The Vaccinia Virus B5 Protein Requires A34 for Efficient Intracellular Trafficking from the Endoplasmic Reticulum to the Site of Wrapping and Incorporation into Progeny Virions

Abstract

The glycoproteins encoded by the vaccinia virus A34R and B5R genes are involved in intracellular envelope virus formation and are highly conserved among orthopoxviruses. A recombinant virus that has the A34R gene deleted and the B5R gene replaced with a B5R gene fused to the enhanced green fluorescent protein (B5R-GFP) gene was created (vB5R-GFP/⌬A34R) to investigate the role of A34 during virion morphogenesis. Cells infected with vB5R-GFP/⌬A34R displayed GFP fluorescence throughout the cytoplasm, which differed markedly from that seen in cells infected with a normal B5R-GFP-expressing virus (vB5R-GFP). Immunofluorescence and subcellular fractionation demonstrated that B5-GFP localizes with the endoplasmic reticulum in the absence of A34. Expression of either full-length A34 or a construct consisting of the lumenal and transmembrane domains restored normal trafficking of B5-GFP to the site of wrapping in the juxtanuclear region. Coimmunoprecipitation studies confirmed that B5 and A34 interact through their luminal domains, and further analysis revealed that in the absence of A34, B5 is not efficiently incorporated into virions released from the cell.

Modified Vaccinia Virus Ankara-Infected Dendritic Cells Present CD4 + T-Cell Epitopes by Endogenous Major Histocompatibility Complex Class II Presentation Pathways

“…Given that B5R and OVA are not expressed upon PUVA treatment, the reduced but still existing response of CD4 B5R after infection of BMDCs with PUVA-treated MVA indicates that the main antigenic source of B5 for the response is virions, and only a small proportion of the response is due to newly synthesized B5. This is conceivable, as B5R is a type I membrane glycoprotein incorporated into the membrane of the enveloped forms of vaccinia virus (intracellular enveloped virus [IEV], cellassociated enveloped virus [CEV], and EEV) (40). In contrast, when PUVA-treated MVA was used for infection, CD4…”

Poxvirus Proteomics and Virus-Host Protein Interactions

“…Since no direct interaction between B5 and A36 has been found, a possible role for the A33-B5 interaction is to incorporate A36 into the complex. The lumenal domains of A34 and B5 are sufficient to mediate their interaction (50,124). This interaction is required for the proper targeting and subsequent incorporation of B5 into EVs since the amount of B5 is greatly reduced in EVs formed in the absence of A34 (50).…”

“…The lumenal domains of A34 and B5 are sufficient to mediate their interaction (50,124). This interaction is required for the proper targeting and subsequent incorporation of B5 into EVs since the amount of B5 is greatly reduced in EVs formed in the absence of A34 (50). The PPI between A34 and A36 has been studied (124,138).…”

Attenuated and Replication-Competent Vaccinia Virus Strains M65 and M101 with Distinct Biology and Immunogenicity as Potential Vaccine Candidates against Pathogens

“…Five proteins of VACV A33, A34, B5, F12, and F13 are present in the envelope of the virus, and the deletion of any of them causes a reduction in plaque size on cell monolayers (42). Among all those proteins, only A34 and F13L presented missense mutations in the genome of M65 and M101 and both proteins were mutated in both viruses.…”

“…M65 and M101 showed one missense mutation in the A34R gene (required for efficient intracellular trafficking from the endoplasmic reticulum to the site of wrapping) (42), in two different positions, S45I and Stop169R, respectively. The mutation in the stop codon adds 6 amino acids (R, Q, Q, K, M, and N) before reaching the next stop codon.…”