SNAP-tagged Chikungunya Virus Replicons Improve Visualisation of Non-Structural Protein 3 by Fluorescence Microscopy

Chikungunya virus (CHIKV), a mosquito-borne alphavirus, causes febrile disease, muscle and joint pain, which can become chronic in some individuals. The non-structural protein 3 (nsP3) plays essential roles during infection, but a complete understanding of its function is lacking. Here we used a microscopy-based approach to image CHIKV nsP3 inside human cells. The SNAP system consists of a self-labelling enzyme tag, which catalyses the covalent linking of exogenously supplemented synthetic ligands. Genetic insertion of this tag resulted in viable replicons and specific labelling while preserving the effect of nsP3 on stress granule responses and co-localisation with GTPase Activating Protein (SH3 domain) Binding Proteins (G3BPs). With sub-diffraction, three-dimensional, optical imaging, we visualised nsP3-positive structures with variable density and morphology, including high-density rod-like structures, large spherical granules, and small, low-density structures. Next, we confirmed the utility of the SNAP tag for studying protein turnover by pulse-chase labelling. We also revealed an association of nsP3 with cellular lipid droplets and examined the spatial relationships between nsP3 and the non-structural protein 1 (nsP1). Together, our study provides a sensitive, specific, and versatile system for fundamental research into the individual functions of a viral non-structural protein during infection with a medically important arthropod-borne virus (arbovirus)

Archival search for historical atypical scrapie in sheep reveals evidence for mixed infections

Flaviviruses are arthropod-borne viruses found worldwide and are responsible for significant human and veterinary diseases, including dengue, Zika, and West Nile fever. Some flaviviruses are insect specific and replicate only in mosquitoes. We report a genetically divergent group of insect-specific flaviviruses from Anopheles mosquitoes that do not replicate in arthropod cell lines or heterologous Anopheles species, exhibiting unprecedented specialization for their host species. Determination of the complete sequences of the RNA genomes of three of these viruses, Karumba virus (KRBV), Haslams Creek virus, and Mac Peak virus (McPV), that are found in high prevalence in some Anopheles mosquito populations and detection of virus-specific proteins, replicative double-stranded RNA, and small interfering RNA responses in the host mosquito species provided strong evidence of a functional replicating virus in the mosquito midgut. Analysis of nucleotide composition in the KRBV and McPV sequences also revealed a pattern consistent with the virus evolving to replicate only in insects. These findings represent a significant advance in our knowledge of mosquito-borne flavivirus ecology, host restriction, and evolution

Proteomic Analysis of Chikungunya Virus Infected Microgial Cells

Chikungunya virus (CHIKV) is a recently re-emerged public health problem in many countries bordering the Indian Ocean and elsewhere. Chikungunya fever is a relatively self limiting febrile disease, but the consequences of chikungunya fever can include a long lasting, debilitating arthralgia, and occasional neurological involvement has been reported. Macrophages have been implicated as an important cell target of CHIKV with regards to both their role as an immune mediator, as well evidence pointing to long term viral persistence in these cells. Microglial cells are the resident brain macrophages, and so this study sought to define the proteomic changes in a human microglial cell line (CHME-5) in response to CHIKV infection. GeLC-MS/MS analysis of CHIKV infected and mock infected cells identified some 1455 individual proteins, of which 90 proteins, belonging to diverse cellular pathways, were significantly down regulated at a significance level of p<0.01. Analysis of the protein profile in response to infection did not support a global inhibition of either normal or IRES-mediated translation, but was consistent with the targeting of specific cellular pathways including those regulating innate antiviral mechanisms

Alphavirus infection: host cell shut-off and inhibition of antiviral responses

Alphaviruses cause debilitating disease in humans and animals and are transmitted by blood-feeding arthropods, typically mosquitoes. With a traditional focus on two models, Sindbis virus and Semliki Forest virus, alphavirus research has significantly intensified in the last decade partly due to the re-emergence and dramatic expansion of chikungunya virus in Asia, Europe, and the Americas. As a consequence, alphavirus-host interactions are now understood in much more molecular detail, and important novel mechanisms have been elucidated. It has become clear that alphaviruses not only cause a general host shut-off in infected vertebrate cells, but also specifically suppress different host antiviral pathways using their viral nonstructural proteins, nsP2 and nsP3. Here we review the current state of the art of alphavirus host cell shut-off of viral transcription and translation, and describe recent insights in viral subversion of interferon induction and signaling, the unfolded protein response, and stress granule assembly

Alphavirus infection: host cell shut-off and inhibition of antiviral responses

Alphaviruses cause debilitating disease in humans and animals and are transmitted by blood-feeding arthropods, typically mosquitoes. With a traditional focus on two models, Sindbis virus and Semliki Forest virus, alphavirus research has significantly intensified in the last decade partly due to the re-emergence and dramatic expansion of chikungunya virus in Asia, Europe, and the Americas. As a consequence, alphavirus-host interactions are now understood in much more molecular detail, and important novel mechanisms have been elucidated. It has become clear that alphaviruses not only cause a general host shut-off in infected vertebrate cells, but also specifically suppress different host antiviral pathways using their viral nonstructural proteins, nsP2 and nsP3. Here we review the current state of the art of alphavirus host cell shut-off of viral transcription and translation, and describe recent insights in viral subversion of interferon induction and signaling, the unfolded protein response, and stress granule assembly

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

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