Experimental Evolution To Isolate Vaccinia Virus Adaptive G9 Mutants That Overcome Membrane Fusion Inhibition via the Vaccinia Virus A56/K2 Protein Complex

Abstract

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.

Structural and functional analysis of vaccinia viral fusion complex component protein A28 through NMR and molecular dynamic simulations

“…Whole cell lysate from transient complementation assays and protein samples after coimmunoprecipitations as described above were resolved in SDS-containing sample buffer, separated on SDS-PAGE gels, and transferred onto nitrocellulose membranes for immunoblot analyses using the following antibodies: anti-A16; anti-G9; anti-L1; anti-G3; anti-L5; anti-O3; anti-F9; anti-H3; and anti-D8 antibodies, which have all been described previously [70]. Anti-A28, anti-H2, anti-J5 and anti-A21 rabbit sera were prepared by immunizing rabbits with soluble recombinant proteins, respectively.…”

“…Cell-cell fusion assays triggered by VACV MV were performed as described previously [70] with slight modification. In brief, GFP-or RFP-expressing HeLa cells were mixed at a 1:1 ratio, seeded in 96-well plates, and cultured overnight.…”

Structural and functional analysis of vaccinia viral fusion complex component protein A28 through NMR and molecular dynamic simulations

“…previously [69]. Anti-A28, anti-H2, anti-J5 and anti-A21 rabbit sera were prepared by immunizing rabbits with soluble recombinant proteins, respectively.…”

“…Cell-cell fusion assays triggered by VACV MV were performed as described previously [69] with slight modification. In brief, equal amounts of the inf/tf lysates harvested from transient complementation assays were applied to confluent GFP-or RFP-expressing HeLa cells mixed at a 1:1 ratio, seeded in 96-well plates, and cultured overnight.…”

Structural and functional analyses of viral H2 protein of the vaccinia virus entry fusion complex

“…Among them, the A16:G9 subcomplex participates in regulating membrane fusion by interacting with the fusion suppressors A26 [34,35] or the A56/K2 complex in an acid-dependent manner [35,36]. Interestingly, evolutionary studies have uncovered that not only poxviruses but also many nucleocytoplasmic large DNA viruses (NCLDV) also host viral genes encoding homologs of some EFC components, implying the presence of a conserved membrane fusion mechanism mediated by these DNA viruses [37].…”