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The causal pathogen of chikungunya virus disease, mosquito-transmitted chikungunya virus (CHIKV), has caused significant global epidemics of arthritic disease over the past two decades. A severe chronic, crippling arthritic condition, CHIKV disease can have a negative impact on a patient's mobility and quality of life. Our earlier research led to the production of CHIKV-NoLS, a live-attenuated CHIKV vaccine candidate that has successfully protected against CHIKV disease in preclinical trials. After receiving one dose, mice immunised with CHIKV-NoLS developed a protective immune response against CHIKV. Additional research has shown the utility of a liposome RNA delivery system for directly delivering the RNA genome of CHIKV-NoLS in vivo and promoting in vivo de novo production of live-attenuated vaccine particles.[...] This thesis provides us with essential information about the development of a new vaccine candidate, CHIKV-NoLS, and illustrates how a clinically relevant booster regimen can overcome the challenges faced by a one dose strategy to provide systemic protection against CHIKV disease. Furthermore, I provide new perspectives on the global progress of CHIKV vaccine development to guide future industrial partnerships and clinical development of the CHIKV-NoLS technology. CHIKV-NoLS may be a promising CHIKV vaccine candidate due to lower reversion risks, reported stability, and longevity of protection against CHIKV infection. CHIKV-NoLS faces a long road ahead because no studies on non-human primates or humans have been conducted. Further research on GM cell lines might be useful to establish universal cell lines for vaccine production., Thesis (PhD Doctorate), Doctor of Philosophy (PhD), School of Pharmacy & Med Sci, Griffith Health, Full Text

The RNA genome of the hepatitis C virus (HCV) establishes a network of long-distance RNA-RNA interactions that direct the progression of the infective cycle. This work shows that the dimerization of the viral genome, which is initiated at the dimer linkage sequence (DLS) within the 3′UTR, is promoted by the CRE region, while the IRES is a negative regulatory partner. Using differential 2′-acylation probing (SHAPE-dif) and molecular interference (HMX) technologies, the CRE activity was found to mainly lie in the critical 5BSL3.2 domain, while the IRES-mediated effect is dependent upon conserved residues within the essential structural elements JIIIabc, JIIIef and PK2. These findings support the idea that, along with the DLS motif, the IRES and CRE are needed to control HCV genome dimerization. They also provide evidences of a novel function for these elements as chaperone-like partners that finetune the architecture of distant RNA domains within the HCV genome.

The RNA genome of the hepatitis C virus (HCV) establishes a network of long-distance RNA-RNA interactions that direct the progression of the infective cycle. This work shows that the dimerization of the viral genome, which is initiated at the dimer linkage sequence (DLS) within the 3′UTR, is promoted by the CRE region, while the IRES is a negative regulatory partner. Using differential 2′-acylation probing (SHAPE-dif) and molecular interference (HMX) technologies, the CRE activity was found to mainly lie in the critical 5BSL3.2 domain, while the IRES-mediated effect is dependent upon conserved residues within the essential structural elements JIIIabc, JIIIef and PK2. These findings support the idea that, along with the DLS motif, the IRES and CRE are needed to control HCV genome dimerization. They also provide evidences of a novel function for these elements as chaperone-like partners that finetune the architecture of distant RNA domains within the HCV genome.

Most HIV-1 virions contain two copies of full-length viral RNA, indicating that genome packaging is efficient and tightly regulated. However, the structural protein Gag is the only component required for the assembly of noninfectious viruslike particles, and the viral RNA is dispensable in this process. The mechanism that allows HIV-1 to achieve such high efficiency of genome packaging when a packageable viral RNA is not required for virus assembly is currently unknown. In this report, we examined the role of HIV-1 RNA in virus assembly and found that packageable HIV-1 RNA enhances particle production when Gag is expressed at levels similar to those in cells containing one provirus. However, such enhancement is diminished when Gag is overexpressed, suggesting that the effects of viral RNA can be replaced by increased Gag concentration in cells. We also showed that the specific interactions between Gag and viral RNA are required for the enhancement of particle production. Taken together, these studies are consistent with our previous hypothesis that specific dimeric viral RNA-Gag interactions are the nucleation event of infectious virion assembly, ensuring that one RNA dimer is packaged into each nascent virion. These studies shed light on the mechanism by which HIV-1 achieves efficient genome packaging during virus assembly.

[EN] Citrus tristeza virus (CTV), a phloem-restricted closterovirus infecting citrus, encodes three different silencing suppressors (p25, p20 and p23), one of which (p23) is a pathogenicity determinant that induces aberrations resembling CTV symptoms when expressed ectopically in transgenic citrus hosts. In this article, the effect of p23 ectopic expression on virus infection was examined in sweet orange (SwO), a highly susceptible host, and sour orange (SO), which severely restricts CTV cell-to-cell movement. Transgenic plants of both species ectopically expressing p23, or transformed with an empty vector, were graft inoculated with the mild CTV isolate T385 or with CTV-BC1/GFP, a clonal strain derived from the severe isolate T36 carrying the gene for the green fluorescent protein (GFP). CTV distribution in infected tissues was assessed by direct tissue blot immunoassay and fluorescence emission, and virus accumulation was estimated by quantitative real-time reverse transcriptase-polymerase chain reaction. CTV accumulation in p23-expressing and control SwO plants was similar, whereas the viral load in transgenic SO expressing p23 was 10-10(5) times higher than in the cognate control plants. Although few infection foci composed of a single cell were observed in the phloem of CTV-infected control SO, the number of foci in p23-expressing plants was higher and usually comprised two to six cells, indicating viral cell-to-cell movement. CTV was detected in mesophyll protoplasts and cells from infected SO and SwO expressing p23, but not in similar protoplasts and cells from infected control plants. Our results show that the ectopic expression of p23 enables CTV to escape from the phloem and, in addition, facilitates systemic infection of the resistant SO host. This is the first report of a viral-encoded protein that enhances virus accumulation and distribution in woody hosts.

The influenza viruses contain a segmented, single-stranded RNA genome of negative polarity. Each RNA segment is encapsidated by the nucleoprotein and the polymerase complex into ribonucleoprotein particles (RNPs), which are responsible for virus transcription and replication. Despite their importance, information about the structure of these RNPs is scarce. We have determined the three-dimensional structure of a biologically active recombinant RNP by cryo-electron microscopy. The structure shows a nonameric nucleoprotein ring (at 12 Å resolution) with two monomers connected to the polymerase complex (at 18 Å resolution). Docking the atomic structures of the nucleoprotein and polymerase domains, as well as mutational analyses, has allowed us to define the interactions between the functional elements of the RNP and to propose the location of the viral RNA. Our results provide the first model for a functional negative-stranded RNA virus ribonucleoprotein complex. The structure reported here will serve as a framework to generate a quasi-atomic model of the molecular machine responsible for viral RNA synthesis and to test new models for virus RNA replication and transcription., [Author summary] The influenza viruses cause annual epidemics of respiratory disease and occasional pandemics that constitute a major public-health issue. The recent spillover of avian H5N1 and H1N1 swine influenza viruses to humans poses a serious threat of a new pandemic. These viruses contain a segmented RNA genome, which forms independent ribonucleoprotein particles including the polymerase complex and multiple copies of the nucleoprotein. Each of these ribonucleoprotein particles are replicated and express the encoding virus genes independently in the virus-infected cells. To better understand how these processes take place we have determined the three-dimensional structure of a model ribonucleoprotein particle that only contains 248 nucleotides of virus RNA but is biologically active in vitro and in vivo. The structure shows a circular appearance and includes 9 nucleoprotein monomers, two of which are associated to the polymerase complex. Docking of the available atomic structures of the nucleoprotein and domains of the polymerase complex has permitted us to propose a quasi-atomic model for this ribonucleoprotein particle and some of the predictions of the model have been confirmed experimentally by site-directed mutagenesis and phenotype analysis in vitro and in vivo.

The simplest known plant pathogens are the viroids. Because of their non-coding singlestranded circular RNA genome, they depend on both their sequence and their structure for both a successful infection and their replication. In the recent years, important progress in the elucidation of their structures was achieved using an adaptation of the selective 2'- hydroxyl acylation analyzed by primer extension (SHAPE) protocol in order to probe viroid structures in solution. Previously, SHAPE has been adapted to elucidate the structures of all of the members of the family Avsunviroidae, as well as those of a few members of the family Pospiviroidae. In this study, with the goal of providing an entire compendium of the secondary structures of the various viroid species, a total of thirteen new Pospiviroidae members were probed in solution using the SHAPE protocol. More specifically, the secondary structures of eleven species for which the genus was previously known were initially elucidated. At this point, considering all of the SHAPE elucidated secondary structures, a classification system for viroids in their respective genera was proposed. On the basis of the structural classification reported here, the probings of both the Grapevine latent viroid and the Dahlia latent viroid provide sound arguments for the determination of their respective genera, which appear to be Apscaviroid and Hostuviroid, respectively. More importantly, this study provides the complete repertoire of the secondary structures, mapped in solution, of all of the accepted viroid species reported thus far. In addition, a classification scheme based on structural hallmarks, an important tool for many biological studies, is proposed.