Respiratory syncytial virus (RSV) is the leading cause of respiratory illness in children worldwide. RSV has a negative sense RNA genome, which is the template for viral mRNA transcription and genome replication, and encodes a polymerase to carry out viral RNA synthesis. The promoters for RSV transcription and genome replication are found in a 44-nucleotide (nt), 3´-extragenic region called the leader (Le). Replication is initiated opposite the first nt of the Le, and transcription of the first gene begins at position +45, at a gene start (GS) sequence. However, transcription is also dependent on sequence within Le1-12. Interestingly, Le nucleotides 3-12 bear strong similarity to a GS signal. We hypothesized that this GS-like sequence is the recruitment site for transcribing polymerase. To test this hypothesis, we examined RNA synthesis events at the Le promoter. We identified a previously undescribed RNA initiation site at Le position +3 (Le+3) that was used frequently during RSV infection. Initiation at Le+3 led to the production of a small ~25 nt RNA. Le+3 initiation was shown to occur independently of replication initiation at +1, indicating it is a bona fide initiation site. Mutation of Le1-12 to increase similarity to a GS resulted in elongation of Le+3 RNA and a decrease in transcription initiation at the GS, demonstrating that the Le initiation sequence alters polymerase processivity and impacts downstream transcription events. Preliminary experiments to determine the function of the small RNA showed that it increased levels of viral RNA replication, suggesting it may be involved in influencing a switch from transcription to replication. These studies suggest a model for RSV transcription initiation, whereby the transcribing polymerase enters at the 3´–end of the genome, initiates RNA synthesis from Le+3 and generates a small RNA, and is then positioned to initiate transcription at the first GS. The small RNA that is generated may act as a feedback molecule to promote RNA replication. These findings provide a greater understanding of polymerase behavior at the promoter and may inform rational drug and vaccine design.

No description available.

Tombusviruses are single, positive strand RNA viruses of plants, often associated with parasitic defective interfering (DI) RNAs. Two viral- coded gene products, namely p33 and p92, are required for tombusvirus replication. The overlapping domains of p33 and p92 contain an arginine/proline-rich (RPR) RNA binding motif. In this study, the role of RPR motif and viral RNA in tombusvirus replication and recombination, as well as involvement of viral RNA in tombusvirus replicase assembly was examined. Using site-directed mutagenesis I generated a series of RPR mutants of Cucumber necrosis tombusvirus (CNV). Analysis of RPR mutants defined that wild type RPR motif, especially two of the four arginines, were required for efficient RNA binding in vitro, for replication of tombusviruses, their associated DI RNAs, subgenomic (sg)RNA synthesis and DI RNA recombination in vivo. Experiments using a two-component tombusvirus replication system showed that RPR motif is critical for functions of both p33 and p92 in replication, but its role in these proteins might not be identical. Recombination studies using a novel tombusvirus three-component system revealed that mutations in RPR motif of p33 replicase protein resulted in an altered viral RNA recombination rate. Identified DI RNA recombinants were mostly imprecise, with recombination sites clustered around a replication enchancer and an additional putative cis-acting element that might facilitate the template switching events by the tombusvirus replicase. To study the role of RNA during the assembly of functional tombusvirus replicase, recombinant CNV replicase that showed similar properties to plant-derived CNV replicase was purified from Saccharomyces cerevisiae. When in addition to p33 and p92 proteins DI RNA was co-expressed in yeast cells, the isolated replicase activity was increased ~40 fold. Further studies defined RNA motifs within two short DI RNA regions that enhanced active CNV replicase formation. In summary, this study showed that the conserved RNA binding motif of the tombusvirus replicase proteins and viral RNA are involved in replicase assembly, viral RNA replication, subgenomic RNA synthesis and RNA recombination. This data shed new light on the complex roles of the viral elements in replication, and will help future studies aimed at interfering with viral infections.

Human metapneumovirus (HMPV) is a respiratory pathogen in Paramyxovirus family that demonstrates extremely high morbidity in the population, with most individuals having been infected by the age of five. Despite the prevalence of this negative-sense RNA virus in the population for decades, it was only identified in 2001. As such, there is currently no specific treatment for HMPV and the potentially severe consequences of infection for elderly and immunocompromised individuals and particularly infants make development of antivirals targeting HMPV of high significance. HMPV constitutes a quarter of all respiratory hospitalizations among infants, placing it second only to RSV, in addition to becoming a greater concern in concentrated populations of seniors. For these susceptible populations, the consequences of infection have a much greater probability of leading to pneumonia, bronchiolitis and even death. These studies investigate events throughout the infectious cycle of HMPV. They describe specific amino acids that modulate the triggering of viral fusion activity in response to low pH. They also include a report on the dynamic and variable control exercised over gene transcription by viral promoters. Finally, the interplay between viral nonstructural proteins and their distinct roles in both replication and assembly are examined. Ultimately, this work seeks to elucidate the goings-on within an HMPV-infected cell at multiple points throughout the process.

The influenza virus causes a disease that kills approximately 500,000 people worldwide each year. Influenza is a negative-sense RNA virus that encodes its own RNA-dependent RNA polymerase. This protein (FluPol) carries out both genome replication and viral transcription. Therefore, like the L-proteins of non-segmented negative-sense RNA (nsRNA) viruses, FluPol also contains mRNA capping and polyadenylation functionality. In FluPol, capping is achieved by snatching cap structures from cellular mRNAs, so requiring cap-binding and endonuclease activities. This makes FluPol a substantial machine. It is a heterotrimeric complex, composed of PB1, PB2 and PA/P3 subunits, with a total molecular weight of 255 kDa. PB1 houses the polymerase active site, whereas PB2 and PA contain, respectively, cap-binding and endonuclease domains. Currently, we only have high resolution structural information for isolated fragments of FluPol. This severely hampers our understanding of influenza replication and consequently inhibits the development of therapies against the virus. In this DPhil project, I have determined a preliminary structure for the heterotrimeric FluPol of influenza C/Johannesburg/1/66, solved by x-ray crystallography to 3.6 Å. Overall, FluPol has an elongated structure with a conspicuous deep groove. PB1 displays the canonical right-hand-like polymerase fold. It sits at the centre of the particle, sandwiched between the two domains of P3, and with PB2 stacked against one side of this dimer. In the structure, the polymerase and endonuclease catalytic sites are both ~40 Å away from the cap-binding pocket. This pocket also faces a tunnel leading to the polymerase core. This suggests a mechanism for how capped cellular mRNAs are cleaved and then fed into the polymerase active site to prime transcription. The structure also hints at a unique trajectory for template RNA, in which the RNA exits at an angle ~180° from which it came in. This provides an explanation for how the polymerases of influenza, and other nsRNA viruses, can copy templates that are packaged into ribonucleoprotein complexes. My work reveals the first molecular structure of any polymerase from an nsRNA virus. It uncovers the arrangement of functional domains within FluPol, illuminating the mechanisms of this and related viral polymerases. This work will help focus future experiments into FluPol biology, and should hopefully spur the development of novel antiviral drugs.

The satellite RNA of Lucerne Transient Streak Virus (LTSV) is a 322-nucleotide, single-stranded circular RNA that has a rod-like structure very similar to that of viroids. As it does not encode any translation products and cannot replicate independently of a helper virus, the satellite RNA is proposed to rely on viral-encoded proteins for the replication and/or cell-to-cell movement that facilitate its systemic infection in a host. To investigate the requirements for replication of the LTSV satellite RNA, transgenic plant systems were generated to express the viral RNA-dependent RNA polymerase and predicted viral transport protein independently as well as in combination. Results of infectivity assays of these transgenic lines demonstrated for the first time that the viral-encoded RNA-dependent RNA polymerase is necessary and sufficient for the replication of LTSV satellite RNA, and that no additional viral proteins are required for its cell-to-cell or systemic transport.

The satellite RNA of Lucerne Transient Streak Virus (LTSV) is a 322-nucleotide, single-stranded circular RNA that has a rod-like structure very similar to that of viroids. As it does not encode any translation products and cannot replicate independently of a helper virus, the satellite RNA is proposed to rely on viral-encoded proteins for the replication and/or cell-to-cell movement that facilitate its systemic infection in a host. To investigate the requirements for replication of the LTSV satellite RNA, transgenic plant systems were generated to express the viral RNA-dependent RNA polymerase and predicted viral transport protein independently as well as in combination. Results of infectivity assays of these transgenic lines demonstrated for the first time that the viral-encoded RNA-dependent RNA polymerase is necessary and sufficient for the replication of LTSV satellite RNA, and that no additional viral proteins are required for its cell-to-cell or systemic transport.

Rapid evolution of RNA viruses with mRNA-sense genomes is a major concern to health and economic welfare due to the devastating diseases these viruses inflict on humans, animals and plants. Rapid viral RNA evolution is frequently due to RNA recombination, which can be facilitated by recombination signals present in viral RNAs. Among such signals are short sequences with high AU contents that constitute recombination hot spots in Brome mosaic virus (BMV) and retroviruses. We have demonstrated that a defective interfering (DI) RNA, a model template associated with Tomato bushy stunt virus (TBSV), a tombusvirus, undergoes frequent recombination in plants and protoplast cells when it carries the AU-rich hot spot sequence from BMV. Similar to the situation with BMV, most of the recombination junction sites in the DI RNA recombinants were found within the AU-rich region. Our results support the idea that common AU-rich recombination signals might promote interviral recombination between unrelated viruses. To test if host genes can affect the evolution of RNA viruses, we used a Saccharomyces cerevisiae single-gene deletion library, which includes ~80% of yeast genes, in RNA recombination studies based on a small viral replicon RNA derived from TBSV. The genome-wide screen led to the identification of five host genes, whose absence resulted in rapid generation of novel viral RNA recombinants. Thus, these genes normally suppress viral RNA recombination, but in their absence hosts become viral recombination hotbeds. Four of the five recombination suppressor genes are likely involved in RNA degradation, suggesting that RNA degradation could play a role in viral RNA recombination. Overall, our results demonstrate for the first time that a set of host genes have major effect on RNA virus recombination and evolution. Replication of the non-segmented, plus-stranded RNA genome of Cucumber necrosis tombusvirus (CNV) requires two essential overlapping viral-coded replication proteins, the p33 replication co-factor and the p92 RNA-dependent RNA polymerase. We have demonstrated that p33 is phosphorylated in vivo and in vitro by a membrane-bound plant kinase. Based on in vitro studies with purified recombinant p33, we show evidence for phosphorylation of threonine and serine residues adjacent to the essential RNA-binding site in p33. Our findings suggest that phosphorylation of threonine/serine residues adjacent to the essential RNA-binding site in the auxiliary p33 protein likely plays a role in viral RNA replication and subgenomic RNA synthesis during tombusvirus infections.

The influenza A virus genome consists of eight single-stranded segments of negativesense viral RNA (vRNA) with highly conserved, partially complementary termini. These termini associate in a double-stranded RNA structure, known as a panhandle, which is bound by the viral RNA-dependent RNA polymerase and can serve as a promoter in both viral transcription and replication. In part A of this thesis, I use a combination of classical biochemistry techniques and fluorescence techniques (both at the ensemble and single-molecule level) for a quantitative investigation of the interaction between purified influenza A RNA polymerase and the individual 5' and 3' conserved termini of the vRNA segments, as well as the double-stranded vRNA promoter. Furthermore, I report the first direct, real-time observation of the promoter changing its structure when bound by the polymerase and show that the structure assumed agrees best with the corkscrew model. In part B of this thesis, I use fluorescence to detect RNA: I design and test a singlemolecule biosensor aimed at probing the presence of influenza A RNA in a sample, on the one hand, and I use click-chemistry to fluorescently label very shorty RNAs (3-25nt) that have been generated in an in vitro transcription reaction, on the other. The biosensing assay I propose can be further developed for diagnostic purposed, while click-chemistry labelling of short RNAs can be optimised and extended such that it becomes a reliable alternative to the use of radiolabels.

Viral RNA-dependent RNA polymerases (RdRps) are enzymes essential for viral multiplication. The general function of RdRp is universal for all RNA viruses: to recognise viral RNA, bind it and synthesize the complementary RNA strand. This series of steps is absolutely crucial for viral infection. It is important to mention that the non-infected cell is incapable of replicating any RNA. The host cell thus does not naturally express any RdRps. I chose RdRps for my research because these enzymes are key to viral replication and thus an excellent target for antivirals. This study characterises polymerases from ​Picornaviridae and Flaviviridae families, in depth. Picornaviral replication takes place in viral-induced membrane structures called Replication Organelles (ROs), where the polymerase is localised to the membrane. In this study, we investigated the recruitment of picornaviral polymerase membrane. Subsequently, we focused on the activation of picornaviral RdRp induced by the insertion of the very first residue into the protein core. Next, we focused on the flaviviral RdRps specifically from yellow fever virus (YFV) and Zika virus (ZIKV). This study reports the first structure of a full length YFV polymerase and a model of ZIKV polymerase in complex with RNA. The model of ZIKV RdRp in complex with...