No description available.

Poxviruses are a diverse family of double-stranded DNA viruses. A characteristic feature of poxviruses is that they express a vast array of immuno-modulatory proteins. Vaccinia virus is the prototypic member of the Orthopoxvirus genus, which also includes variola virus, the causative agent of smallpox. The vaccinia E3 protein is required for virus replication in vivo and in numerous cell culture systems. Although E3 function has received considerable study, many aspects of E3 biology remain to be addressed. While E3 can inhibit cytokine expression, the pathways targeted by E3 to block cytokine expression have not been identified. Furthermore, the factor(s) which stimulate innate immune responses during vaccinia infection are not known. In this study, E3 was found to target PKR and RIG-I-like receptor mediated signal transduction to differentially block expression of IFN-β, TNF-α and IL-6 in HeLa cells. RNA species generated in vaccinia infected cells were identified as pathogen-associated molecular patterns capable of inducing cytokine expression and activating apoptosis. Furthermore, PKR, RIG-I and MDA5 play non-overlapping and essential roles in mediating the innate immune response to these RNA species. Orthologues of E3 are encoded by all poxviruses which infect vertebrate animals except the Avipoxviruses and molluscum contagiosum virus. However, orthologues of vaccinia E3 remain essentially uncharacterized. A comparative analysis of the ability of E3 orthologues encoded by sheeppox, yaba monkey tumour, swinepox and myxoma virus to complement deletion of E3 was performed. E3 orthologues of myxoma virus and swinepox virus suppress PKR activation and interferon induced antiviral activity and restore the host range function of E3 in culture. In contrast, the E3 orthologues of sheeppox virus and yaba monkey tumour virus are unable to inhibit PKR activation. While the sheeppox orthologue cannot restore the host range function of E3, the yaba monkey tumour virus orthologue partially restores E3 deficient vaccinia replication. However, none of these E3 orthologues restore pathogenicity to E3 deficient vaccinia in vivo. In summary, these results highlight the role of the vaccinia virus E3 protein and its poxvirus orthologues in suppressing innate immune responses activated by RNA-based pathogen-associated molecular patterns

Poxviruses are a diverse family of double-stranded DNA viruses. A characteristic feature of poxviruses is that they express a vast array of immuno-modulatory proteins. Vaccinia virus is the prototypic member of the Orthopoxvirus genus, which also includes variola virus, the causative agent of smallpox. The vaccinia E3 protein is required for virus replication in vivo and in numerous cell culture systems. Although E3 function has received considerable study, many aspects of E3 biology remain to be addressed. While E3 can inhibit cytokine expression, the pathways targeted by E3 to block cytokine expression have not been identified. Furthermore, the factor(s) which stimulate innate immune responses during vaccinia infection are not known. In this study, E3 was found to target PKR and RIG-I-like receptor mediated signal transduction to differentially block expression of IFN-β, TNF-α and IL-6 in HeLa cells. RNA species generated in vaccinia infected cells were identified as pathogen-associated molecular patterns capable of inducing cytokine expression and activating apoptosis. Furthermore, PKR, RIG-I and MDA5 play non-overlapping and essential roles in mediating the innate immune response to these RNA species. Orthologues of E3 are encoded by all poxviruses which infect vertebrate animals except the Avipoxviruses and molluscum contagiosum virus. However, orthologues of vaccinia E3 remain essentially uncharacterized. A comparative analysis of the ability of E3 orthologues encoded by sheeppox, yaba monkey tumour, swinepox and myxoma virus to complement deletion of E3 was performed. E3 orthologues of myxoma virus and swinepox virus suppress PKR activation and interferon induced antiviral activity and restore the host range function of E3 in culture. In contrast, the E3 orthologues of sheeppox virus and yaba monkey tumour virus are unable to inhibit PKR activation. While the sheeppox orthologue cannot restore the host range function of E3, the yaba monkey tumour virus orthologue partially restores E3 deficient vaccinia replication. However, none of these E3 orthologues restore pathogenicity to E3 deficient vaccinia in vivo. In summary, these results highlight the role of the vaccinia virus E3 protein and its poxvirus orthologues in suppressing innate immune responses activated by RNA-based pathogen-associated molecular patterns

No description available.

No description available.

Vaccinia virus E3 protein is known to bind double-stranded RNA and mediate interferon resistance. Alanine scanning mutagenesis was performed on its dsRNA binding domain, sufficient for wild-type tropism and immune suppression in vitro, and dsRNA binding and host range function assayed. Residues involved in dsRNA binding were required for host range function; however, seven dsRNA binding mutants were unable to rescue ΔE3L replication. Utilizing recombinant viruses, non-rescue mutants were unable to inhibit protein Kinase R phosphorylation despite dsRNA binding. Furthermore, host range was found to correlate with cytokine suppression and replication in IFN stimulated Huh7R cells. Additionally, no direct association was found between dsRNA binding and PKR interaction, refining the suppression model. Novel protein-protein interactions were discovered between E3 and cellular proteins via differential gel electrophoresis. This study represents the first full mapping of E3 residues involved in dsRNA binding and tropism, forming a basis for future study.

No description available.

Vaccinia virus E3 protein is known to bind double-stranded RNA and mediate interferon resistance. Alanine scanning mutagenesis was performed on its dsRNA binding domain, sufficient for wild-type tropism and immune suppression in vitro, and dsRNA binding and host range function assayed. Residues involved in dsRNA binding were required for host range function; however, seven dsRNA binding mutants were unable to rescue ΔE3L replication. Utilizing recombinant viruses, non-rescue mutants were unable to inhibit protein Kinase R phosphorylation despite dsRNA binding. Furthermore, host range was found to correlate with cytokine suppression and replication in IFN stimulated Huh7R cells. Additionally, no direct association was found between dsRNA binding and PKR interaction, refining the suppression model. Novel protein-protein interactions were discovered between E3 and cellular proteins via differential gel electrophoresis. This study represents the first full mapping of E3 residues involved in dsRNA binding and tropism, forming a basis for future study.

No description available.

The significance of poxvirus manipulation of the host ubiquitin proteasome system has become increasingly apparent. Ubiquitin is post-translationally added to target proteins by a highly conserved enzymatic cascade, typically resulting in protein degradation via the 26S proteasome. The highly conserved poxvirus protein, p28, is a functional ubiquitin ligase and a critical virulence factor. Here, we investigate the relationship between p28 and ubiquitination. We observed that the KilA-N DNA binding domain in p28 targeted p28 to viral factories, where p28 co-localized with conjugated ubiquitin. Furthermore, we determined that p28 is highly regulated by ubiquitination and proteasomal degradation. Disruption of p28 ubiquitin ligase activity revealed that p28 is regulated through auto-ubiquitination and ubiquitination by an additional unknown ubiquitin ligase. Moreover, we observed Lysine-48 ubiquitin linkages, Lysine-63 ubiquitin linkages and a proteasomal subunit co-localizing with p28 at the viral factory, suggesting an intricate relationship between p28 and proteasomal degradation. / Virology