Generation and characterization of a large panel of murine monoclonal antibodies against vaccinia virus

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A group of vaccinia virus (VACV) proteins, including A11, L2, and A6, are required for biogenesis of the primary envelope of VACV, specifically, for the acquisition of viral membrane precursors. However, the interconnection among these proteins is unknown and, with the exception of L2, the connection of these proteins with membranes is also unknown. In this study, prompted by the findings that A6 coprecipitated A11 and that the cellular distribution of A11 was dramatically altered by repression of A6 expression, we studied the localization of A11 in cells by using immunofluorescence and cell fractionation analysis. A11 was found to associate with membranes and colocalize with virion membrane proteins in viral replication factories during normal VACV replication. A11 partitioned almost equally between the detergent and aqueous phases upon Triton X-114 phase separation, demonstrating an intrinsic affinity with lipids. However, in the absence of infection or VACV late protein synthesis, A11 did not associate with cellular membranes. Furthermore, when A6 expression was repressed, A11 did not colocalize with any viral membrane proteins or associate with membranes. In contrast, when virion envelope formation was blocked at a later step by repression of A14 expression or by rifampin treatment, A11 colocalized with virion membrane proteins in the factories. Altogether, our data showed that A11 associates with viral membranes during VACV replication, and this association requires A6 expression. This study provides a physical connection between A11 and viral membranes and suggests that A6 regulates A11 membrane association.

Section: Methodsmentioning

confidence: 82%

“…MAbs against A11 (10G11) and A6 (10F1) were developed from mice immunized with recombinant A11 or A6 protein, respectively. The hybridomas were generated as described previously (16).…”

Section: Methodsmentioning

confidence: 99%

Vaccinia Virus Virion Membrane Biogenesis Protein A11 Associates with Viral Membranes in a Manner That Requires the Expression of Another Membrane Biogenesis Protein, A6

Meng

Yan

et al. 2012

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“…Murine anti-A33 MAb (KA10 IgG2a isotype) was generated from a mouse immunized with VACV as described previously (74). Additional unpublished VACV-specific MAbs, including 12D4.1 (IgG1), were gen-erated similarly.…”

Section: Methodsmentioning

confidence: 99%

Unusual Features of Vaccinia Virus Extracellular Virion Form Neutralization Resistance Revealed in Human Antibody Responses to the Smallpox Vaccine

Benhnia

Maybeno

Blum

et al. 2013

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“…Cells were infected with VACV at a multiplicity of infection (MOI) of 5 for 8 h. The cells were then lysed for Western blot analysis as described previously (14). The detection antibodies were mouse monoclonal antibodies (MAbs) against V5 (Sigma-Aldrich), Hsp70 (Santa Cruz), and WR148 (15) and rabbit polyclonal antibodies against VACV D12 (1).…”

Section: Construction Of Recombinant Vaccinia Viruses Vk1lmentioning

confidence: 99%

C7L Family of Poxvirus Host Range Genes Inhibits Antiviral Activities Induced by Type I Interferons and Interferon Regulatory Factor 1

Meng

Schoggins

Rose

et al. 2012

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cVaccinia virus (VACV) K1L and C7L function equivalently in many mammalian cells to support VACV replication and antagonize antiviral activities induced by type I interferons (IFNs). While K1L is limited to orthopoxviruses, genes that are homologous to C7L are found in diverse mammalian poxviruses. In this study, we showed that the C7L homologues from sheeppox virus and swinepox virus could rescue the replication defect of a VACV mutant deleted of both K1L and C7L (vK1L ؊ C7L ؊ ). Interestingly, the sheeppox virus C7L homologue could rescue the replication of vK1L ؊ C7L ؊ in human HeLa cells but not in murine 3T3 and LA-4 cells, in contrast to all other C7L homologues. Replacing amino acids 134 and 135 of the sheeppox virus C7L homologue, however, made it functional in the two murine cell lines, suggesting that these two residues are critical for antagonizing a putative host restriction factor which has some subtle sequence variation in human and murine cells. Furthermore, the C7L family of host range genes from diverse mammalian poxviruses were all capable of antagonizing type I IFN-induced antiviral activities against VACV. Screening of a library of more than 350 IFN-stimulated genes (ISGs) identified interferon-regulated factor 1 (IRF1) as an inhibitor of vK1L ؊ C7L ؊ but not wild-type VACV. Expression of either K1L or C7L, however, rendered vK1L ؊ C7L؊ resistant to IRF1-induced antiviral activities. Altogether, our data show that K1L and C7L antagonize IRF1-induced antiviral activities and that the host modulation function of C7L is evolutionally conserved in all poxviruses that can readily replicate in tissue-cultured mammalian cells. Poxviruses are a family of complex DNA viruses that replicate entirely in the cytoplasm and dedicate a substantial amount of their genome coding capacity for modulating host immune responses (16). Based on differences in genetics and host range, poxvirus family members that infect mammalian hosts are classified into seven genera: Orthopoxvirus, Parapoxvirus, Capripoxvirus, Leporipoxvirus, Suipoxvirus, Molluscipoxvirus, and Yatapoxvirus (16). Vaccinia virus (VACV), a member of the Orthopoxvirus genus, serves as the vaccine for smallpox and is the prototypical poxvirus. VACV has a broad host range in vivo and in cell lines, as it is capable of entering nearly all cell types examined to date and initiating the synthesis of the early set of viral proteins (11). However, whether viral replication proceeds to completion or not depends on the host cell type and often requires virus-carried host range genes, including K1L and C7L (11). VACV requires either K1L or C7L for productive replication in most mammalian cells (3, 17), but it requires neither for replication in avian cells and in a few specific mammalian cell lines, such as human hepatoma Huh7 cells (13). When Huh7 cells are pretreated with type I interferon (IFN), however, K1L or C7L is required for replication beyond early gene expression (13), suggesting that K1L and C7L support VACV replication by antagonizing some antivira...