There is an A33-dependent mechanism for the incorporation of B5-GFP into vaccinia virus extracellular enveloped virions

Abstract

Orthopoxviruses produce two, antigenically distinct, infectious virions, intracellular mature virions and extracellular virions (EV). A33 and B5 are found on EV but not intracellular mature virions. To investigate the function of A33, a recombinant virus that has A33R deleted and expresses B5R-GFP (vB5R-GFP/ΔA33R) was generated. A comparison of vB5R-GFP/ΔA33R to an analogous virus (vΔA33R) revealed an additional defect in infectious EV production that was not apparent when A33R was present. Characterization of these recombinants revealed that EV produced in the absence of A33 had undetectable levels of B5-GFP. Both recombinants released similar amounts of EV but there were differences in their infectivity. Approximately equal numbers of virions produced by these recombinants were able to bind cells even though EV produced by vB5R-GFP/ΔA33R do not contain B5. These results suggest that in the absence of A33, the cytoplasmic tail of B5 contributes to its incorporation into the envelope of progeny virions.

Increased Interaction between Vaccinia Virus Proteins A33 and B5 Is Detrimental to Infectious Extracellular Enveloped Virion Production

“…The B5 protein has been reported to interact with A33, A34, and F13 (3,5,6,10,(24)(25)(26)31). B5R encodes a 42-kDa type I glycoprotein that is involved in EV formation (11,12,18,47).…”

“…It has been shown to form homodimers that can be linked by interprotein disulfide bonds (3,30). A33 has been shown to be required for efficient target cell binding by EV, and deletion of A33R results in the release of more EV from the cell surface with reduced infectivity (5,30). During morphogenesis, A33 interacts with IEV protein A36 through the cytoplasmic tail (46,49).…”

“…The following day, cells were harvested by scraping, washed once in PBS, and lysed in radioimmunoprecipitation assay (RIPA) buffer (0.5ϫ PBS, 0.1% sodium dodecyl sulfate, 1% Triton X-100, 1% NP-40, 0.5% sodium deoxycholate) containing protease inhibitors as previously described (5). Immunoprecipitation was performed using an anti-HA MAb (Santa Cruz Biotechnology) as previously described (10).…”

“…The binding assay has been described previously (5). Briefly, RK 13 cells were infected with vB5R-GFP, vA33R…”

Interaction of Poxvirus Intracellular Mature Virion Proteins with the TPR Domain of Kinesin Light Chain in Live Infected Cells Revealed by Two-Photon-Induced Fluorescence Resonance Energy Transfer Fluorescence Lifetime Imaging Microscopy

“…Later in the infection, it disrupts microtubule organization, centrosome function, and actin organization and polymerizes actin beneath membrane-bound, enveloped virus particles to assist dispersal (11). During morphogenesis, various different types of VACV particles are observed within the infected cell: immature virions (IV), intracellular mature virus (IMV; also known as mature virions), intracellular enveloped virus (IEV; also known as wrapped virions), cell-associated enveloped virus (CEV), and extracellular enveloped virus (EEV); the last two particle types are also known collectively as extracellular virions (EV) (9,32,37,46). These particles display different VACV proteins on their surfaces, dependent primarily on the nature and origins of the particular outer membrane that surrounds the nucleic acid-containing core.…”