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

Chan, Winnie; Ward, Brian M.

doi:10.1128/jvi.00253-12

Cited by 11 publications

(15 citation statements)

References 48 publications

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“…We also found it interesting that coimmunoprecipitation of cells infected with vA34 1-130 -V5 precipitated more B5 than that of cells infected with vA34R-V5. An identical result was reported for certain charge-to-alanine mutations in A33 (58). The mutants also resulted in a small-plaque phenotype.…”

Section: Discussionsupporting

confidence: 83%

“…The mutants also resulted in a small-plaque phenotype. Further analysis determined that as a result of this stronger interaction, A33 and B5 exist as a detectable complex on the surface of EV and resulted in a reduction in the ability of EV to bind cells (58). It has also been reported that A34 and B5 are present as a complex on the surface of EV during infection (46), and the addition of glycosaminoglycans (GAGs) resulted in a reduction in complex formation.…”

Section: Discussionmentioning

confidence: 99%

“…Alternatively, the defect in EV cell binding for our recombinant viruses could be indirectly due to the decrease in A33 and B5 incorporation (Fig. 9), as our laboratory has shown that both an A33 deletion virus (vΔA33R) and a B5 deletion virus (vΔB5R) exhibit a defect in the ability of EV to bind cells (51,58). A33 and A34, both of which independently interact with B5, could indirectly form a complex on the surface of EV and mediate cooperative cell binding.…”

Section: Discussionmentioning

confidence: 99%

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The Ectodomain of the Vaccinia Virus Glycoprotein A34 Is Required for Cell Binding by Extracellular Virions and Contains a Large Region Capable of Interaction with Glycoprotein B5

Monticelli

Earley

Tate

et al. 2019

J Virol

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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.

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Section: Discussionsupporting

confidence: 83%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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The Ectodomain of the Vaccinia Virus Glycoprotein A34 Is Required for Cell Binding by Extracellular Virions and Contains a Large Region Capable of Interaction with Glycoprotein B5

Monticelli

Earley

Tate

et al. 2019

J Virol

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“…These data show that EEV lacking F13 do not exhibit a defect in their ability to bind cells. It has been shown that the presence of A33 and B5 on EEV is required for efficient binding to cells (43,44). Furthermore, the deletion of A34 greatly reduces incorporation of B5 into the EV membrane and drastically reduces infectivity (45,46).…”

Section: Resultsmentioning

confidence: 99%

Vaccinia Virus Phospholipase Protein F13 Promotes Rapid Entry of Extracellular Virions into Cells

Bryk

Brewer

Ward

2018

J Virol

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The vaccinia virus protein F13, encoded by the F13L gene, is conserved across the subfamily and is critical among orthopoxviruses to produce the wrapped form of virus that is required for cell-to-cell spread. F13 is the major envelope protein on the membrane of extracellular forms of virus; however, it is not known if F13 is required in steps postwrapping. In this report, we utilize two temperature-sensitive vaccinia virus mutants from the Condit collection of temperature-sensitive viruses whose small plaque phenotypes have been mapped tothe F13L gene. Despite the drastic reduction in plaque size, the temperature-sensitive viruses were found to produce levels of extracellular virions similar to those of the parental strain, Western Reserve (WR), at the permissive and nonpermissive temperatures, suggesting that they are not defective in extracellular virion formation. Analyses of extracellular virions produced by one temperature-sensitive mutant found that those produced at the nonpermissive temperature had undetectable levels of F13 and bound cells with efficiency similar to that of WR but displayed delayed cell entry kinetics. Additionally, low-pH treatment of cells bound by extracellular virions produced at the nonpermissive temperature by the temperature-sensitive reporter virus was unable to overcome a block in infection by bafilomycin A1, suggesting that these virions display increased resistance to dissolution of the extracellular virion envelope. Taken together, our results suggest that F13 plays a role both in the formation of extracellular virions and in the promotion of their rapid entry into cells by enhancing the sensitivity of the membrane to acid-induced dissolution. Vaccinia virus (VACV) is an orthopoxvirus and produces two infectious forms, mature virions (MV) and extracellular virions (EV). EV are derived from MV and contain an additional membrane that must first be removed prior to cell entry. F13 is critical for the formation of EV, but a postenvelopment role has not been described. Here, two temperature-sensitive VACV mutants whose deficiencies were previously mapped to the F13L locus are characterized. Both viruses produced EV at the nonpermissive temperature at levels similar to those of a virus that has F13L, yet they had a small plaque phenotype and rate of spread similar to that of an F13L deletion virus. F13 was undetectable on the EV membrane at the nonpermissive temperature, and these EV exhibited delayed cell entry kinetics compared to EV containing F13. This study is the first to conclusively demonstrate a novel role for F13 in cell entry of the EV form of the virus.

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“…As this interaction is dependent on the coiled-coiled domain being anchored to a membrane, its role in adhesion across the viral envelope membranes is unclear and may represent an independent role for A33 in incorporation of B5 into the envelope, or vice versa [63], [64], [65]. Intriguingly, mutations that increase B5–A33 adhesion result in reduced binding of EEV to host cells suggesting a cis interaction in the membrane between these proteins and raising the possibility that A33 might mask and/or regulate the availability of the adjacent SCR4 [66]. Further characterisation of the nature of these interactions may clarify how adhesion between the outer viral membranes is achieved and regulated.…”

Section: Discussionmentioning

confidence: 99%

A36-dependent Actin Filament Nucleation Promotes Release of Vaccinia Virus

et al. 2013

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Cell-to-cell transmission of vaccinia virus can be mediated by enveloped virions that remain attached to the outer surface of the cell or those released into the medium. During egress, the outer membrane of the double-enveloped virus fuses with the plasma membrane leaving extracellular virus attached to the cell surface via viral envelope proteins. Here we report that F-actin nucleation by the viral protein A36 promotes the disengagement of virus attachment and release of enveloped virus. Cells infected with the A36YdF virus, which has mutations at two critical tyrosine residues abrogating localised actin nucleation, displayed a 10-fold reduction in virus release. We examined A36YdF infected cells by transmission electron microscopy and observed that during release, virus appeared trapped in small invaginations at the plasma membrane. To further characterise the mechanism by which actin nucleation drives the dissociation of enveloped virus from the cell surface, we examined recombinant viruses by super-resolution microscopy. Fluorescently-tagged A36 was visualised at sub-viral resolution to image cell-virus attachment in mutant and parental backgrounds. We confirmed that A36YdF extracellular virus remained closely associated to the plasma membrane in small membrane pits. Virus-induced actin nucleation reduced the extent of association, thereby promoting the untethering of virus from the cell surface. Virus release can be enhanced via a point mutation in the luminal region of B5 (P189S), another virus envelope protein. We found that the B5P189S mutation led to reduced contact between extracellular virus and the host membrane during release, even in the absence of virus-induced actin nucleation. Our results posit that during release virus is tightly tethered to the host cell through interactions mediated by viral envelope proteins. Untethering of virus into the surrounding extracellular space requires these interactions be relieved, either through the force of actin nucleation or by mutations in luminal proteins that weaken these interactions.

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

Cited by 11 publications

References 48 publications

The Ectodomain of the Vaccinia Virus Glycoprotein A34 Is Required for Cell Binding by Extracellular Virions and Contains a Large Region Capable of Interaction with Glycoprotein B5

The Ectodomain of the Vaccinia Virus Glycoprotein A34 Is Required for Cell Binding by Extracellular Virions and Contains a Large Region Capable of Interaction with Glycoprotein B5

Vaccinia Virus Phospholipase Protein F13 Promotes Rapid Entry of Extracellular Virions into Cells

A36-dependent Actin Filament Nucleation Promotes Release of Vaccinia Virus

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