Acidic residues in the membrane-proximal stalk region of vaccinia virus protein B5 are required for glycosaminoglycan-mediated disruption of the extracellular enveloped virus outer membrane

2010

Virology

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.

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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

2010

Virology

“…Previous studies have shown that the cytoplasmic tail (25) and a large portion of the extracellular domain of B5 (14) are not required for EV formation, indicating an important role for the transmembrane domain and the coiled-coil structure of B5. In addition, the coiled-coil structure, or stalk, has been reported to be critical for ligand-induced rupture of the outer EEV membrane (34). The fact that A33-HA interacts with ⌬/cc/G/⌬-Strep, a construct that has only the coiled-coil domain of B5 and the transmembrane domain of VSVG, suggests that the coiled-coil domain of B5 is sufficient for interaction with A33 (Fig.…”

Section: Discussionmentioning

confidence: 99%

The A33-Dependent Incorporation of B5 into Extracellular Enveloped Vaccinia Virions Is Mediated through an Interaction between Their Lumenal Domains

2012

J Virol

There are two mechanisms for the incorporation of B5 into the envelope of extracellular virions produced by orthopoxviruses, one that requires A33 and one that does not. We have hypothesized that the A33-dependent mechanism requires a direct interaction between A33 and B5. In this study, chimeric constructs of A33 and B5/B5-green fluorescent protein (GFP) were used to show that the two proteins interact through their lumenal domains and that the coiled-coil domain of B5 is sufficient for an interaction with A33. Furthermore, our experiments reveal that a transmembrane domain, not necessarily its own, is requisite for the lumenal domain of B5 to interact with A33. In contrast, the lumenal domain of A33 is sufficient for interaction with B5. Furthermore, the lumenal domain of A33 is sufficient to restore the proper localization of B5-GFP in infected cells. Taken together, our results demonstrate that the lumenal domains of A33 and B5 interact and that the interaction is required for the incorporation of B5-GFP into extracellular virions, whereas the incorporation of A33 is independent of B5. These results suggest that viral protein incorporation into extracellular virions is an active process requiring specific protein-protein interactions. R emarkably, orthopoxviruses produce two infectious forms that are morphologically and antigenically distinct (1, 35). Viral replication occurs entirely in the cytoplasm of infected cells in a specialized area known as the viral factory, where the first form of infectious virions, termed intracellular mature virions (IMV), is produced (7, 26). IMV represent the majority of progeny virions and are released only if the cell is lysed. A subset of IMV is transported along microtubules to the site of wrapping and obtains an additional double membrane envelope derived from the trans-Golgi network or early endosome (15,40,45). These wrapped virions are termed intracellular enveloped virions (IEV). IEV are transported along microtubules to the plasma membrane and released from the cytoplasm by fusion of their outermost membrane with the plasma membrane (13,17,33,52). Virions retained on the cell surface are termed cell-associated enveloped virions (CEV). Some CEV are propelled away from infected cells by actin tails, a process that requires the viral A36 protein (16,37,44,56). When CEV are released from the cell surface, they become extracellular enveloped virions (EEV). CEV are required for efficient cell-to-cell spread (43, 52), while EEV are required for longrange dissemination of virus (1, 30). CEV and EEV are collectively termed extracellular virions (EV) (27).Eight proteins encoded by vaccinia virus, A33 (36), A34 (8), A36 (48), A56 (42), B5 (11, 55), F12 (47, 58), F13 (2), and K2 (46, 49), have been shown to be specific for IEV. Deletion of the gene that encodes any of these proteins, with the exception of A56 and K2, results in a small-plaque phenotype, implying that these proteins have a role in EV morphogenesis and/or infectivity. Several interactions among these proteins hav...

“…This raises the possibility that B5 and A33 mediate cooperative target cell binding. A34 and B5 have been shown to be present as a complex on the surface of EEV (27). It has also been reported that in the absence of B5, A34 is present at a reduced level (2).…”

Section: Figmentioning

confidence: 97%

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

2012

J Virol

Two mechanisms exist for the incorporation of B5 into extracellular virions, one of which is dependent on A33. In the companion to this paper (W. M. Chan and B. M. Ward, J. Virol. 86:8210 -8220, 2012), we show that the lumenal domain of A33 is sufficient for interaction with the coiled-coil domain of B5 and capable of directing B5-green fluorescent protein (GFP) into extracellular virions. Here, we have created a panel of charge-to-alanine mutations in the lumenal domain of A33 to map the B5 interaction site. While none of these mutations abolished the interaction with B5, a subset displayed an increased interaction with both B5 and B5-GFP. Both B5 and B5-GFP recombinant viruses expressing these mutant proteins in place of normal A33 had a small-plaque phenotype. The increased interaction of the mutant proteins was detected during infection, suggesting that normally the interaction is either weak or transient. In addition, the increased A33-B5 interaction was detected on virions produced by recombinant viruses and correlated with reduced target cell binding. Taken together, these results show that both B5 and B5-GFP interact with A33 during infection and that the duration of this interaction needs to be regulated for the production of fully infectious extracellular virions. (22). Replication in the cytoplasm results in two infectious forms, intracellular mature viruses (IMV) and extracellular viruses (EV) (7, 28). EV arise from IMV that have been wrapped in additional membranes derived from the transGolgi complex or early endosome (16,32,36) and are actively released from infected cells to spread infection. The process of wrapping IMV to form EV produces intermediate forms called intracellular enveloped virions (IEV) that contains two more membranes than IMV and one less membrane than the released EV form. The vaccinia virus proteins A33 (29), A34 (8), A56 (33), B5 (11, 47), F13 (1), and K2 (37, 40) are found on both the IEV and EV forms, whereas F12 (38, 50) and A36 (39) are found only on the IEV form. Deletion of any of the genes corresponding to these proteins, except for A56R and K2L, results in a small-plaque phenotype, indicating that they are involved in IEV formation, egress, and/or infectivity of EV.Multiple interactions between the EV and IEV proteins have been demonstrated. The B5 protein has been reported to interact with A33, A34, and F13 (3, 5, 6, 10, 24-26, 31). B5R encodes a 42-kDa type I glycoprotein that is involved in EV formation (11,12,18,47). The lumenal domain of B5 contains four short consensus repeats (SCRs), which represent most of the extracellular domain, followed by a predicted coiled-coil domain that precedes the transmembrane domain and a short cytoplasmic tail (11, 18). The coiled-coil domain is sufficient for interaction with the lumenal domain of A33 (4), while deletion of either the cytoplasmic tail or the four SCRs of B5 does not affect EV formation (15,21).A33R is predicted to encode a 21-kDa type II glycoprotein (29) with a predicted C-type lectin-like domain (35). It has b...