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Association of vaccinia virus fusion regulatory proteins with the multicomponent entry/fusion complex.

ABSTRACT: The proteins encoded by the A56R and K2L genes of vaccinia virus form a heterodimer (A56/K2) and have a fusion regulatory role as deletion or mutation of either causes infected cells to form large syncytia spontaneously. Here, we showed that syncytia formation is dependent on proteins of the recently described entry fusion complex (EFC), which are also required for virus-cell fusion and low-pH-triggered cell-cell fusion. This finding led us to consider that A56/K2 might prevent fusion by direct or indirect interaction with the EFC. To test this hypothesis, we made a panel of recombinant vaccinia viruses that have a tandem affinity purification tag attached to A56, K2, or the A28 EFC protein. Interaction between A56/K2 and the EFC was demonstrated by their copurification from detergent-treated lysates of infected cells and identification by mass spectrometry or Western blotting. In addition, a purified soluble transmembrane-deleted form of A56/K2 was shown to interact with the EFC. Tagged A56 did not interact with the EFC in the absence of K2, nor did tagged K2 interact with the EFC in the absence of A56. The finding that both A56 and K2 are required for efficient binding to the EFC fits well with prior experiments showing that mutation of either A56 or K2 results in spontaneous fusion of infected cells. Because A56 and K2 are located on the surface of infected cells, they are in position to interact with the EFC of released progeny virions and prevent back-fusion and syncytia formation.

SUBMITTER: Wagenaar TR

PROVIDER: S-EPMC1900102 | biostudies-literature | 2007 Jun

REPOSITORIES: biostudies-literature

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Association of vaccinia virus fusion regulatory proteins with the multicomponent entry/fusion complex.

Wagenaar Timothy R TR Moss Bernard B

Journal of virology 20070404 12

The proteins encoded by the A56R and K2L genes of vaccinia virus form a heterodimer (A56/K2) and have a fusion regulatory role as deletion or mutation of either causes infected cells to form large syncytia spontaneously. Here, we showed that syncytia formation is dependent on proteins of the recently described entry fusion complex (EFC), which are also required for virus-cell fusion and low-pH-triggered cell-cell fusion. This finding led us to consider that A56/K2 might prevent fusion by direct ...[more]

PMID: 17409143

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Project description:For cell entry, vaccinia virus requires fusion with the host membrane via a viral fusion complex of 11 proteins, but the mechanism remains unclear. It was shown previously that the viral proteins A56 and K2 are expressed on infected cells to prevent superinfection by extracellular vaccinia virus through binding to two components of the viral fusion complex (G9 and A16), thereby inhibiting membrane fusion. To investigate how the A56/K2 complex inhibits membrane fusion, we performed experimental evolutionary analyses by repeatedly passaging vaccinia virus in HeLa cells overexpressing the A56 and K2 proteins to isolate adaptive mutant viruses. Genome sequencing of adaptive mutants revealed that they had accumulated a unique G9R open reading frame (ORF) mutation, resulting in a single His44Tyr amino acid change. We engineered a recombinant vaccinia virus to express the G9H44Y mutant protein, and it readily infected HeLa-A56/K2 cells. Moreover, similar to the ΔA56 virus, the G9H44Y mutant virus on HeLa cells had a cell fusion phenotype, indicating that G9H44Y-mediated membrane fusion was less prone to inhibition by A56/K2. Coimmunoprecipitation experiments demonstrated that the G9H44Y protein bound to A56/K2 at neutral pH, suggesting that the H44Y mutation did not eliminate the binding of G9 to A56/K2. Interestingly, upon acid treatment to inactivate A56/K2-mediated fusion inhibition, the G9H44Y mutant virus induced robust cell-cell fusion at pH 6, unlike the pH 4.7 required for control and revertant vaccinia viruses. Thus, A56/K2 fusion suppression mainly targets the G9 protein. Moreover, the G9H44Y mutant protein escapes A56/K2-mediated membrane fusion inhibition most likely because it mimics an acid-induced intermediate conformation more prone to membrane fusion.IMPORTANCE It remains unclear how the multiprotein entry fusion complex of vaccinia virus mediates membrane fusion. Moreover, vaccinia virus contains fusion suppressor proteins to prevent the aberrant activation of this multiprotein complex. Here, we used experimental evolution to identify adaptive mutant viruses that overcome membrane fusion inhibition mediated by the A56/K2 protein complex. We show that the H44Y mutation of the G9 protein is sufficient to overcome A56/K2-mediated membrane fusion inhibition. Treatment of virus-infected cells at different pHs indicated that the H44Y mutation lowers the threshold of fusion inhibition by A56/K2. Our study provides evidence that A56/K2 inhibits the viral fusion complex via the latter's G9 subcomponent. Although the G9H44Y mutant protein still binds to A56/K2 at neutral pH, it is less dependent on low pH for fusion activation, implying that it may adopt a subtle conformational change that mimics a structural intermediate induced by low pH.

Project description:Poxviruses are known to encode a set of proteins that form an entry-fusion complex (EFC) to mediate virus entry. However, the diversity, evolution, and origin of these EFC proteins remain poorly understood. Here, we identify the EFC protein homologs in poxviruses and other giant viruses of the phylum Nucleocytoviricota. The 11 EFC genes are present in almost all poxviruses, with the two smallest, G3 and O3, being absent in Entomopoxvirinae and basal lineages of Chordopoxvirinae. Five of the EFC genes are further grouped into two families, A16/G9/J5 and F9/L1, which are widely distributed across other major lineages of Nucleocytoviricota, including metagenome-assembled genomes, but are generally absent in viruses infecting algae or nonamoebozoan heterotrophic protists. The A16/G9/J5 and F9/L1 families cooccur, mostly as single copies, in 93% of the non-Poxviridae giant viruses that have at least one of them. Distribution and phylogenetic patterns suggest that both families originated in the ancestor of Nucleocytoviricota. In addition to the Poxviridae genes, homologs from each of the other Nucleocytoviricota families are largely clustered together, suggesting their ancient presence and vertical inheritance. Despite deep sequence divergences, we observed noticeable conservation of cysteine residues and predicted structures between EFC proteins of Poxviridae and other families. Overall, our study reveals widespread distribution of these EFC protein homologs beyond poxviruses, implies the existence of a conserved membrane fusion mechanism, and sheds light on host range and ancient evolution of Nucleocytoviricota. IMPORTANCE Fusion between virus and host membranes is critical for viruses to release genetic materials and to initiate infection. Whereas most viruses use a single protein for membrane fusion, poxviruses employ a multiprotein entry-fusion complex (EFC). We report that two major families of the EFC proteins are widely distributed within the virus phylum Nucleocytoviricota, which includes poxviruses and other double-stranded (dsDNA) giant viruses that infect animals, amoebozoans, algae, and various microbial eukaryotes. Each of these two protein families is structurally conserved, traces its origin to the root of Nucleocytoviricota, was passed down to the major subclades of Nucleocytoviricota, and is retained in most giant viruses known to infect animals and amoebozoans. The EFC proteins therefore represent a potential mechanism for virus entry in diverse giant viruses. We hypothesize that they may have facilitated the infection of an animal/amoebozoan-like host by the last Nucleocytoviricota common ancestor.

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Association of vaccinia virus fusion regulatory proteins with the multicomponent entry/fusion complex.

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Association of vaccinia virus fusion regulatory proteins with the multicomponent entry/fusion complex.

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