Structural divergence creates new functional features in alphavirus genomes

Alphaviruses are mosquito-borne pathogens that cause human diseases ranging from debilitating arthritis to lethal encephalitis. Studies with Sindbis virus (SINV), which causes fever, rash, and arthralgia in humans, and Venezuelan equine encephalitis virus (VEEV), which causes encephalitis, have identified RNA structural elements that play key roles in replication and pathogenesis. However, a complete genomic structural profile has not been established for these viruses. We used the structural probing technique SHAPE-MaP to identify structured elements within the SINV and VEEV genomes. Our SHAPE-directed structural models recapitulate known RNA structures, while also identifying novel structural elements, including a new functional element in the nsP1 region of SINV whose disruption causes a defect in infectivity. Although RNA structural elements are important for multiple aspects of alphavirus biology, we found the majority of RNA structures were not conserved between SINV and VEEV. Our data suggest that alphavirus RNA genomes are highly divergent structurally despite similar genomic architecture and sequence conservation; still, RNA structural elements are critical to the viral life cycle. These findings reframe traditional assumptions about RNA structure and evolution: rather than structures being conserved, alphaviruses frequently evolve new structures that may shape interactions with host immune systems or co-evolve with viral proteins

Uudsed vaated alfaviiruste poolt kodeeritud nsP2 valgu funktsioonide kohta

Väitekirja elektrooniline versioon ei sisalda publikatsiooneAlfaviirused (sugukond Togaviridae) on sfäärilise ümbrisega virionide ja positiivse polaarsusega RNA genoomiga viirused, mis enamasti levivad lülijalgsete vektorite vahendusel. Paljud alfaviirused on olulised inimeste patogeenid, sealhulgas ulatuslikke puhanguid põhjustav Chikungunya viirus (CHIKV), Austraalias leviv Ross River viirus (RRV), Ameerikas leviv ida hobuste entsefaliidi viirus (EEEV) ja ka Eestis leiduv Sindbis viirus (SINV). Alfaviiruste põhjustatud haigused sõltuvad viiruse liigist ja selle päritolust,kus üldistatult põhjustavad Vana Maailma alfaviirused palaviku, löövet ja artriiti, samal ajal kui Uue Maailma alfaviirused on sageli kõrge patogeensusega põhjustades neuroloogilisi haiguseid, sealhulgas entsefaliiti. Alfaviiruste RNA genoom kodeerib nelja viiruse RNA replikaasi allühikut, mis toodetakse liitvalgust eellase (polüproteiini) kujul. Liitvalk lõigatakse neljaks valmis replikaasi valguks viiruse nsP2 proteaasi abil. Viimastel aastakümnetel läbiviidud uurimistööd on võimaldanud välja selgitada viiruse replikaasi kompleksi kui ka selle komponentide peamised funktsioonid ning meie arusaamine viiruse valkude struktuuridest on oluliselt täienenud. Ka need uurimised on näidanud, et alfaviiruse infektsiooni üks esimesi ja kõige olulisemaid sündmusi on replikaasi valkude eellase lõikamine valmis valkudeks. Eelvalgu lõikamised toimuvad kindlas järjekorras ja onkindlalt ajastatud, mis määrab ära selle, kas vabanevad valgud moodustavad toimiva RNA replikaasi või mitte. Seega kujutab eelvalgu lõikamise protsessi läbi viiv nsP2 endast RNA replikatsiooni käivitamise ja efektiivsuse regulaatorit. Samas on tegemist multifunktsionaalse valguga, mis on lisaks RNA sünteesi reguleerimisele oluline nii raku viirusvastaste kaitsereaktsioonide aktiveerimisel kui ka nende mahasurumiseks. Sellest tulenevat mõjutavad muudatused nsP2 valgus, sealhulgas punktmutatsioonid valgu funktsionaalselt olulistes regioonides, oluliselt viiruse bioloogilisi omadusi. Seejuures ei ole mitmete nsP2 aktiivsuste taga seisvad molekulaarsed mehhanismid lõpuni selged. Küll aga on selgunud, et nsP2 funktsioone mõjutavad interaktsioonid alfaviiruse ülejäänute replikaasi valkude nsP1, nsP3 ja nsP4-ga. Käesoleva uurimistöö raames selgitati välja ja analüüsiti mitmeid nsP2 valgu ja RNA replikaasi seni tundmatuid funktsioone. Töö peamised tulemused saab kokku võtta järgnevalt: 1. alfaviiruste genoomide alusel konstrueeritud trans-replikatsiooni süsteemid võimaldavad uurida nsP2 valgu funktsioone, mis on seotud viiruse RNA replikaasi kompleksi moodustamisega, aga ka selle moodustamise blokeerimise ja/või selle aktiivsuse mahasurumisega. Viimati nimetatud aktiivsused on olulised viiruste superinfektsiooni blokeerimisel ja tõenäoliselt ka viiruse replikatsiooni ajalisel reguleerimisel. CHIKV ja SINV trans-replikatsiooni süsteeme kasutades tehti kindlaks, et superinfektsiooni blokeerimise puhul on võtmesündmuseks ühe viiruse replikaasi eellase lõikamine teise viiruse nsP2 poolt ja et seda mõjutavad mutatsioonid nii sihtmärk-liitvalgus kui ka nsP2 valgus; 2. leiti, et individuaalse nsP2 süntees sääse (vektorputuka) rakkudes surub maha alfaviiruse replikatsiooni aktiivsust. Seda nähtust ei põhjusta ainult nsP2 proteaasne aktiivsus – olulised on ka nsP2 proteaassest aktiivsusest sõltumatud mehhanismid. See, milline on konkreetsete mehhanismide osakaal, sõltub nii viiruse RNA replikaasi kui ka selle moodustamist inhibeeriva nsP2 valgu päritolust. Homoloogse viiruse replikatsiooni mahasurumisel domineerib nsP2 proteaassest aktiivsustest sõltuv mehhanism, kus nsP2 poolt teostatud replikaasi eelvalgu lõikamine 2/3 saidist välistab funktsionaalse RNA replikaasi moodustumise. Mõne alfaviiruse (SINV) puhul on nsP2 proteaassest aktiivsusest sõltuv mehhanism ka peamiseks mehhanismiks millega surutakse alla heteroloogsete alfaviiruste replikaaside moodustamist; 3. nii SINV kui ka CHIKV nsP2 valk takistab heteroloogsete alfaviiruste RNA replikatsiooni ka proteaassest aktiivsusest sõltumatute mehhanismide abil, kus CHIKV nsP2 puhul on need peamisteks meetoditeks heteroloogsete alfaviiruste replikatsiooni mahasurumisel. Selle inhibeerimise täpne põhjus on hetkel teadmata, küll aga näitasime, et erinevad mutatsioonid nsP2 valgus mõjutavad taolise inhibeerimise efektiivsust. Need andmed viitavad sellele, et proteaassest aktiivsusest sõltumatu replikatsiooni inhibeerimine saavutatakse tõenäoliselt mitmete paralleelsete mehhanismide abil; 4. väga patogeensele ja seetõttu vähe uuritud EEEV-le konstrueeritud trans-replikastiooni süsteem osutus kõrgelt efektiivseks nii inimese kui ka sääse rakkudes. Samuti osutus võimalikuks EEEV replikaasi eelvalku (P1234) kodeeriva järjestuse jagamine kaheks (P123 + nsP4) ja kolmeks (nsP1+P23+nsP4) komponendiks. See võimaldas uurida kas ja kuidas replikaasi komponentide vahekord mõjutab EEEV RNA replikaasi aktiivsust. Läbiviidud katsed näitasid, et erinevalt seni uuritud alfaviirustest sõltub EEEV RNA replikaasi aktiivsus teda moodustavate komponentide optimaalsest vahekorrast ja et RNA replikaasi katalüütilise allühiku (nsP4) ülehulk vähendab EEEV RNA replikaasi aktiivsust. Selline aktiivsuse langus tuleneb sellest, et väheneb nii rakkude hulk milles RNA replikatsioon aktiveeritakse, kuid ka RNA replikatsiooni aktiivsus nendes rakkudes. Hetkel pole veel selge kas tegemist on EEEV RNA replikaasi unikaalse omadusega või leidub ka teisi sarnaste omadustega alfaviiruste replikaase; 5. uuriti RRV loodusliku isolaadi RRV 2528 omadusi. Leiti, et see viiruse isolaat põhjustab väga tugevat tüüp-I interferoonide vastust. Viiruse genoomi analüüs tõi välja, et RRV 2528 erineb teistest vähemal määral interferoonide tootmist indutseerivatest RRV isolaatidest mitmete mutatsioonide poolest, millest paljud paiknevad just nsP2 valgus Näitasime, et need mutatsioonid mõjutavad RRV nakkuse käigus toimuvat interferoonide tootmist, kuid ei mõjuta viiruse replikaasi eelvalgu protsessimist valmis valkudeks, viiruse võimet suruda maha raku valgusünteesi ega viiruse struktuurvalkude sünteesi. Seega ei tulene suurenenud tüüp-I interferoonide vastus defektist RRV 2528 RNA replikatsioonikomplekside moodustamisel või võimetusest maha suruda raku üldist geeniekspressiooni.Over the last decades, the intensive studies of factors/activities responsible for multiple aspects of alphavirus infection have been performed. In particular, our understanding about structures and functions of viral RNA replicase and its components has significantly increased. Novel findings emphasize that one of the first and most essential event in alphavirus infection is processing of the ns polyprotein carried out by its nsP2 region and an individual nsP2; this process not only ensures the release of functional replicase subunits but also determines would these proteins form the active RCs or not. Thus, it is increasingly evident that nsP2 is one of the “main driving forces” of successful RNA replication. Furthermore, due to its versatile functions and various activities nsP2 is involved in other aspects of infection. It is one of the key determinants associated with activation as well as counteracting of antiviral response in infected cells. Therefore, different modifications of the protein, including point mutations, often have drastic impact on alphavirus infection. However, much of the precise mechanisms of P2 action remain enigmatic. What is clear is that nsP2 does not act alone, its activities are modulated by other components of viral replicase. The current study allowed us to identify and confirm new functions and properties of nsP2 and alphavirus RNA replicase. The general conclusions of this study can be presented as follows: 1. Alphavirus trans-replicase systems can be applied as a tool for studies of functions of nsP2 associated with inhibition of RC formation/activity. These functions are essential for SIE and are likely related to these used to regulate RNA replication in the alphavirus infected cells. Using trans-replicase systems of CHIKV and SINV, it was found that the key event in SIE is targeting of replicase precursor (P1234) by an individual nsP2 protein and that this ability of nsP2 can be altered by mutations present in its functionally important regions. 2. It was found that synthesis of an individual (free) nsP2 in mosquito cells has an inhibitory effect on the alphavirus RC formation/functionality. This is not, however, a result of a single mechanism but results from combination of nsP2 protease-activity dependent mechanism and protease-activity independent mechanisms. The level and dominant mode of inhibition of alphavirus RNA replication depends form the virus, source of free nsP2 and substitutions present in this protein. The protease-activity mediated mechanism is important for suppression of replication of matching virus and relies mostly on ability of nsP2 to cleave 2/3 site in ns polyprotein. For some viruses such as SINV it is also dominant mechanism used to suppress formation of RNA replicases of heterologous alphaviruses. 3. nsP2 of SINV and CHIKV can inhibit formation/activity of RNA replicase of heterologous alphavirus using protease-activity independent mechanisms; for nsP2 of CHIKV this is the dominant mechanism to suppress activity of RNA replicases of heterologous alphaviruses. The precise details of protease-activity independent mode of action of nsP2 remain unknown; however, it is clear that this property can be enhanced by introduction of certain mutations into nsP2. It is likely that the protease-activity independent inhibitory effect originates not from a single mechanism but from several mechanisms. 4. Trans-replicase of highly pathogenic EEEV was found to be highly active in human and mosquito cells. Splitting of construct of EEEV P1234 expression into two (P123 and nsP4) or three (nsP1, P23 and nsP4) expression construct allowed analysis of requirements of active RC formation. It was found that activity of EEEV RNA replicase depends from correct ratio of P123 (or nsP1+P23) component to nsP4 component and that in contrast to previously studied alphaviruses an excess of nsP4 reduced activity of EEEV RNA replicase. The reduction was due to the decrease of a number of cells were RNA replication was initiated as well as to the reduced RNA replicase activity in such cells. It remains unclear, is this property unique for EEEV RNA replicase. 5. Natural isolate RRV 2528 was found to be a prominent inducer of type-I IFN expression. This property was associated with specific amino acid substitutions in the nsP2 encoded by this isolate. None of these substitutions or their combination affected ability of RRV to induce shutdown of cellular protein synthesis or level of viral structural proteins expression. Similarly, no effect on the processing of P1234 was detected. Combined, these findings indicate that the excessive type-I IFN induction was not due to the lack of the ability to induce general shutdown of cellular gene expression or due to the defects in RC formation.https://www.ester.ee/record=b553517

The role of RNA structure in Chikungunya virus early replication events

Chikungunya virus (CHIKV) is a pathogenic, single-stranded, positive-sense RNA virus transmitted to humans by Aedes spp. mosquitoes. After decades of low-level endemic circulation, CHIKV has re-emerged to establish local transmission on five continents, infecting upwards of 3,000,000 people. There are no currently available vaccines or direct-acting anti-viral therapeutic agents. A greater understanding of the CHIKV replication cycle is essential, as much of what is known about the replication cycle is assumed from studies of related but divergent viruses, which have provided conflicting reports. Preliminary work carried out by A. Tuplin (University of Leeds) suggested a highly ordered structured region at the 5′ end of the CHIKV genome, spanning ~300 nt including the 5′ untranslated region (UTR) and the 5′ coding sequence of nsp1. The aim of this project was to determine the phenotypic importance of secondary structure in this region for the CHIKV lifecycle in human and mosquito cells at multiple stages of viral replication and to investigate the sequence and structure requirements for functional interactions. This study represents the first investigation of functional elements within the 5′ UTR and adjacent nsP1-coding region in CHIKV. Taking a structure-led reverse genetic approach, in both infectious virus and sub-genomic replicon systems, the wild-type secondary structure of the 5′ 300 nt of the CHIKV genome was found to be essential for genome replication in human- and Ae. albopictus-derived cells. Six RNA stem-loops were determined to individually enhance CHIKV genome replication - including novel structures analysed for the first time in this study. Comparative analysis in human and mosquito-derived cell lines revealed that the novel stem-loop SL47 in the 5′ UTR functions in a host-independent manner while stem-loops in nsp1 function in a host-dependent manner. Stem-loops were demonstrated to function within the positive-strand genomic RNA, via predominantly structure-dependent mechanisms. Furthermore, single-host passaging studies suggested strong selection pressure to regenerate secondary structures and highlighted potential differences in translational recoding between host species. Finally, the potential for tertiary structure formation was explored. In addition to furthering knowledge of fundamental aspects of the molecular virology of this important human pathogen, this study will inform rational design of a genetically stable attenuated vaccine

USING SHAPE-MaP TO IDENTIFY FUNCTIONAL RNA SECONDARY STRUCTURES IN RNA VIRUSES

Alphaviruses are a genus of arboviruses often transmitted by mosquitos. Alphaviruses are responsible for multiple outbreaks over the last two decades and continue to pose a serious threat to human health. The 2013 outbreak of chikungunya virus (CHIKV), the most notable alphavirus, caused over one million infections. Despite the frequency with which these viruses re-emerge, there are no effective therapies or vaccines against alphaviruses. Like other RNA viruses, alphavirus genomes contain functionally important RNA secondary structures that contribute to immune evasion, RNA transcription, RNA translation, and virion assembly. However, very little of alphavirus genomes have been characterized due to a previous inability to accurately and quickly model long RNAs. This work used the RNA structure probing technique SHAPE-MaP to produce experimentally informed RNA secondary structure models of multiple RNA virus genomes. We probed genomes of closely related alphaviruses to identify conserved structured and unstructured regions. We found that alphaviruses are structurally unique and most conserved structured regions fold into distinct RNA secondary structures. After we identified a novel functionally important RNA secondary structure specific to Sindbis virus, we revised our approach to identify regions within each virus likely to fold into a specific conformation. We identified 23 regions of the CHIKV genome that were specifically structured. The four previously known RNA secondary structures were included in the 23 regions identified, validating the approach. Further, we demonstrated that one of the uncharacterized structured regions enhanced virus replication. Lastly, we demonstrated our approach to structure identification and testing was applicable to RNA viruses beyond the alphavirus genus using Zika virus, a flavivirus responsible for a large outbreak in 2015. For each of our studies we used silent structure disrupting mutations to assess RNA structure without affecting protein coding sequence, so structures could be assessed in the context of infection. These findings improve our understanding of known pathogenic RNA viruses and provide an approach to quickly study and assess future emerging RNA viruses. A more comprehensive knowledge of functionally important RNA structures in viruses could be used to design safer live attenuated vaccines or develop new RNA-binding small molecule therapies.Doctor of Philosoph

Venezuelan equine encephalitis virus nonstructural protein 2 in the host cell

Venezuelan equine encephalitis virus (VEE) is a naturally emerging disease threat and a previously developed biological weapon, making it an important pathogen of the Alphavirus family. Unfortunately, little is known about how the viral proteins interact with the host cell at the molecular level. The viral nonstructural protein 2 (nsP2), a required member of the alphavirus replication complex, has undefined activities that are critical in determining the outcome of alphavirus infection. To gain insight into auxiliary functions of nsP2, cellular proteins in the mammalian host cell with which nsP2 interacted were examined. In VEE and other alphaviruses, nsP2 is associated with ribosomal protein S6 (RpS6), an essential component of the 40S ribosomal subunit. The interaction of RpS6 with nsP2 occurred throughout the course of infection and did not require the presence of the other viral proteins. Moreover, reducing the cellular level of RpS6 protein caused diminished expression from alphavirus subgenomic messages, whereas it did not dramatically alter cellular translation. The subcellular localization of VEE nsP2 was examined in order to gain insight into its activity in the mammalian host cell. VEE nsP2 was found both in the cytoplasm and the nucleus of mammalian cells during infection and also when expressed in the absence of other viral proteins. The C-terminal third of VEE nsP2 can localize EGFP exclusively to the nucleus of mammalian cells, and contains the sequence PGKMV, which is homologous to the nuclear localization signal of Semliki Forest virus nsP2. Mutation in this sequence diminished, but did not eliminate, localization to the nucleus, suggesting that this sequence contributes to the nuclear localization of VEE nsP2. Furthermore, VEE nsP2 specifically interacted with the nuclear import protein karyopherin-α 1, suggesting that during infection nsP2 is transported to the nucleus by karyopherin-α 1. Additionally, a novel leucine-rich nuclear export signal was identified in VEE nsP2, and nuclear export mediated by this signal occurred via the CRM1 pathway. Taken together, these results establish that nsP2 interacts with essential cellular components and is actively directed to multiple subcellular compartments, suggesting that nsP2 mediates a critical interplay of the virus with the host cell

Defining the importance of the hnRNP I interaction to the Sindbis virus subgenomic viral RNA using an innovative tethering approach.

Old World alphaviruses cause significant outbreaks of illness and debilitating multi-joint arthritis for prolonged periods. Currently, there are no FDA approved vaccines or antiviral therapies; and thus, there is a critical need to identify and characterize the molecular biology of alphaviruses. Alphaviruses rely on the host cell machinery to complete the viral lifecycle and are dependent on interactions with host RNA binding proteins. Accordingly, several host heterogenous nuclear ribonucleoprotein proteins (hnRNPs) have been found to bind to the Sindbis virus (SINV) RNAs. Disrupting the interaction sites in the viral RNAs of these RNA:Protein interactions results in decreased viral titers in tissue culture models of infection. Nonetheless, whether the observed phenotypes were due to loss of hnRNP binding, or the incorporation of polymorphisms into the primary nucleotide sequence of SINV remained unknown. To determine if the loss of hnRNP binding was the primary cause of attenuation, or if the disruption of the RNA sequence itself was responsible for the observed phenotypes, we utilized an innovative protein tethering approach to vi restore the binding of a candidate hnRNP protein in the absence of the native interaction site. Specifically, we reconstituted the hnRNP I interaction with the viral RNA by replacing the native interaction site with the 20nt Bovine Immunodeficiency virus Transactivation RNA Response element (BIV-TAR). Importantly, the BIV-TAR element will bind with high specificity to proteins tagged with a TAT peptide. Reestablishment of the hnRNP I:vRNA interaction via the BIV-TAR / TAT tethering approach restored the phenotype to wild-type like levels. As the reconstitution of the hnRNP I interaction in the absence of the native interaction site repaired the mutant phenotype we can conclude that hnRNP I binding, and not primary sequence, is responsible for the observed mutant phenotype following the loss of the native interaction site. Further examinations of the mutant phenotype revealed that the increased structural protein expression observed following the loss of hnRNP I binding led to an apparent overwhelming of the host glycosylation machinery which in turn caused poor viral particle function as manifested by decreased specific infectivity

Alfaviiruse nsP2 valk biokeemilisest vaatekohast: lugu mitmedomäänse valgu ensümaatilisest analüüsist

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Chikungunya viirus (CHIKV) on suure meditsiinilise tähtsusega viirus mis kuulub alfaviiruste perekonda sugukonnas Togaviridae. Pregusel ajal ei ole CHIKV vastast vaktsiini ega spetsiifilist ravi. CHIKV paljunemine nakatatud rakkudes sõltub tema RNA genoomilt sünteesitavatest replikaasi valkudest. NsP2 on nelja replikaasi valgu seas suurim ja tal on palju teadaolevaid või eeldatavaid aktiivsuseid. Käesolevas töös näidati eksperimentaalselt, et CHIKV nsP2 omab kaheahelalist RNAd lahtiharutavat aktiivsust ning võib läbi viia ka vastupidist protsessi – soodustada kaheahelalise RNA moodustamist. Mõlemad need funktsioonid on olemas ainult täispikal nsP2 valgul. Peale selle näidati, et nsP2 omab ka NTPaset ja proteaaset aktiivsust. Kuna ka need aktiivsused on kõige tugevamad täispikal valgul siis saab saadud tulemustest järeldada, et CHIKV nsP2 toimib kui üks tervik: valgu erinevad osad seonduvad omavahel ja mõjutavad vastastikku üksteise aktiivsusi. Lisaks sellele leiti, et nsP2 aktiivsuseid mõjutavad ka mutatsioonid, mis on seotud mitte-tsütotoksilise fenotüübiga st. viiruse võimetusega maha suruda raku biosünteese ja põhjustada raku surma. Siiski ei saa kogutud andmeist teha järeldust milline või millised defektid nsP2 funktsioonides seda fenotüüpi põhjustavad. Lisaks nsP2 funktsionaalsele analüüsile viidi läbi ka muude CHIKV replikaasi valkude ekspresserimine ja puhastamine. Saadud kõrge kvaliteediga valke kasutati efektiivsete polüklonaalsete antiseerumite saamiseks. Nüüdseks on need töövahedid kasutusel paljudes laborites üle maailma ja on võimaldanud välja selgitada palju uusi fakte CHIKV (ja alfaviiruste üldse) molekulaarbioloogia kohta.Chikungunya virus (CHIKV, genus Alphavirus, family Togaviridae) has a positive sense RNA genome with length approximately 12 kb. It codes for four nonstructural (ns) proteins designated as nsP1, nsP2, nsP3 and nsP4 and for five or six structural proteins. Ns-proteins are involved in replication of virus RNA, in addition they also have functions unrelated to RNA replication. Out of all the nsPs, nsP2 plays a pivotal role towards regulation of CHIKV RNA replication. The protein was known to have NTPase, RTPase and protease activity and its N-terminal region was presumed to have helicase activity. Out of the enzymatic activities of nsP2 the helicase related functionalities were most insufficiently studied. Further, it was not known why two different and seemingly disconnected functional entities such as protease and helicase are present on a single polypeptide and what could be the importance of N terminal most part of nsP2 on the helicase activity. The bioinformatical, biochemical and biophysical approaches were employed to characterize the helicase related activities and to reveal the apparent minimal requirements for these activities. The bioinformatics platform suggests that the 3D-structure of the first 470 aa of nsP2 resembles the fold pattern of ToMV helicase which is a superfamily 1 of helicase. In particular, this fragment was predicted to consist from three domains. From these the extreme N terminal domain appears to be disordered while the other two domains possess RecA-like fold which is commonly found in NTPases. The biochemical analysis, carried out with purified full length and manipulated versions of nsP2, revealed that the C-terminal part of nsP2, which was known to have protease activity, is also essential for RNA helicase activity. Thus, the presence of protease region in nsP2 is clearly not accidental and these different functional domains are co-evolved to accomplish more significant tasks. The use of biophysical method (CD spectroscopy) confirmed that secondary structures of wt and manipulated versions of nsP2 are comparable; this indicates that functional defects detected in various enzymatic activities did not result from misfolding of mutant proteins. This also applies to forms of nsP2 which were engineered to contain mutations associated with noncytotoxic (NCT) phenotype of CHIKV replicons. It was found, all analyzed nsP2 enzymatic activities (protease, NTPase and helicase activities) were invariably affected by the NCT related mutations. In general, however, there was no significant correlation observed between extent of enzymatic defect(s) of nsP2 and phenotype of corresponding replicon. Thus, the development of NCT phenotype is apparently more complicated and could involve a number of underneath viral replication related functionalities. Finally, a number of ns-proteins from different alphaviruses were expressed, purified to raise polyclonal sera. These represent tools for detection of viral proteins using different immunological, such as western blot and immunofluorescence, methods. Similarly, the standardized enzymatic assays of nsP2 represent platform for screening and analysis of potential inhibitors of CHIKV infection. Taken together, these works elevated general understanding of nsP2 from a biochemical perspective and provided useful tools for studies aiming to understand molecular biology of alphaviruses

Characterisation of the response of Aedes mosquito cells to Semliki Forest virus infection

Arboviruses are transmitted to vertebrates by arthropod vectors such as mosquitoes or ticks. The replication of Semliki Forest virus (SFV) (Togaviridae; Alphavirus) in vertebrate cells is well established and triggers cell death. SFV infection of Aedes albopictus mosquito cells was characterised. Virus growth curves were compared in three cell lines. Infection of U4.4 cells was persistent and did not affect growth of the culture. In contrast, infection of C6/36 and C7-10 cells resulted in a static culture with no cell division and no cell death. The response of U4.4 cells was characterised in greater detail using viruses containing fluorescent or luciferase markers within the replicase or structural open reading frame of the virus genome. Activation of the STAT/IMD pathway prior to SFV infection significantly reduced virus driven luciferase expression and virus production. Activation of the Toll pathway prior to SFV infection had no effect. However, activation of Toll in addition to STAT/IMD had a cumulative effect on luciferase expression and virus production. viRNAs were characterised by Illumina Solexa sequencing. Two percent of the small RNA species found in virus infected cells were derived from virus RNA. These were predominantly 21 nt long and mapped along the entire SFV genome and genome complementary RNAs. Generation of these viRNAs was not random. Some areas produced high frequencies and others no or very few; hot and cold spots respectively. There were no correlations between viRNA frequency and base pairing or secondary structures predictions. Cold spot-derived viRNAs were more effective than hot-spot viRNAs in inhibiting virus replication. Similar results were observed in Aedes aegypti-derived cells. Attempts were made to investigate the source of these viRNAs using a virus containing an IRES element which had been reported to prevent virus replication in insect cells but which did not efficiently do so in this study. A virus containing the RNAi inhibitor p19 was characterised and shown to increase virus production. Techniques for infecting mosquitoes via a blood meal feed were established. No infection was observed with virus replicon particles carrying a fluorescent marker gene. Infection was established using virus containing p19

Inhibition of type I and type II interferon signaling by alphavirus nonstructural proteins

Alphaviruses are mosquito-borne pathogens that represent an emerging threat due their capacity to cause large outbreaks of infectious disease ranging from severe arthritis, as observed with the current chikungunya virus outbreak in the Indian Ocean, to potentially fatal encephalitis, as witnessed by periodic outbreaks of Venezeulan equine encephalitis virus (VEEV). Type I interferon (IFN) plays a crucial role in limiting alphavirus replication and pathogenic strains must employ mechanisms to downregulate this response. In these studies, we have identified a previously unrecognized mechanism by which VEEV and Sindbis virus (SINV) inhibit the response to both type I and type II IFNs, which presumably would limit the antiviral effects of these cytokines within infected cells. Studies with VEEV, as well as propagation-defective replicon particles devoid of the viral structural genes, indicated that the viral nonstructural proteins (nsPs) disrupt critical signaling events downstream of type I and type II IFN receptor activation, as indicated by failed activation and nuclear localization of STAT1, a transcription factor central to multiple signaling pathways. Notably, these inhibitory events occurred upstream and independently of global transcriptional shutoff, which was previously proposed to be the mechanism by which alphaviruses downregulate IFN induction and signaling. Our subsequent studies with SINV demonstrated that other alphaviruses also antagonize Jak/STAT activation and that the efficiency of this inhibition correlated tightly with the relative virulence of SINV strains. Importantly, we were able to map this effect to a single amino acid determinant that was required for efficient STAT inhibition by AR86, an adult mouse neurovirulent SINV strain. This determinant, threonine at nsP1 538, was critical for adult mouse neurovirulence and could rescue efficient STAT inhibition when introduced into an avirulent virus. These studies strongly suggest that STAT signaling inhibition by alphaviruses plays an important role in their capacity to cause disease, and they set the stage for future in vivo studies designed to assess the role that Jak/STAT signaling inhibition plays during alphavirus pathogenesis