVB14 [129].
Capsid functions The capsid is a protein shell that surrounds the viral genome in the virion. It has two main functions, firstly to protect the genome, and secondly to attach to the surface of the host cell, facilitating entry. This requires that the capsid is robust enough to protect the RNA, although still able to release it into the cytosol at the appropriate time. An additional requirement is specificity, allowing binding to the receptors of a suitable host cell, thus playing an important part in tissue tropism and pathogenicity. The physiochemical properties of the virion are dependent on the capsid and have a decisive influence on Figure 6. Electron microscopy image of crystallized poliovirus [130].
27 what tissue is infected and by which route. Many enteroviruses primarily infect via the intestine, which means that they have to withstand the acidity of the stomach. Rhinoviruses, which primarily infect the respiratory tract, do not need protection from acidity and thus, their capsids are not acid resistant [77].
The capsid structure The capsid of an enterovirus is approximately 30 nm in diameter and has an icosahedral shape, composed of the four proteins of the P1 region, VP1-4. These form a protomer of triangular shape with VP1-3 on the outside and VP4 on the inside of the capsid. Five of these identical protomers form a pentamer, and twelve pentamers form the icosahedral capsid (Figure 7A). In total, there are 60 copies of each protein in each capsid [131]. The proteins exposed on the surface of the capsid, VP1-3, all have the same basic structure; an eight-stranded anti-parallel beta-barrel with two flanking helices forming a wedge shaped core [128]. The major difference between these proteins are the carboxyl- and amino-terminals, as well as the loops in-between the beta-sheets. The structural flexibility of these loops provides the different morphological and antigenic properties of the virions [71]. VP4 has a more extended conformation, and, together with extensions of the amino-terminals of VP1-3, it lines the inside of the capsid [128]. At the amino-terminal of the VP4 a myristic acid is bound covalently. This is thought to play a role in virion assembly and during the entry process [132]. A B
VP1 VP2
VP3
VP4
Figure 7. The capsid. A) Schematic illustration of the organization of the capsid proteins. B) Model of the virion of RVB3 [133] made by Jean-Yves Sgro using Qutemol. Blue areas are VP1, green are VP2 and brown VP3. Used with permission.
28 The capsid surface The topography of the capsid consists of valleys and ridges that form star shaped and triangular plateaus (Figure 7B). In the center of the ‘star plateau’ there is a five-fold axis surrounded by five copies of the VP1 protein, and at the center of the triangular plateaus there is a three-fold axis surrounded by alternating copies of VP2 and three VP3 proteins. In-between these there are depressions, usually referred to as canyons, the depth of which varies between viruses [131]. Beneath the canyon floor enteroviruses have hydrophobic pockets, mainly formed by the VP1 protein. These contain lipid ‘pocket factors’, which stabilize the capsid and are thought to play a role in the entry process [134].
29 THE INFECTIOUS CYCLE
The members of the Enterovirus species infect through the fecal-oral route, passing through the acidity of the stomach and entering through the epithelium of the intestine. The Rhinovirus species infect through aerosol droplets and generally cause infections in the respiratory tract. Despite differences in tissue tropism, the viral lifecycle of the members of the Enterovirus genus follow the same basic principles (Figure 8). The duration of an enteroviral replication cycle varies between 5 an 10 hours depending on an array of factors including the type of virus, temperature, pH and number of virus particles [131].
Attachment The first step of the enteroviral replication cycle is attachment to a susceptible cell. This is achieved through interactions between the viral capsid and receptors expressed on the surface of the host cell. Consequently, the structure of the capsid surface is a very important determinant of host range, tissue tropism and pathogenesis. The members of the Enterovirus genus have evolved to use a wide range of cell surface molecules as receptors, including integrins and proteins belonging to the immunoglobulin-like family [135] (Table II). For many enteroviruses the receptor-binding site is located in the canyon surrounding the five-fold axis. Intercellular adhesion molecule 1 (ICAM-1), coxsackie- and adenovirus receptor (CAR) and poliovirus receptor (PVR) are all examples of receptors that bind in the canyon. Other receptors, such as the decay-acceleration factor (DAF or CD55) and the low-density lipoprotein receptor (LDLR), bind outside the canyon. For some enteroviruses, such as poliovirus, only one receptor is needed to facilitate attachment and initiation of the replication cycle. For others, for example CVA21, more than one receptor is needed. The virus attaches to the cell via the DAF, but interaction with ICAM-1 is also required for cell entry [136].
30
Figure 8. The enteroviral replication cycle. (1) The virus capsid attaches to the cell via receptors. (2) The virion enters the cell and the viral genome is delivered. (3) After removal of the VPg protein the viral genome is translated by host cell ribosomes. (4) A polyprotein is formed. (5) The polyprotein auto-cleaves into three smaller proteins. (6) Proteins are processed further and the proteins needed for the replication of the viral genome are transported to replication vesicles formed from the organelle membranes. (7) The capsid is formed by the structural proteins. (8) The viral genome is transported to the replication vesicles. (9) The viral genome is copied into double stranded RNA intermediate. (10) The negative-stranded genome copy is used as a template for the formation of new positive- stranded genomes. (11) Some of the newly formed positive stranded genomes are used for replication and translation (12) The viral genome is encapsidated. (13) The newly formed virion is released from the cell [131].
31 Table II. Examples of receptors used by enteroviruses. Adapted from [131]. Receptor Viruses Ref PVR (CD155) Poliovirus [137] ICAM-1 Major group of rhinoviruses [138-140] Coxsackievirus A13, A18, A21 [135] Integrins Echovirus 1, 22 [141, 142] Coxsackievirus A9 [143, 144] DAF (CD55) Echovirus 3 6, 7, 11, 12, 13, 21, 24, 29, 30 and 33 [145-148] Coxsackievirus A21 [149] Coxsackievirus B1, B3, B5 [150] Enterovirus 70 [151] CAR Coxsackievirus B1-B6 [152, 153] LDLR Minor group of rhinoviruses [154] Sialic acid Rhinovirus 87 [155] Enterovirus 68, 70 [156, 157] Heparan sulfate Echovirus 5, 6 [158, 159] Coxsackievirus B3 variant PD [160] SCARB2 Enterovirus 71 [161] Coxsackievirus A16 [161] Abbreviations: PVR, poliovirus receptor; ICAM-1, intracellular adhesion molecule 1; DAF, decay- accelerating factor; CAR, coxsackie- and adenovirus receptor; LDLR, low-density lipoprotein receptor; SCARB2, Scavenger receptor B2
Independent of the division into species, rhinoviruses are also divided into the major and minor groups of rhinoviruses, depending on their receptor interactions. The vast majority of rhinoviruses binds the ICAM-1 receptor and is thereby classified as belonging to the major group of rhinoviruses. Most of the remaining rhinoviruses with known receptor usage are in the minor group and bind the low-density lipoprotein receptor (LDLR) [155]. Recently it has been reported that enteric viruses are helped by interactions with the microbiota in the gastrointestinal tract [162, 163]. Both in vivo and in vitro evidence show that severity of poliovirus infections is coupled via the interaction of the virus with the polysaccharides on the surface of intestinal bacteria. These interactions have been proposed to make the virus more thermostable and mediate attachment to the cell surface [162].
Entry Although attachment of the virus to the susceptible cell is a crucial first step of the replication cycle, it is not the only role of the receptors. They also need to facilitate release of the viral RNA into the cytoplasm and transmission of intracellular signals that are important for infection. Enteroviruses have evolved different mechanisms for translocation of their RNA into the cytoplasm [164]. For example, the poliovirus is internalized after binding to the PVR. The binding of the receptor also
32 mediates conformational changes in the capsid that allows for release of the viral RNA. The capsid is referred to as an altered particle after this conformational change. The altered particle lacks the internal VP4 and has externalized amino-terminals of the VP1. These terminals are hydrophobic, increasing the affinity for the membrane as compared to the unaltered virion. The VP1 amino-terminals and VP4 create a pore-like structure in the cell membrane through which the viral RNA can pass into the cytoplasm, leaving an empty capsid [165, 166]. Exactly how the RNA is released into the cytoplasm is not known. Rhinoviruses use another mechanism. They utilize endosomes for entry. Since they are not acid-stable, the low pH of the endosomes causes conformational changes in the capsid, allowing for RNA release [167]. The minor group of rhinoviruses form a specific pore in the endosomal membrane through which the RNA passes into the cytoplasm [168], while the conformational change in the major group triggers lysis of the endosome and release of both the empty capsid and the RNA into the cytosol [169].
Translation Once inside the cytoplasm of the host cell, the viral genome needs to be translated in order for the infectious cycle to continue. The viral proteins coded by the RNA genome are crucial for replication of the genome; hence, translation is the first process to occur in an infected cell. Compared to mRNA translation the major differences are that the viral genome is translated cap-independently, and that the translation complex forms at the IRES element in the 5’UTR [131]. Prior to translation, a cellular protein cleaves off the VPg protein [170]. Thereafter, the cellular 40S ribosomal subunit binds the IRES, the translation complex is formed, and the ribosome scans the RNA until it reaches the start codon, where translation starts. The AUG start codon for enteroviruses is situated 20-150 nt downstream of the IRES element and the genome is translated as one ORF into a polyprotein [171]. The effects of a frameshift mutation introduced shortly after the start codon is studied in Paper II. Many enteroviruses have evolved to take advantage of the fact that cellular mRNAs need other initiation factors to form a translation initiation complex. By cleaving the eukaryotic initiation factor (eIF) 4G and the poly(A)-binding protein (PABP), the 2Apro and 3Cpro inhibit, or even completely shut off host cell cap-dependent translation. This allocates cellular recourses to translation of viral proteins instead [82].
33 RNA replication The genome is not only the template for translation, but also for RNA replication. This takes place in the replication vesicles formed by viral proteins and derived from the membranes of cellular organelles such as the ER, Golgi and lysosomes (Figure 8, step 6) [172]. Replication of the genome is primer- dependent and performed by the 3Dpol. However, viral replication complexes are composed of several viral and host proteins, as well as structures in the viral RNA necessary for replication [131]. Since the genome is single stranded, a replicative intermediate is required to produce RNA with the correct polarity. With the genome as a template, a minus-stranded genome is made, which in turn is used to make plus-stranded genome copies. To achieve replication, an elaborate interplay between the viral proteins, cellular proteins, replication vesicles and the viral genome is required. Minus-stranded RNA synthesis starts with the circularization of the plus- stranded RNA. This is facilitated via interaction with the poly(C) binding protein (PCBP) and/or the 3AB precursor bound to the oriL and the PABP, which associates with the poly(A)-tail of the genome. After circularization the VPg protein is uridylylated with oriI as the template, yielding VPgpUpU, which is transferred to the 3’ end of the poly(A)-tail. The uridylylated VPg is used by the 3Dpol as a primer. The next step of RNA replication is elongation of the primed minus strand on the plus-stranded template, resulting in a double stranded intermediate. The VPg is moved to the 3’ end of the minus strand and the double-stranded RNA is unwound. The polymerase binds to the VPgpUpU primer and starts elongation of the plus-stranded RNA. Multiple copies of plus-stranded RNA genomes are synthesized simultaneously on the same minus-stranded template [173]. The newly formed plus-stranded RNA is used either for translation, RNA replication or as a genome in the viral progeny.
Assembly Assembly is the process by which the structural proteins form the capsid and the VPg-linked RNA is encapsidated. Although the general outline of this process has been known since the 1970s [174], many of the specific details remain unknown. Only newly synthesized RNA genomes are encapsidated, suggesting that this process is tightly linked to the formation of the plus- stranded genomes [175]. When the P1 region is cleaved from the polyprotein, the 3’end is covalently linked to myristic acid [132, 176], and cellular chaperon Hsp90 assists in the change to a processing-competent conformation [177]. Once this has occurred, the 3CDpro cleaves the P1 precursor into VP0, VP3 and VP1, which immediately form a protomer. Five
34 of these protomers form a pentamer, which is recruited to the RNA replication complex by the 2C protein [81, 178]. Twelve pentamers form the procapsid and the newly formed VPg-linked viral RNA genome is encapsidated. The last step of assembly is the RNA-dependent autocatalytic maturation cleavage of VP0 to VP2 and VP4. This stabilizes the capsid and the new virion is thus formed [179].
Release The newly formed virions must exit the host cell in order to infect new cells. Two strategies are used for this phase of the virus life cycle. The first is cell lysis, often referred to as cytopathic effect (CPE); this is generally considered to be the most common mechanism of virion release. In a monolayer cell culture, CPE is clearly detectable using light microscopy. Typically, cells lose their outstretched morphology and become rounded. This causes them to disassociate from the culture surface and the surrounding cells (Figure 9). The underlying mechanism behind CPE is not entirely understood, and it is likely that several different strategies may be employed. One suggested mechanism is lysosomal leakage [180]. Another is the use of apoptotic pathways of the host cell. During translation and RNA replication, the virus is dependent on the cell and expresses antiapoptotic proteins [94]. When enough virions have formed, the virus regulates the protein expression so that proapoptotic mechanisms are activated [85-87]. The second mechanism is release of virion without lysis of the cell, a so-called persistent or non-cytolytic infection. The way in which virions are released in such infections is even more obscure. It has been suggested that 2B may facilitate release of virions by making the plasma membrane permeable [91]. It has also been proposed that the release of virions from persistently infected cells is facilitated through autophagy [181, 182].
Infected cells Control
Figure 9. Cell lysis of Rhabdomyosarcoma (RD) cells induced by an infection with CVB2 Ohio VP1Q164K mutant. The control was incubated without virus. The infected cells are rounded up, while the mock-infected cells maintain a spread-out morphology.
35 EVOLUTION OF ENTEROVIRUSES
Evolution is essential for viruses in order to adapt to new conditions and counter host evolution of resistance. The ability to adapt largely depends on the genetic diversity of a species and therefore the mutation and recombination rates. In sexually reproducing species, the offspring’s genome is a combination of the genomes of the two parents, whereby each parent contributes 50 % of their genetic material to their progeny. For viruses with fragmented genomes that infect the same host cell, a somewhat similar process can occur, whereby the viral progeny gain gene fragments from different viral ’parents’. This type of diversification is not possible for the members of the Enterovirus genus due to the non-segmented organization of their genomes; therefore, they must rely on other mechanisms to evolve.
Mutations The 3Dpol, like other RNA-dependent RNA polymerases, lacks proofreading and is therefore error-prone. On average, there are 10-4 mutations per nucleotide per generation [183], which for enteroviruses means approximately one introduced mutation per copied genome. The mutation rate, in combination with the short replication time and large quantity of viral progeny, leads to a ‘heterogeneous cloud’ of viruses referred to as quasispecies. The viruses of the quasispecies differ from each other by one or more nucleotides in the genome. This diversity greatly contributes to the adaptability of the viruses and improves the chances for survival, as a quasispecies, in changing environments [184].
Recombination Recombination produces recombinants, which for enteroviruses are a combination of two, or more, genomes. Recombination in enteroviruses was first reported by Hirst in 1962 in a study involving polioviruses [185]. Since
36 then, recombination has been found in all enteroviruses studied to date [186], as well as numerous other viruses infecting both animals and plants. However, the frequency of recombination differs between viral species [187]. Recombination contributes to the genetic diversity of a virus species and is an important factor in enteroviral evolution. Natural recombination in enteroviruses most commonly arises through a phenomenon called ‘copy choice’, whereby the 3Dpol detaches from the minus stranded RNA of one virus during replication, and then continues elongation on the minus RNA strand of another virus, in cells infected with more than one virus type or species [188]. A non-replicative model has also been proposed where recombination occurs through the combination of two broken RNA fragments. However, this is considered to be a rare event in the history of enteroviruses [189].
Selective pressure Adaptation occurs via selective pressure on the existing individuals of a population. For enteroviruses, the main sources of new genetic diversity are mutations and recombination. Some of the resulting viruses will have decreased fitness due to the presence of deleterious mutations, and will have less opportunity to produce viral progeny. Others will have increased fitness due to the presence of beneficial mutations, and produce more and/or fitter offspring. Over time, via the process of positive natural selection, the fittest variants will increase in frequency in the population. The determinants of what makes for a fit virus varies in space and time and the selective pressure could for example be antiviral agents or the immune response of the host [190]. Selective pressures are not constant, and this is one reason why it is necessary for viruses to have high adaptability.
37 WORKING WITH ENTEROVIRUSES
Enteroviruses are convenient to work with, partly because most grow very well in cultured cells. Furthermore, their genome organization makes them easy to manipulate through cloning. In this chapter, some of the methods used in the studies in this thesis will be described.
Cell culturing Immortal cell lines At the beginning of 1951, George Gey recognized that cell samples taken from a cervical cancer patient behaved in a way that had not been reported earlier. Up until that point attempts to culture human cells had resulted in the cells dying after a few passages. But these cells did not die [191]. The cells were named HeLa after Henrietta Lacks, the woman who had unknowingly donated them [192]. This was the first of many immortal cell lines and HeLa cells can now be found in laboratories all over the world. Immortal cell lines, also known as standard cell lines, have become an invaluable tool for researchers studying viruses. For instance, HeLa cells were used by Jonas Salk to develop the first poliovirus vaccine at the beginning of the 1950s [193]. Immortal cell lines generally stem from extremely aggressive tumors and the advantage of working with these cells is that they are easy to culture, homogenous, are readily available and relatively cheap. A disadvantage is that they might respond differently than healthy cells [194]. Regardless, immortal cell lines are required for a number of different experiments where large virus quantities are needed. RV-C does not grow in immortal cell lines, which is an obstacle for the development of antiviral agents, vaccines and for further research into the virus. In Paper IV, restrictions of replication in immortal cell lines were investigated.
38 An alternative to standard cell lines is to use primary cells, which are cells taken from tissue. These can generally only be sustained for a few passages and are difficult to grow into large quantities. The short lifespan of primary cells can also be a problem when repeating experiments, since the same cells cannot be used. In this thesis, three standard cell lines have been used: HeLa, green monkey kidney (GMK), and rhabdomyosarcoma (RD) cells.
Propagating the virus There are several ways to propagate a virus (Figure 10). Most straightforward is the use of viral particles to infect cells grown in monolayer cell cultures. However, transfection may be preferable in cases where alterations in the genome prior to infection are required, or if homogenous viral RNA sequences are prioritized.
Figure 10. Methods of virus culturing. To generate viral progeny viruses can be added to a cell culture, which will result in infected cells. Another strategy is to extract the viral RNA and, via cDNA synthesis, clone the genome into a plasmid vector. The vector can either be used to transfect the cells directly, or in vitro transcribed RNA can be synthesized and used for transfection. All of these methods, if performed successfully, will generate viral progeny.
39 Passaging of virus When growing larger amount of viruses or studying them in cell culture over time, the virus is ‘passaged’. This means transferring the virus-containing supernatant from a previous infection and adding it to a culture of uninfected cells. After a set amount of time, the cultures are freeze-thawed, which releases the virions still trapped inside cells out into the medium. The medium can then be used for the next passage. Consecutive passages are usually referred to as passage 1, 2, 3 and so on. Transfection Transfection is the act of introducing nucleic acid into the cell by non- infectious means and the method was first described in 1973 [195]. By use of transfection, the attachment and entry stages of the infectious cycle are circumvented. Due to the organization of the enterovirus genome, translation and replication will take place, providing that the nucleic acid is delivered into the cytoplasm of a susceptible cell. Normally, this would occur through the virion attaching to cell surface receptors and delivering the genome into the cytoplasm, but it can also be achieved synthetically. In Papers I-IV, transfection was performed with a formulation called Lipofectamine® 2000 (Life Technologies). Lipofectamine® 2000 contains cationic and neutral lipid components, able to form liposomes in an aqueous environment. The positively charged surface of the liposomes allows them to bind the negatively charged nucleic acid and the cell surface. The neutral co-lipids of the liposome mediate fusion with the plasma membrane and thereby facilitate passage of the nucleic acid through the plasma membrane [196]. Reverse genetics One of the most important tools used in the projects of this thesis is the infectious cDNA clone. They were used in Papers I-IV, as well as in the study preceding Paper V. The first cDNA clone containing an enterovirus was described in 1981 [197]. RNA extracted from viruses is used as a template for cDNA synthesis. The cDNA is, in turn, inserted into a plasmid through use of restriction enzymes and ligases and an infectious cDNA clone is created. The viral genome inside the clone can then be altered to the specifications of the project design. The plasmids are amplified in E. coli cultures. The virus is replicated as a DNA molecule by an organism with a much less error-prone polymerase, and therefore a homogeneous set of virus genomes are created as opposed to the heterogeneous sequence cloud of virions. If the plasmid has a promotor for a bacteriophage RNA polymerase, RNA copies can be created from the cDNA clone. With the use of the promotor, typically T3, T7 or SP6, in vitro transcription can be performed, creating RNA copies of the infectious clone. Both the cDNA clone and the in
40 vitro transcribed RNA can be used to transfect susceptible cells, although transfection with in vitro transcribed RNA is more effective [198]. The effect on RNA replication initiation, when the additional nucleotides of the promotor are cleaved off after transcription, was investigated in Paper III.
Quantifying viruses and determining their presence In molecular virology there are a number of methods to quantify and detect viruses (Figure 11). In this thesis, viruses have been quantified by plaque assay, tissue culture infectious dose 50 % (TCID50) and qPCR, and detected using immunofluorescence staining (IF) and Western Blot analysis. Although the latter three methods can also be used for detecting effects of the virus, rather than the virus itself, this has not been the case for the work described in this thesis.
Figure 11. Biomolecular methods used for virus research. The amount of viruses in a sample can be determined through qPCR, while PFU and TCID50 defines the number of infectious units. Western Blot and IF are other methods that can be used for detection of viruses. They can also be used for studying the effects of viruses on host cells.
41 Plaque assay Usually, more than one virion is needed to achieve a successful infection. Therefore, it is sometimes more appropriate to determine the amount of ‘infectious units’ rather than the exact number of virions. With plaque assay, the number of plaque-forming units (PFU) is determined. The first plaque assay performed on animal cells was reported by Dulbecco in 1952 [199]. To determine the number of PFU in Paper I, confluent monolayers of cells were infected with virus in a serial dilution, after which they were covered with a semi-solid medium before incubation. The high viscosity of the medium prevented the diffusion of released viruses, and thereby only allowed spreading to nearby cells. After incubation, the cells were washed and stained with an ethanol-crystal violet mixture. The uninfected cells were fixed and stained while the cells killed by viruses were washed away, leaving a plaque. The number of plaques in a given dilution corresponds to the number of PFU. An obvious drawback with plaque assays is that not all viruses are able to form plaques, particularly those that cause persistent, non-cytolytic infections.
Tissue culture infectious dose 50 % Like PFU, tissue culture infectious dose 50 % (TCID50) is a measure of infectious units, not virions. The method has the same basis as other end- point titration, such as median lethal dose (LD50) [200]. Cells are seeded in a multi-well plate and infected with a 10-fold serial dilution, with at least four replicates per dilution. As the name indicates, TCID50 is an assay designed to calculate the infectious dose needed to kill 50 % of the cultures in a given dilution. It is an endpoint titration and is not analyzed until the infections have run their course. The theoretical relationship between PFU and TCID50 is 0.69×PFU = 1×TCID50 based on Poisson distribution; however, in practice, the relationship is more complicated than that. Infectivity is affected by a number of variables, such as cell line, virus, culturing medium and temperature, and the different natures of the assays mean that the relationship can change with each altered variable. Although TCID50 can be determined for more viruses compared to PFU, the assay is based on the virus killing the cell; hence, non-cytolytic viruses cannot be analyzed in this way. qPCR Polymerase chain reaction (PCR) revolutionized molecular biology in the 1980s-90s and is now an indispensible tool [201]. It is a method by which
42 millions of copies of a given DNA sequence can be generated. The method is based on repeated cycles of three stages of different temperatures: denaturation (the DNA strands separate), annealing (two primers, that define the area to be amplified, bind the DNA) and elongation (a thermo-stable polymerase copies the DNA strand). A version of PCR that can be used for simultaneous detection and quantification of RNA is quantitative reverse transcription real-time PCR (qRT-PCR). The RNA is copied into cDNA and the cDNA is amplified in the same manner as in a regular PCR. The difference lies in the detection. To detect DNA amplified by regular PCR, gel electrophoresis in combination with a fluorescent intercalating dye is used. With qPCR (and qRT-PCR), a fluorescent intercalator is included in the PCR mix and facilitates detection of the amplified product in real-time, via laser detection in the qPCR machine. The first PCR machine capable of this was described in 1992 [139]. By determining the number of cycles required for the pool of amplicons to reach a set threshold value (the cycle threshold, or Ct value), the starting amount of DNA can be calculated via comparison to a standard curve. The quality of the product can be detected by adding an additional dissociation step, often known as a melt curve analysis. The intercalating substance displays stronger fluorescence when bound to double-stranded DNA. After completion of the final qPCR cycle, the temperature is lowered to ensure all amplicons are double stranded, and then slowly raised again. When the temperature reaches the precise melting temperature of the amplicons, the DNA strands will separate which is reflected by weaker fluorescence. Pure samples, where amplicons have identical sequence, will have the same melting temperature, thus exhibiting a synchronized drop in fluorescence. Immunofluorescence staining The location of a virus or a protein inside an infected cell can be determined with immunofluorescence staining (IF). It is an immunological method based on the immunohistochemistry staining method described by Albert Coons in 1941 [202]. Immunological detection methods are based on the use of antibodies specifically binding to the target protein or nucleic acid. The cells are fixed on a non-fluorescent surface and incubated with the specific antibodies. For visualization, secondary antibodies that bind the specific antibodies are added. Attached to the secondary antibodies are fluorescent molecules that can be detected by a fluorescence microscope. In order to assess the location of the targeted protein/nucleic acid, the cell nuclei are often detected using DAPI (4',6-diamidino-2-phenylindole; Paper I) or propidium iodide (Paper IV), which fluoresce when bound to A-T rich regions of double stranded DNA [203] or the G-C bond in nucleic acid, respectively [204].
43 Western Blot analysis In 1975 Edward Southern developed a technique for visualizing DNA fragments. The method involved separation of the DNA fragments using electrophoresis, blotting the DNA to a membrane and detection by specific radioactive probes. The method was named Southern Blot and inspired George Stark to develop a method that, by similar means, facilitated detection of RNA. He named this method Northern Blot. In the following years, a similar assay for visualizing proteins was developed, and named Western Blot [205]. This technique is often used to study protein expression, as well as to detect modifications and protein cleavage. The proteins are separated according to size using SDS-PAGE gel electrophoresis. Following separation, the proteins are blotted onto a membrane. As with IF, a specific primary antibody is used as a first step for detection. In the Western Blot analysis utilized in Paper I, a secondary antibody conjugated with horseradish peroxidase was used for detection in a chemiluminescence detection system.
44 AIMS AND OBJECTIVES
The overall aim of this thesis was to develop a more detailed understanding of enteroviral replication. The included studies have been aimed at specifically investigating the possibilities of recombination, the ability of the viruses to adapt, the RNA replication initiation in transfected cells, and the differences in cytolytic and persistent infections. The included papers address the following specific aspects of enteroviral replication:
I. Whether the replicative backbone of CVB5D can produce viral proteins and RNA without the structural protein-coding region and if the backbone can replicate with the structural protein-coding region of other members of the EV-B species.
II. Whether an enterovirus can overcome a frameshift mutation at the beginning of the protein-coding region.
III. The efficiency of viral RNA replication activation when transfecting colon cancer cell line HT29 with in vitro transcribed RNA with an authentic genomic 5’ end. Additionally, to investigate the lytic effect on colon cancer cell lines, using E5N as a prototype.
IV. The replication capacity of RV-C in immortal cell lines using the RV-C34 prototype strain pico58.
V. The differences in gene expression in RD cells infected with cytolytic and non-cytolytic variants of CVB2 Ohio (CVB2O), and thus, narrow down the causes of different replication strategies.
45 RESULTS AND DISCUSSION
Efficient replication of Enterovirus B recombinant (PAPER I) Independent evolution of genomic regions Sequence analyses of picornaviruses reveal that the structural and non- structural parts of the genome can be regarded as two separate units evolving independently, where the structural part is type-specific and the non- structural part is species-specific, without correlation to specific virus types [206]. They also evolve at different rates, the capsid-coding region changes more frequently, likely due to evolutionary pressure [207]. There are very few reports of recombination within the P1-coding region, while it is a common occurrence in the remainder of the genome [206, 208]. This apparently independent evolution might be facilitated by the very distinctive roles of the different regions in the enteroviral life cycle [186, 209]. Genome composition and sequence identity are the main determinants of successful recombination. Therefore, recombination within a species is frequent, whereas recombination between species is a much less common event, which has only been observed in the highly conserved genomic regions [210, 211]. The replication competent backbone The seemingly independent evolution of the structural and non-structural protein-coding regions prompted investigation of the relationship between the different genomic parts. In a previous study [212], it was concluded that the structural coding region of different CVB5 strains formed replication competent complexes with the backbone of CVB5D. To facilitate that study, an infectious cDNA clone containing the complete genome of CVB5D was altered to become a ‘cassette vector’ (Figure 12), where the structural protein- coding region could be removed and replaced with the corresponding region
46 5’UTR Structural proteins Non-structural proteins 3’UTR
Poly(A)
q
q SalI (757) ClaI (3340) Figure 12. The cassette vector. The structural protein-coding part of the genome can be removed and/or exchanged for the corresponding region of another virus, through the use of the restriction enzymes indicated at nucleotide position 757 and 3340. of another virus. In the present study, the cassette vector was used to investigate whether the non-structural protein-coding region of CVB5D could replicate with a structural-protein coding region from other types of the same species. Furthermore, a clone without the structural protein-coding region was constructed to study whether RNA replication could occur without structural proteins. Confirmation of replication GMK cells were transfected with RNA transcripts of three chimeric constructs to ascertain whether they could initiate viral replication. CPE was detected in all transfected cell cultures and the viruses were passaged five times. To further compare the viral progeny of the constructs to the CVB5D positive control, a plaque assay was performed on transfected cells, as well as on cells infected with supernatant from passage five. In addition, TCID50 was performed using supernatants of passage 5. Both the TCID50 and the plaque assay showed similar results for the construct progeny and CVB5D, although plaque morphology differed. Moreover, results from qRT-PCR performed on RNA extracted from passages 1, 3 and 5 showed comparable values. There appear to be no obstacles for efficient replication with chimera viruses of CVB5D containing the structural-protein coding region of other EV-B types. Replication of RNA transcripts from the cassette vector without a structural protein-coding region was not measured with the methods described above, since no virions can be formed without the structural proteins. Therefore, immunological methods were used for detection of translation and RNA replication. The absence of the VP1 protein and presence of the 3Dpol in cells transfected with RNA transcripts from the deletion mutant were confirmed with the use of Western Blot. Furthermore RNA replication was confirmed by IF using an antibody specific to double stranded RNA, thereby confirming the presence of replication intermediates in the cells.
47 Conclusion The cassette vector supports the replication of CVB5D chimera viruses derived from clones containing the structural-coding region of other EV-B. Moreover, translation and RNA replication occurs in cells transfected with in vitro transcribed RNA derived from the cassette vector without a structural protein-coding region.
Initiation of enterovirus translation (PAPER II)
In Paper I, we demonstrated that the P1 region of an EV-B genome could be replaced with the corresponding region of another virus of the same species. In the process of constructing the chimeras used in Paper I, a clone with the P1-coding region of E30 Bastiani (E30B) of the EV-B species was accidentally created with two additional nucleotides (AC) 22 nt downstream of the translation initiation site. In addition to the frameshift clone (pCasE30Bins, where ins stands for insertion), a construct with the correct reading frame was produced (pCasE30B). The ability of the virus to adapt and circumvent the frameshift was tested with RNA transcripts from pCasE30Bins, with pCasE30B as a positive control.
Measuring infectivity GMK cells were transfected in triplicate with in vitro transcribed RNA derived from the two E30B clones and incubated until complete CPE, which occurred within 2 days for viral progeny of pCasE30B (CasE30B) and 3 days for pCasE30Bins (CasE30Bins). The virus was passaged five times and the incubation time needed to reach complete CPE remained unchanged. To measure virus replication and evaluate infectivity, qRT-PCR and TCID50 were performed on passages 1 and 5. In passage 1 the Ct value and TCID50 were significantly lower for CasE30Bins than for the control. However, at passage 5 no significant differences were observed between the viruses with either of the methods. Hence, both methods indicated an adaptation of CasE30B from passage 1 to 5.
Sequence analysis To determine the cause of the adaptation, sequence analysis of passage 5 of CasE30Bins was performed, which showed that the virus had reverted back to the original sequence by excluding the inserted nucleotides. Since Sanger sequencing was used, only the consensus sequence was obtained. To ascertain whether the insert was present in earlier passages, further sequence analysis was performed and it was concluded that genomes that had reverted to the
48 original reading frame were in majority and there were no signs of the inserted nucleotides in the sequence data obtained from any of the triplicate samples. An alignment of almost all types of Enterovirus was performed, revealing a high degree of conservation following translation initiation. Among the 95 EV-B types included, only 7 deviated from the consensus sequence in the first 15 amino acids in the polyprotein. The sequence data clearly indicates a strong evolutionary pressure to conserve the sequence just downstream of the translation initiation site.
Alternative means of translation initiation To initiate RNA replication, a number of viral proteins are needed. Consequently, successful translation of the viral genome is required for RNA replication. The reading frame of pCasE30Bins is only 18 codons in length and does not code for a single viral protein. Thus, initiation of translation occurs in a non-traditional manner. As shown in Paper I, the structural proteins are not needed for RNA replication; therefore, it should not matter if part of the VP4 protein is missing at the initial stage of the replication cycle. One potential way of obtaining the correct reading frame is a so-called ‘slippery sequence’, where the ribosome moves one or more steps up- or downstream [213]. Another possibility is that translation initiation occurs at another suitable AUG codon. One such AUG codon is present in the genome 180 nt downstream of the classic initiation site. A third option is the use of a different start codon than AUG for initiation of translation. These three alternative mechanisms are based on the assumption that the adaptation of the genome sequence occurs during replication of the RNA. Regardless of what mechanism enabled translation of the genome, a, for us, hitherto unknown mechanism for enteroviral initiation of translation would be utilized. However, the reversal to the correct reading frame might possibly be due to recombination events between nicked or broken RNA-strands [189], thereby restoring the correct reading frame before translation. Although, this mechanism is highly unlikely since it would require the RNA strands to break and in a manner that would specifically eliminate the inserted nucleotides.
Conclusion RNA transcripts with a frameshift mutation 22 nt downstream of the translation initiation site still initiate translation of viral proteins. Furthermore, our data indicates strong evolutionary pressure to conserve the sequence at the beginning of the protein-coding region.
49 Increased efficiency of RNA replication activation (PAPER III)
Cancer is the leading cause of death worldwide and was responsible for 8.2 million deaths in 2012. Of those, 694,000 deaths were caused by colorectal cancer, the forth most common type of cancer [214]. A promising method to treat cancer is oncolytic virotherapy (or viral oncolysis). The treatment is based on viruses that specifically target cancer cells. Both genetically modified and naturally occurring strains from several virus genera are currently at different stages of clinical trials [35, 36, 215]. Two of these are the picornaviruses CVA21 and Seneca valley viruses. Additional picornaviruses may also be candidates. In this project, after having shown lytic capacity in colon cancer cell lines and artificial tumors, echovirus 5 Noyce (E5N) was used as a prototype for evaluating the benefits of the infectious RNA with an authentic 5’end. Lytic ability of E5N in colon cancer cell lines A panel of colon cancer cell lines were transfected with in vitro transcribed RNA derived from an E5N cDNA clone. Out of the six tested cell lines, only one was not lysed by transfection with E5N; the remaining five cell lines exhibited complete CPE within three days of transfection. Furthermore, HT29 spheroids were transfected to investigate whether the virus could replicate and spread in a more tumor-like environment. Like the monolayer cell culture, the spheroids underwent cell lysis and were completely disintegrated six days post transfection. These data, together with previously reported findings of low- or non-pathogenic echo viruses being lytic in colon cancer cell lines [216], suggest that echoviruses might be candidates for oncolytic virotherapy. Oncolytic virotherapy with infectious nucleic acid Recently it was shown in mice with xenograft tumors that administration of infectious nucleic acid was as efficient in causing tumor regression as treatment with virus particles [217]. These are very interesting results given the benefits of using nucleic acid over virions when performing oncolytic virotherapy: i) the use of a homologous virus sample where the sequence of the utilized virus is known; ii) the extracellular immune response of the patient is not in contact with the viral proteins until oncolysis is initiated and the first round of infectious cycles is complete; iii) reduced risks of handling a highly concentrated virus for medical staff during treatment; iv) it is easier and
50 cheaper to manufacture infectious nucleic acid than to propagate and purify viruses to a clinical grade purity. The importance of an authentic 5’end The use of a promotor sequence is necessary for in vitro transcription; leading to additional nucleotides at the 5’end of the genome. The correct sequence is restored during viral replication, but there is a delay in initiation [217, 218]. If a sequence of a cis-acting ‘hammerhead’ ribozyme is placed before the virus sequence, the extra nucleotides are automatically removed when the RNA is transcribed. This was previously shown with a clone containing the genome of a poliovirus [219]. In this study a hammerhead sequence was introduced to investigate whether an authentic 5’ end could increase the replication initiation efficiency for the oncolytic prototype. Transfection of HT29 cells with infections RNA The efficiency of replication initiation in HT29 cells was tested for in vitro transcribed RNA from three different clones: with an authentic 5’ end (rib(+)E5-RNA; +0 extra nt at 5’end), with the T7 promotor sequence (E5N- RNA; +24 extra nt at 5’end) and with the sequences of both the T7 promotor and a non-functional hammerhead ribozyme (rib(-)E5-RNA; +71 extra nt at 5’end). An assay was performed to determine the shortest treatment with the transfection mixture and the lowest amount of RNA that could be used while still obtaining complete CPE within 24 hours. A dilution of the transfection mixture was added to monolayers of HT29 cells at different time points and incubated for 24 hours. After evaluation of the assay, the RNA amount was set to 0.5 µg/well and the treatment time to 30 minutes. Immediately after the transfection mixture was added, cells were frozen to be used for normalization of the virus amount post incubation. After 30 minutes of treatment another set of samples were frozen. The appearance of the remaining cell cultures was documented and cells were regularly frozen until clear signs of CPE were observed. Twenty-two hours post transfection, ‘plaques’ of dead cells, indicative of CPE, had formed in cultures transfected with rib(+)E5-RNA. To a lesser extent, plaques were also visible in the E5N-RNA transfected cells, however no signs of CPE were observed in the cells transfected with rib(-)E5-RNA. A qRT-PCR was performed to measure the production of viral RNA in the cell cultures. After 9 hours of incubation post transfection, the viral RNA levels were 20 times higher in the rib(+)E5-RNA transfection as compared to E5N- RNA. Little or no increase in viral RNA titers from 30 minutes to 9 hours was detected for the rib(-)E5-RNA. At 22 hours after transfection the difference remained, but an increase in newly produced RNA in the rib(-)E5- RNA transfection was detected. Hence, an increased number of extra
51 nucleotides at the 5’ end seems to affect the efficiency of replication initiation negatively, which is in accordance with data produced by van der Werf et al. [218]. However, this was not observed with transcripts from the poliovirus clones [219]. Conclusion E5N can lyse multiple colon cancer cell lines cultured in monolayers as well as tumor-like spheroids of HT29 cells. When transfected into HT29 cell culture the efficiency of replication initiation is 20-fold higher if the genomic RNA has an authentic 5’end.
Towards the propagation of Rhinovirus C in standard cell culture (PAPER IV)
Rhinoviruses can be found globally and have great impact on our society, both medically and economically [67]. There are three species of Rhinovirus, A, B and C. Although the first definition of rhinoviruses was made in 1963 [220], the third species of rhinovirus (RV-C) was not identified until 2007 [221]. RV-C has circulated for many years [222], and the late discovery is very likely due to the difficulty of growing the virus in standard (immortal) cell cultures. The first successful attempts to grow the virus were performed in organ culture [223] and later RV-C was grown in differentiated primary cells [224, 225], both by using virus particles and cDNA-derived in vitro transcribed RNA. Despite this progress, more efficient ways of culturing RV-C are necessary. To learn more about the virus, for vaccine production and testing of antiviral agents, large quantities of virions are needed, and for that to be possible growth in immortal cell culture is necessary [226]. Detection of the RV-C34 type and construction of a cDNA clone The virus used in our studies of RV-C replication is pico58, the prototype strain of RV-C34. It was discovered in a sequence library constructed from sequences obtained from nasopharyngeal aspirates from children with severe respiratory tract infections. The assembled virus was determined to be a new RV-C type. However, when compared to other RV-C genomes, it was observed that approximately 167 nucleotides were missing at the 5’ end of the genome sequence. The cloverleaf structure at the 5’ end of the 5’UTR has a vital role in viral RNA replication and the genomic sequence was therefore appended with 167 nucleotides, corresponding to the missing region, from the (at the time) most similar RV-C genome available in Genbank. A viral cDNA
52 clone was synthesized (Genescript) with the addition of sequences of a T7 RNA polymerase promotor and a cis-acting hammerhead ribozyme. The T7 promotor enabled in vitro transcription of the cDNA clone and the hammerhead ribozyme gave the RNA an authentic viral 5’ end, which has previously been shown to increase the efficiency of RNA replication initiation in picornaviruses (Paper III and [219]). Transfections of immortal cells and attempts to passage pico58 Three different cell lines were used to test the ability of pico58 to replicate in immortal cells; GMK, HeLa and RD cells. Cells were transfected with in vitro transcribed pico58 RNA and incubated until complete CPE or a maximum of 6 days. The transfection of RD cells was very efficient and complete CPE was visible within 24 hours. CPE was visible in the remaining two cell lines as well, but to a much lesser extent. After 6 days of incubation they had still not reached complete CPE. Because of the rapid onset of CPE in the transfected RD cells, most of the subsequent efforts to passage the virus were performed with RD cells. In the attempts to passage pico58, a variety of parameters were changed; from incubation time and temperature, to the amount of supernatant added and whether or not the supernatant was removed and the cells washed before incubation. In spite of our efforts, no CPE was visible and no replication could be detected either with PCR or qRT-PCR. Viral translation and RNA replication To further investigate the transfected cells, immunofluorescence staining was carried out using the monoclonal antibody J2, specific to double-stranded RNA (dsRNA), as the primary antibody. To stain the nucleus and obtain a double staining of the dsRNA, a mounting medium containing propidium iodide was used. The presence of dsRNA was observed in all three transfected cell lines, indicating that viral RNA replication occurred. This in turn demonstrates the occurrence of translation, since the viral RNA replication is dependent on both host and viral proteins. Conclusion Transfection of cells with in vitro transcribed pico58 RNA caused CPE and the viral genome was both translated and replicated. While this does not conclusively prove formation of virions, these results make it highly likely. Hence, the most probable explanation for the lack of successful propagation of the virus is in the attachment and entry stage of the viral life cycle. The immortal cell lines used might lack appropriate receptors, co-factors and/or endosomal mechanisms that are present in the primary cell lines where propagation is possible.
53 Effects of viral replication on the host cell (Paper V)
Most enteroviruses are easily grown in cell culture and generally the newly formed virions leave the cell through CPE. However, this is not always the case and several viruses have been observed to cause persistent infections, where the virions escape the cells without killing them [91]. The mechanisms for viral exit are poorly understood and we aimed to discover to what extent virions were continuously released during infection. Moreover, we sought to investigate differences between lytic and non-lytic virus infections in this regard. Experimental set-up Next generation sequencing (NGS) was used to determine differences in gene expression of infected cells. This study was a continuation of a previous project in which reverse genetics was used to determine that a single surface- exposed amino acid mutation in the VP1 protein (Q164K) caused the CVB2 prototype Ohio (CVB2O) to change from a non-cytolytic to a cytolytic infection in RD cells. This alteration did not, however, change the efficiency of virus generation [227]. The current study was carried out using RNA extractions from cells infected with the non-cytolytic (CVB2Owt) virus, and the cytolytic variant (vVP1Q164K) as well as on mock-infected cells, that is, with no added virions. Triplicates were collected from cells infected with vVP1Q164K at 24 and 48 hours and from CVB2Owt and mock at 48 hours, producing a total of 12 samples. The obtained sequences were annotated through comparison with the human and CVB2O genomes. Differences in intracellular viral load Approximately 35,000 expressed genes were found and annotated in the analyzed samples. In addition to information on specific genes, the annotation of genes when compared to the human and CVB2O genomes also provided an estimate of the relative number of expressed human genes and viral genomes. In the cells incubated with vVP1Q164K for 24 hours, 13.4 % of the RNA was of viral origin, while for the cells incubated for 48 hours that number had risen to 17.0 %, indicating a build-up of viruses inside the cells. In contrast, only 1.3 % of the RNA in the CVB2Owt-infected cells was viral at 48 hours post infection. Since the efficiency of the infection and amount of produced virions were comparable, the difference in intra-cellular viral RNA clearly indicates that CVB2Owt is efficiently released from the cell during infection, while the variant vVP1Q164K is not.
54 Differentially expressed genes When comparing the vVP1Q164K infections at the two time points, almost 1000 genes were found to be expressed at significantly different levels. The two vVP1Q164K samples showed similar results when compared to the other samples, although with a lower number of differentially expressed genes in the sample incubated for 24 hours. Roughly 4000 genes were differentially expressed when comparing vVP1Q164K to either CVB2Owt or mock, a sharp contrast to the approximately 400 differences found in gene expression between CVB2Owt and mock. The vVP1Q164K variant clearly has a much greater influence on gene expression in the host cell than the prototype CVB2Owt. Annotated genes All differentially expressed genes were submitted to the PANTHER database to obtain the putative gene functions. The genes were found to correspond well with the human genome as a whole, consequently [228, 229], no particular gene function stood out as being of additional interest. The 20 most differentially expressed genes, in the comparison of the vVP1Q164K-infected cells and the CVB2Owt-infected cells, comprised 12 protein-coding genes, 3 pseudogenes and 8 non-coding RNA-genes. Furthermore, the three most differentially expressed genes over all were also protein-coding. Conclusion To the best of our knowledge, this is the first time that the gene expression of cells, infected with a cytolytic and a non-cytolytic variant of the same virus, have been studied with NGS. A single amino acid change causes the CVB2O variant vVP1Q164K to have a cytolytic infection cycle. Virions were found to be efficiently released from CVB2Owt-infected cells, but not from cells infected with vVP1Q164K, where the newly formed virions build up inside the cell. Additionally, the mutation dramatically altered the effect on the gene expression of the host cell. The lytic variant had an almost 10 times larger impact on gene expression of the host cell than the prototype strain CVB2Owt, affecting a wide range of functions. Further research is required to identify the specific mechanism that enables CVB2Owt to be released from the infected cells.
55 CONCLUDING REMARKS
The projects presented in this thesis rely on reverse genetics, whereby viral genomes have been altered in order to study specific aspects of the enteroviral replication cycle. In Paper I, limitations of recombination and the independence of the non-structural part of the genome were investigated. The adaptability of two different viruses was tested in Papers II and IV. In Paper III, the efficiency of virus activation after transfection with in vitro transcribed RNA with an authentic 5’end was examined, as was the lytic effect of E5N on colon cancer cell lines. In Paper V, the impact on cells infected with lytic and non-lytic variants of CVB2O was investigated through NGS. The major findings of these five papers are summarized below.
I. The replicative backbone of CVB5D is able to produce viral proteins and RNA without the structural region, and to replicate with the structural proteins of other members of the EV-B species.
II. The E30B chimera virus is able to overcome a frameshift mutation at the beginning of the protein-coding region, and thereby rapidly restore efficient replication.
III. E5N is able to lyse five out of six colon cancer cell lines as well as HT29 spheroids. Furthermore, the efficiency of viral activation after transfection is greatly improved with an authentic 5’end of the in vitro transcribed RNA.
IV. No signs of replication were detected in the attempts to passage the virus. However, the genome of pico58 was translated and the RNA replicated after transfection.
V. Newly formed virions are built up inside cells infected with the lytic variant, while the non-lytic virions are efficiently released. The cytolytic variant has an almost 10 times larger impact on gene expression of the host cell than the non-cytolytic virus, affecting a wide range of functions.
56 Future perspectives The cassette vector used in Paper I to study the compatibility of the structural and non-structural genomic regions of types from the same species could in the future be used to study restrictions in recombination of genomic regions from different species [207]. Since the replicative backbone is able to initiate viral translation and RNA replication, the restriction should either lie in the assembly of the capsid or in the encapsidation of the RNA genome. The next natural step after Paper II is to determine by which means the virus reverted to the original sequence. By further reverse genetic studies, the suggested alternative means of translation can be confirmed or disproved. The use of oncolytic virotherapy will hopefully be a widely used and effective cancer treatment, thus our findings in Paper III showing increased efficiency of viral activation with an authentic 5’end may contribute to making those treatments safer and more efficient. A potential next step is to test the system in colon cancer xenografts. In order to study a number of different aspects of viruses, a system where large quantities of the virus can be grown is necessary. This requires propagation of viruses in standard cell lines. With our results in Paper IV, the restriction of RV-C replication in cell lines has been clarified. Most likely, the reason for the inability of RV-C to complete replication cycles in cell lines lies in the attachment or entry stages. Future studies should focus on differences in potential receptors expressed in primary cells, where replication is possible, which are not present in standard cell lines. NGS is a powerful new tool that can be used for comparison of gene expression in primary cells, which support replication, and standard cell lines. The solution might also be obtained through pull-down assays [230] with the viral capsid proteins and membrane proteins of the primary cells. With NGS, vast amounts of data are obtained and a study such as the one presented in Paper V can be seen as a puzzle where the first pieces have been put together. To complete the puzzle, further studies are needed. Such data has the potential to reveal how CVB2Owt is able to escape from the host cell without destroying it. By further exploring the differences in expression and by modifications of the gene expression, the answer will hopefully be found. A complementary approach could be to express the VP1 protein of the two CVB2O variants and perform a pull-down assay [230] with RD cell lysate. As a whole, the projects included in this thesis have contributed to a deeper understanding of different stages of enteroviral replication. They have provided several promising new avenues of research and the findings will hopefully be useful in further investigations into theses viruses as well as in development of vaccines, anti-viral drugs and oncolytic virotherapies.
57 ACKNOWLEDGEMENTS
I would like to start by thanking my supervisor Michael Lindberg for giving me the opportunity to study viruses. You have always been generous with your time and expertise and it has been clear that you have always wanted the best for me.
Next I would like to thank my assistant supervisors Björn Andersson, Jonas Waldenström and Peter Gierow as well as my examiner Sven Tågerud for their support.
Thanks to present and past members of the group. Thanks to Kjell Edman for always making time to help me with my problems. Thanks to Nina for taking the time to help me with everything, from showing me the ropes in the lab to brainstorming. Thanks to Stina and Maria for your ideas and helpful suggestions. Thank you Conny for not hesitating to help me when I asked for it.
Thanks to Anki who has been an excellent office mate and friend. Thank you for inviting me to dinner the first week I was here and didn’t know anybody in town. Thanks to Kim for showing me around in Kalmar so that I always knew where to go and thanks for being one of my first friends here.
Thanks to Camilla, Dieter, Elina, Elinor, Gustaf, Lasse, Louise, Malin, Per and Stina for showing me that it will probably work out in the end, no matter how stressed you are about your thesis and dissertation. And thank you for all the laughs and the wonderful atmosphere you contribute to along with Anna, Berit, Björn, Jenny, Jesper, Johanna, John, Karin, Kerstin, Lilita, Stefan, Thomas, Ulrika and all the other past and present Norrgård people. I would like to say extra thanks to Johanna for the “2014 will be better” chocolate - it warmed my heart.
58 A place like Norrgård could not work without the help of the cleaning ladies, lab service and the technicians. You made every day easier.
I would also like to thank ‘the harbor people’: Alexis, Andreas, Carina, Felix, Jo, Michelle, Mireia, Sandra and Stephan. You have been awesome! Friday beers and our little gatherings have made my time in Kalmar so much better. Extra thanks goes to Michelle for inviting me into this little group and to Jo for reading my thesis.
Thanks to my family; to västgötar, skåningar, sävnebor and lyrenäsor, your support has been invaluable.
Thanks to my outside-of-work friends Anders, Anna, Annika, Cissi, Jenny, Jessi, Linda and Sabina.
Thank to Erik and Sara in particular and the Andersson clan in general for welcoming me into their family; you have been so very kind to me.
I am grateful to my mother whom I will miss till the day I die. She was the best woman I knew, the most wonderful mother imaginable and a great source of inspiration. Thanks to my father, who all my life, have loved me, cared, and sometimes worried about me. Thanks for teaching me to value things and for supporting me in my aspirations. Thanks to my brother Erik who has always been a step ahead of me, showing me the way.
Håkan, thank you for reading this thesis and all my papers. Even though you didn’t always understand, you always gave me good suggestions. Thank you for making me dinner and cheering me on when things have been too hectic to think. We have been through some rough times together and I don’t think that I would have made it without you. Thank you for all your love and for being my home base. I love you.
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