The role of ZAP and OAS3/RNAseL pathways in the attenuation of an RNA virus with elevated frequencies of CpG and UpA dinucleotides.

Zinc finger antiviral protein (ZAP) is a powerful restriction factor for viruses with elevated CpG dinucleotide frequencies. We report that ZAP similarly mediates antiviral restriction against echovirus 7 (E7) mutants with elevated frequencies of UpA dinucleotides. Attenuation of both CpG- and UpA-high viruses and replicon mutants was reversed in ZAP k/o cell lines, and restored by plasmid-derived reconstitution of expression in k/o cells. In pull-down assays, ZAP bound to viral RNA transcripts with either CpG- and UpA-high sequences inserted in the R2 region. We found no evidence that attenuation of CpG- or UpA-high mutants was mediated through either translation inhibition or accelerated RNA degradation. Reversal of the attenuation of CpG-high, and UpA-high E7 viruses and replicons was also achieved through knockout of RNAseL and oligodenylate synthetase 3 (OAS3), but not OAS1. WT levels of replication of CpG- and UpA-high mutants were observed in OAS3 k/o cells despite abundant expression of ZAP, indicative of synergy or complementation of these hitherto unconnected pathways. The dependence on expression of ZAP, OAS3 and RNAseL for CpG/UpA-mediated attenuation and the variable and often low level expression of these pathway proteins in certain cell types, such as those of the central nervous system, has implications for the use of CpG-elevated mutants as attenuated live vaccines against neurotropic viruses.

Dynamic network approach for the modelling of genomic sub-complexes in multi-segmented viruses.

Viruses with segmented genomes, including pathogens such as influenza virus, Rotavirus and Bluetongue virus (BTV), face the collective challenge of packaging their genetic material in terms of the correct number and types of segments. Here we develop a novel network approach to predict RNA-RNA interactions between different genomic segments. Experimental data on RNA complex formation in the multi-segmented BTV genome are used to establish proof-of-concept of this technique. In particular, we show that trans interactions between segments occur at multiple specific sites, termed segment assortment signals (SASs) that are dispersed across each segment. In order to validate the putative trans acting networks, we used various biochemical and molecular techniques which confirmed predictions of the RNA network approach. A combination of mutagenesis and reverse genetics systems revealed that the RNA-RNA interacting sites identified are indeed responsible for segment assortment and complex formation, which are essential criteria for genome packaging. This paves the way for their exploitation as novel types of drug target, either to inhibit assembly, or for designing defective interfering particles containing an incomplete set of genomic segments.

CpG and UpA dinucleotides in both coding and non-coding regions of echovirus 7 inhibit replication initiation post-entry.

Most vertebrate and plant RNA and small DNA viruses suppress genomic CpG and UpA dinucleotide frequencies, apparently mimicking host mRNA composition. Artificially increasing CpG/UpA dinucleotides attenuates viruses through an entirely unknown mechanism. Using the echovirus 7 (E7) model in several cell types, we show that the restriction in E7 replication in mutants with increased CpG/UpA dinucleotides occurred immediately after viral entry, with incoming virions failing to form replication complexes. Sequences of CpG/UpA-high virus stocks showed no evidence of increased mutational errors that would render them replication defective, these viral RNAs were not differentially sequestered in cytoplasmic stress granules nor did they induce a systemic antiviral state. Importantly, restriction was not mediated through effects on translation efficiency since replicons with high CpG/UpA sequences inserted into a non-coding region were similarly replication defective. Host-cells thus possess intrinsic defence pathways that prevent replication of viruses with increased CpG/UpA frequencies independently of codon usage.

Generation of infectious RNA complexes in Orbiviruses: RNA-RNA interactions of genomic segments.

Viruses with segmented RNA genomes must package the correct number of segments for synthesis of infectious virus particles. Recent studies suggest that the members of the Reoviridae family with segmented double-stranded RNA genomes achieve this challenging task by forming RNA networks of segments prior to their recruitment into the assembling capsid albeit direct evidence is still lacking. Here, we investigated the capability of virus recovery by preformed complexes of ten RNA segments of Epizootic Haemorrhagic Disease Virus (EHDV), a Reoviridae member, by transcribing exact T7 cDNA copies of genomic RNA segments in a single in vitro reaction followed by transfection of mammalian cells. The data obtained was further confirmed by RNA complexes generated from Bluetongue virus, another family member. Formation of RNA complexes was demonstrated by sucrose gradient ultracentrifugation, and RNA-RNA interactions inherent to the formation of the RNA complexes were demonstrated by electrophoretic mobility shift assay. Further, we showed that disruption of RNA complex formation inhibits virus recovery, confirming that recruitment of complete RNA networks is essential for packaging and consequently, virus recovery. This efficient reverse genetics system will allow further understanding of evolutionary relationships of Reoviridae members and may also contribute to development of antiviral molecules.

Generation of virus-like particles for emerging epizootic haemorrhagic disease virus: Towards the development of safe vaccine candidates.

Epizootic haemorrhagic disease virus (EHDV) is an insect-transmitted pathogen which causes high mortality in deer populations and may also cause high morbidity in cattle. EHDV belongs to the Orbivirus genus and is closely related to the prototype Bluetongue virus (BTV). To date seven distinct serotypes have been recognized. However, a live-attenuated vaccine is commercially available against only one serotype namely EHDV-2, which has been responsible for multiple outbreaks in North America, Canada, Asia and Australia. Here we expressed four major capsid proteins (VP2, VP3, VP5 and VP7) of EHDV-1 using baculovirus multiple gene expression systems and demonstrated that three-layered VLPs were assembled mimicking the authentic EHDV particles but lacking the viral genomic RNA segments and the transcriptase complex (TC). Antibodies generated with VLPs not only neutralized EHDV-1 infection in cell culture but also showed cross neutralizing reactivity against two other serotypes, EHDV-2 and EHDV-6. For proof of concept, we demonstrated that EHDV-2 VLPs could be generated rapidly by expressing the EHDV-2 variable outer capsid proteins (VP2, VP5) together with EHDV-1 VP3 and VP7, the two inner capsid proteins, which are highly conserved among the 7 serotypes. Data presented in this study validate the VLPs as a potential vaccine and demonstrate that a vaccine could be developed rapidly in the event of an outbreak of a new serotype.