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.

An interaction between the orthopoxvirus glycoproteins A34 and B5 has been reported. The transmembrane and ectodomain of A34 are sufficient for interaction with B5, localization of B5 to the site of intracellular wrapping, and subsequent incorporation into the envelope of released extracellular virions. Several mutagenic approaches were undertaken to better define the B5 interaction domain on A34. A set of C-terminal truncations in A34 identified residues 1 to 80 as sufficient for interaction with B5. Additional truncations identified residues 80 to 130 of A34 as sufficient for interaction with B5. To better understand the function of this region, a set of recombinant viruses expressing A34 with the full, partial, or no B5 interaction site (residues 1 to 130, 1 to 100, and 1 to 70, respectively) was constructed. All the recombinants expressing truncations of A34 incorporated B5 into extracellular virions but had a small-plaque phenotype similar to that of a virus with the A34R gene deleted (vΔA34R). Further characterization indicated that the small-plaque phenotype exhibited by these viruses is due to a combination of abrogated actin tail formation, reduced cell binding, and a defect in polyanion-induced nonfusogenic dissolution. Taken together, these results suggest that residues 80 to 130 of A34 are not necessary for the proper localization and incorporation of B5 into extracellular virions and, furthermore, that the C-terminal residues of A34 are involved in cell binding and dissolution. IMPORTANCE Previous studies have shown that the vaccinia virus glycoproteins A34 and B5 interact, and in the absence of A34, B5 is mislocalized and not incorporated into extracellular virions. Here, using a transient-transfection assay, residues 80 to 130 of the ectodomain of A34 were determined to be sufficient for interaction with B5. Recombinant viruses expressing A34 with a full, partial, or no B5 interaction site were constructed and characterized. All of the A34 truncations interacted with B5 as predicted by the transient-transfection studies but had a small-plaque phenotype. Further analysis revealed that all of the recombinants incorporated detectable levels of B5 into released virions but were defective in cell binding and extracellular virion (EV) dissolution. This study is the first to directly demonstrate that A34 is involved in cell binding and implicate the ectodomain in this role.

Orthopoxviruses produce two, antigenically distinct, infectious enveloped virions termed intracellular mature virions and extracellular virions. Extracellular virions are required for cell-to-cell spread and pathogenesis. Specific to the extracellular virion membrane, glycoproteins A33, A34, and B5 are highly conserved among orthopoxviruses and have roles during extracellular virion formation and subsequent infection. B5 is dependent on an interaction with either A33 or A34 for localization to the site of intracellular envelopment and incorporation into the envelope of released extracellular virions. In this report we show that an interaction between A33 and A34 can be detected in infected cells. Furthermore, we show that a three-protein complex between A33, A34, and B5 forms in the endoplasmic reticulum (ER) that disassociates post ER export. Finally, immunofluorescence reveals that coexpression of all three glycoproteins results in their localization to a juxtanuclear region that is presumably the site of intracellular envelopment. These results demonstrate the existence of two previously unidentified interactions: one between A33 and A34 and another simultaneous interaction between all three of the glycoproteins. Furthermore, these results indicate that interactions among A33, A34, and B5 are vital for proper intracellular trafficking and subcellular localization. IMPORTANCE The secondary intracellular envelopment of poxviruses at the trans-Golgi network to release infectious extracellular virus (EV) is essential for their spread and pathogenesis. Viral glycoproteins A33, A34, and B5 are critical for the efficient production of infectious EV and interactions among these proteins are important for their localization and incorporation into the outer extracellular virion membrane. We have uncovered a novel interaction between glycoproteins A33 and A34. Furthermore, we show that B5 can interact with the A33-A34 complex. Our analysis indicates that the three-protein complex has a role in ER exit and proper localization of the three glycoproteins to the intracellular site of wrapping. These results show that a complex set of interactions occur in the secretory pathway of infected cells to ensure proper glycoprotein trafficking and envelope content, which is important for the release of infectious poxvirus virions.

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