Rotaviruses are the single most common cause of fatal and severe childhood diarrhoeal illness worldwide (>125 million cases annually). Rotavirus shares structural and functional features with many viruses, such as the presence of segmented double-stranded RNA genomes selectively and tightly packed with a conserved number of transcription complexes in icosahedral capsids. Nascent transcripts exit the capsid through 12 channels, but it is unknown whether these channels specialise in specific transcripts or simply act as general exit conduits; a detailed description of this process is needed for understanding viral replication and genomic organisation. To test these opposing models, a novel single-molecule assay was developed for the capture and identification (CID) of newly synthesised specific RNA transcripts. CID combines the hybridisation of transcripts with biotinylated and FRET compatible labelled ssDNAs with the implementation of recent developments in single molecule fluorescence such as alternating laser excitation (ALEX) and total internal reflection fluorescence (TIRF) microscopy. CID identifies and quantifies specific transcripts of rotavirus based on a FRET/Stoichiometry (E*/S) value of the hybridised labelled probes. I used CID to pull down the capsid on the surface slide and identify partially extruded transcripts of three different segments 2, 6 and 11. The findings presented in this thesis support a model in which each channel specialises in extruding transcripts of a specific segment, that in turn is linked to a single transcription complex. The method can be extended to study other transcription systems including E.coli, and can be further developed as a potential diagnostic tool.

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 98-115).

Peste des petits ruminants virus (PPRV) belongs to the family paramyxoviridae which comprises non segmented negative sense RNA viruses including measles and rinderpest virus. PPRV is the causative agent of peste des petits rumaninats disease (also known as sheep or goat plague disease) in small ruminants. The viral genome contains a non segmented negative sense RNA encapsidated by viral encoded nucleocapsid protein (N-RNA). Viral transcription is carried out by the virus encoded RNA dependent RNA polymerase complex represented by the large protein L and phosphoprotein P. Viral transcription begins at the 3’ end of the genome synthesising all the viral transcripts (3’-N-P-M-F-HN-L-5’). A remarkable feature common to all members of Paramyxoviridae family is the gradient of transcription from 3’ end to the 5’ end due to attenuation of polymerase transcription at each gene junction. The objectives of the present study are characterization of peste des petits ruminants virus transcription and the associated activities required for post transcriptional modification of viral mRNA. In addition, an attempt has been made to develop in vitro transcription with heterologous combination of PPRV and RPV polymerase proteins. The first reaction in capping involves removal of γ-phosphate from triphosphate ended precursor mRNA by RNA triphosphatase. The domain having RNA triphosphatase activity within the L protein has been identified and expressed independently in E. coli. The details of the objectives are presented below. 1. Development of in vitro transcription system for PPRV mRNA synthesis In order to develop an in vitro transcription reconstitution system for PPRV, the viral RNP complex comprising large (L), phospho (P) and N protein encapsidating viral genomic RNA was purified from virus infected Vero cells. The in vitro transcription reconstituted system with RNP complex was able to synthesise all the viral mRNA as analysed by RT-PCR. As a control, total RNA from virus infected cells was isolated and analysed by RT-PCR. In order to refine the in vitro transcription system, separately expressed recombinant polymerase complex was used to reconstitute transcriptional activity in vitro. For this,viral genomic RNA (N-RNA) was purified from PPRV infected cells using CsCl density gradient centrifugation. The recombinant baculovirus for PPRV P protein was earlier generated in the lab. A recombinant baculovirus harbouring the L gene of PPRV was generated in the present study (described in part one). The viral RNA polymerase consisting of L-P complex was expressed in Sf21 insect cells and partially purified by ultra centrifugation on 5-20% glycerol gradient. Glycerol gradient fraction containing the L-P complex was found to be active in the in vitro transcription reconstitution system. Further quantitation of transcripts made in vitro and in infected cells has been carried out by real time PCR. Notably, the gradient of polarity of transcription of viral mRNA observed in vitro with the partially purified recombinant L-P complex was similar to the gradient observed in infected cells. Host proteins have been shown to modulate the transcription of many paramyxoviruses. In order to test the role of host factors, uninfected cell lysate of Vero cells was added to the in vitro transcription reaction and the transcript level was measured by real time PCR. The result showed an increase in the transcription by addition of host proteins suggesting the involvement of host factors in viral transcription. Further, the newly developed in vitro reconstitution system was used to test if recombinant L and P proteins of RPV can functionally replace PPRV L and P protein in the in vitro transcription complementation assay. The result presented in part one indicates that the L or P protein of PPRV can be replaced by RPV L and P protein in heterologous transcription reconstitution system ,with a reduced efficiency. However, the homologous polymerase complex of RPV failed to recognise the N-RNA genomic template of PPRV. 2. RNA triphosphatase activity of PPRV L protein and identification of RNA triphosphatase domain Post transcriptional modification of mRNA such as capping and methylation determines the translatability of viral mRNA by cellular ribosome. In negative sense RNA viruses, synthesis of viral mRNA is carried out by the viral encoded RNA polymerase in the host cell cytoplasm. Since the host capping and methylation machinery is localized to the nucleus, viruses should either encode their own mRNA modification enzymes or adopt alternative methods as has been reported for orthomyxoviruses (cap snatching) and picornaviruses (presence of IRES element). In order to test, if PPRV RNA polymerase possesses any of the capping activities, the RNP complex containing the viral N-RNA and RNA polymerase (L-P) were purified from virion. Using the purified RNP complex, the first activity required for mRNA capping, RNA triphosphatase was tested and the results are described in part two. RNP complex purified from virion showed both RNA triphosphatase (RTPase) activity. The RNA triphosphatase from viruses, fungi and other eukaryotes have been classified into two groups, metal dependent and metal independent. The cleavage of the γ-phosphate from triphosphate ended precursor mRNA by L protein of PPRV was found to be metal dependent. So, by the metal dependency of the RTPase reaction, PPRV L protein was assigned to the metal dependent RTPase tunnel family. One of the key features of metal dependent RTPase group members is the ability to hydrolyse γ-β phosphoanhydride bond of NTPs. PPRV L protein associated with RNP complex also was also able to cleave γ-β phosphoanhydride bond of NTPs. Owing to the large size of L protein (240 KDa), it is conceivable that the L protein functions in a modular fashion for different activities pertaining to mRNA synthesis and post transcriptional modification. Sequence comparison of L proteins from different morbilliviruses revealed the presence of three conserved domains namely domain I (aa 1-606), domain II (aa 650-1694) and domain III (aa 1717-2183). Domain II has the catalytic motif for viral RNA dependent RNA polymerase. Multiple sequence alignment of PPRV L protein with known RNA triphosphatases predicted a two hundred amino acid long region on L protein comprising the C terminus of domain II and N terminus of DIII as a possible candidate for RNA triphosphatase domain. The above predicted domain was cloned and expressed in E. coli. The ability of the purified recombinant RTPase domain to cleave γ-β phosphoanhydride bond of RNA was tested. The results described in part two suggest that the predicted RTPase domain has RNA triphosphatase activity. In addition to RNA triphosphatase, the RTPase domain also has the NTPase activity. The RNA triphosphatase of DNA viruses, yeasts and other fungi have three motifs essential for enzyme activity. Motif A and motif C are rich in glutamate and are involved in metal binding. Motif B is rich in basic amino acids and forms the centre for catalysis. The glutamate residue (E1647) of motif A of PPRV L protein RTPase domain was converted to alanine and the loss of RTPase activity was assessed. The results summarised in appendix 1 shows that the E1647A mutant has reduced RNA triphosphatase and NTPase activity.

Morbilliviruses belong to the family Paramyxoviridae of the Mononegavirale order of viruses. The Mononegavirale order contains viruses which contain negatively-polar, non-segmented and single stranded RNA genomes. This order contains some of most lethal pathogens known to the humankind. Ebola virus and Marburg virus are perhaps the most lethal human pathogens. Rinderpest virus, declared eradicated in 2011, was known to be the most significant cattle killer. Similarly the Canine distemper virus and Rabies virus, two topmost canine pathogens belong to this order. The L protein in the viruses of Morbillivirus genus harbours the viral RNA-dependent RNA polymerase that replicates and transcribes the viral genome and also all the mRNA capping enzymes, viz. RNA 5’ triphosphatase, guanylyltransferase, RNA (guanine-7-)methyltransferase and RNA 5’ cap-dependent (2’-oxo-)methyltransferase. Moreover this protein can act as a protein kinase that can regulate the function of P protein which serves as a switch between transcription and replication. mRNA capping is necessary for the virus for the purpose of exploiting host cellular machinery towards viral protein synthesis. The Rinderpest virus L protein serves as a model to study the capping enzymes of Morbillivirus. RNA triphosphatase (RTPase), the first enzyme of the capping cascade had earlier been located on the L protein. The RTPase minimal domain on the L protein was identified earlier by sequence homology studies done with RTPase proteins of Baculovirus and Vaccinia virus and cloned. The bacterially expressed recombinant domain was shown to possess RTPase activity. The enzymatic activity was characterized and the RTPase was found to be a metal-dependent enzyme which is highly specific to capping viral mRNA. Further characterization of the domain revealed that the domain also possesses nucleotide triphosphatase (NTPase), tripolyphosphatase and pyrophosphatase activities. Two site-directed mutants in motif-A of the domain: E1645A and E1647A were also tested and were found to be essential for the RTPase and NTPase activity. It was also recognized through these mutant studies that the active sites of RTPase and NTPase activities are partially overlapping. Earlier work done with Vesicular stomatitis virus capping enzymes showed that the Rhabdoviridae family of viruses follow unconventional capping pathway utilizing an enzyme polyribonucleotidyltransferase (PRNTase) which transfers GDP to 5’-monophosphated RNA. Characterization of the RTPase activity which converts 5’-triphosphated RNA into 5’-diphosphated RNA is an evidence for the morbilliviruses utilizing the conventional eukaryotic capping cascade. The results show that Paramyxoviridae do not follow unconventional capping pathway for the mRNA capping as has been the paradigm in the past decade.

Malgré les améliorations significatives apportées par la thérapie antirétrovirale à la durée et à la qualité de vie des personnes vivant avec le VIH, elle ne permet pas de complètement éliminer le virus de l’organisme. La persistance du virus est due à l’existence de réservoirs viraux, des cellules infectées de manière latente par le VIH. Ces réservoirs nécessitent un traitement antirétroviral à vie, car le virus réapparait en cas d’interruption du traitement, signifiant que l’immunité des cellules T spécifiques du VIH n’est pas restaurée. Bien que cela soit théoriquement possible, seule une fraction de personne vivant avec le VIH, appelée Contrôleurs Élites, parvient à contrôler le virus en absence de traitement. Pour la majorité des individus, l’infection par le VIH entraîne une évasion virologique ainsi qu’un épuisement et une altération des réponses cellulaires spécifiques au VIH. À ce jour, les stratégies thérapeutiques visant à éliminer les réservoirs viraux ont échoué, en partie en raison de la présence de provirus principalement défectifs dans ces réservoirs. Dans cette thèse, nous avons identifié et caractérisé les provirus défectifs latents du VIH pouvant être transcrits et/ou traduits, ainsi que la relation entre ces réservoirs et les réponses immunitaires spécifique du virus. Dans un premier temps, nous avons montré que bien que défectifs et potentiellement incapable de donner lieu à la réplication virale, ces provirus peuvent être transcrits et traduits soit par réactivation à l’aide d’agents de réversion de la latence, soit de manière spontanée. Ces réservoirs donnent lieu à plusieurs populations de réservoirs, en fonction de la présence ou de l’absence certains gènes viraux. Nous avons déterminé que ces différentes populations sont régies par le profil génomique des cellules infectées. Les provirus identifiés étaient très rarement intacts, mais l’intégrité du génome était associée à la processivité de la transcription et de la traduction. Dans un second objectif, nous avons caractérisé les réponses T CD4+ et CD8+ spécifiques du VIH avant et après le début du traitement antirétroviral. Nous avons observé que les réponses T CD4+ spécifiques étaient comparables pendant l’infection chronique et après le traitement. En revanche, les réponses T CD8+ diminuaient considérablement après l’initiation de la thérapie antirétrovirale. Nous avons également constaté que la taille du réservoir traductionnellement actif pendant le traitement antirétroviral était négativement associée aux réponses T CD8+ spécifiques avant le début de la thérapie, tandis que le réservoir incapable de traduire les protéines du VIH subsistait. Ces observations mettent en évidence le rôle des cellules T CD8+ dans le contrôle de l’infection par le VIH, comme nous l’avons observé chez les Contrôleurs Élites. Nos travaux contribuent à une meilleure compréhension des réservoirs viraux du VIH, qui pourraient potentiellement être impliqués dans l’inflammation chronique et la dysfonction immunitaire associé à la pathogénèse du VIH. / Despite the significant improvement brought by antiretroviral therapy in the duration and quality of life for people living with HIV, it does not completely eliminate the virus from the body. The persistence of the virus is due to the existence of viral reservoirs, which are cells latently infected with HIV. These reservoirs require lifelong antiretroviral treatment because of the viral rebound reoccurring in case of treatment interruption. This suggests that HIV-specific T cell immunity is not restored. Although theoretically possible, only a fraction of people living with HIV, known as Elite Controllers, are able to control the virus in the absence of treatment. For the majority of individuals, HIV infection leads to virologic escape, as well as exhaustion and altered cellular responses to HIV. To date, therapeutic strategies aimed at eliminating viral reservoirs have failed, partly due to the presence of predominantly defective proviruses in these reservoirs. In this thesis, we have identified and characterized latent defective proviruses of HIV that can be transcribed and/or translated. We also have characterized the relationship between these reservoirs and the specific immune responses to the virus. Firstly, we have shown that although defective and potentially replication-incompetent, these proviruses can be transcribed and translated either through reactivation using latency reversal agents or spontaneously. These reservoirs give rise to several populations of reservoirs, depending on the presence or absence of certain viral genes. We have determined that these different populations are governed by the genomic profile of infected cells. The identified proviruses were rarely intact, and genome integrity was associated with the processivity of transcription and translation. Then, we characterized the specific CD4+ and CD8+ T cell responses to HIV before and after the initiation of antiretroviral treatment. We observed that specific CD4+ T cell responses were comparable during chronic infection and after treatment. However, CD8+ T cell responses decreased significantly after the initiation of antiretroviral therapy. We also found that the size of the translationally active reservoir during antiretroviral treatment was negatively associated with the specific CD8+ T cell responses prior to treatment initiation, while the translation-incompetent cells persisted. These observations highlight the role of CD8+ T cells in the control of HIV infection, as observed in Elite Controllers. Our work contributes to a better understanding of HIV viral reservoirs, which could potentially be involved in chronic inflammation and immune dysfunction associated with HIV pathogenesis.