Entry of Vaccinia Virus and Cell-Cell Fusion Require a Highly Conserved Cysteine-Rich Membrane Protein Encoded by the A16L Gene

Abstract

The vaccinia virus A16L open reading frame encodes a 378-amino-acid protein with a predicted C-terminal transmembrane domain and 20 invariant cysteine residues that is conserved in all sequenced members of the poxvirus family. The A16 protein was expressed late in infection and incorporated into intracellular virus particles with the N-terminal segment of the protein exposed on the surface. The cysteine residues were disulfide bonded via the poxvirus cytoplasmic redox system. Unsuccessful attempts to isolate a mutant virus with the A16L gene deleted suggested that the protein is essential for replication. To study the role of the A16 protein, we made a recombinant vaccinia virus that has the Escherichia coli lac operator system regulating transcription of the A16L gene. In the absence of inducer, A16 synthesis was repressed and plaque size and virus yield were greatly reduced. Nevertheless, virus morphogenesis occurred and normal-looking intracellular and extracellular virus particles formed. Purified virions made in the presence and absence of inducer were indistinguishable, though the latter had 60-to 100-fold-lower specific infectivity. A16-deficient virions bound to cells, but their cores did not penetrate into the cytoplasm. Furthermore, A16-deficient virions were unable to induce low-pH-triggered syncytium formation. The phenotype of the inducible A16L mutant was similar to those of mutants in which synthesis of the A21, A28, H2, or L5 membrane protein was repressed, indicating that at least five conserved viral proteins are required for entry of poxviruses into cells as well as for cell-cell fusion.Vaccinia virus (VACV) is a member of the poxvirus family of large, double-stranded DNA viruses that replicate entirely in the cytoplasm (17). VACV strain Western Reserve (WR), the prototype member of the orthopoxvirus genus, contains approximately 200 genes of which 44 have a putative transmembrane domain (Poxvirus Bioinformatics Resource Center [www.poxvirus.org]). VACV membrane proteins have diverse essential and nonessential roles in virus-host interactions, virion assembly, intracellular movement, and cell-to-cell spread. A novel class of poxvirus membrane proteins, required for cell entry but not for virion assembly, was recently identified (20-22, 27, 28). These four VACV proteins (A21, A28, H2, and L5) are conserved in all sequenced poxviruses, have common features including N-terminal or near N-terminal transmembrane domains and cysteines that form two to four intramolecular disulfide bonds, and are located on the surfaces of infectious intracellular mature virions (IMVs). Each of the four proteins is also required for cell-cell fusion, implying that cell entry involves a related or identical fusion mechanism. Fusion of IMVs with the plasma membrane was previously suggested on the basis of biochemical and microscopic studies (4,7,8,11,15). Cellular receptor proteins that participate in cell entry, however, remain to be identified.Following virus morphogenesis, IMVs are completely enveloped by an additio...

Poxvirus Proteomics and Virus-Host Protein Interactions

“…The intermediate component, A2.5, forms disulfide-linked complexes with E10 and G4 (147). Oxidized G4 can then catalyze disulfide bond formation on a number of different poxvirus proteins such as A16, A21, A28, F9, H2, L1, and L5 (13,122,145,147,164,165). Proteins of the ERV/ALR family are encoded by all poxviruses and are characterized by a ϳ100-residue domain that is adapted for the catalysis of disulfide bond formation in various organelles and biological settings.…”

“…To allow for the formation of stable disulfide bonds in this cytoplasmic compartment, poxviruses exploit a novel virusspecified cytoplasmic redox pathway (147,148). Studies of this pathway revealed that the key virus-encoded components can be divided into two groups, viral redox-active proteins such as E10, A2.5, and G4 (146,149,174) and viral substrate proteins such as A16, A21, A28, F9, G9, H2, J5, L1, and L5 (13,122,145,164,165) (Table 3). Three viral redox-active proteins (E10, A2.5, and G4) play a role in the cytoplasmic redox pathway by transferring oxidizing potential from E10 through A2.5 to G4 (147).…”

Vaccinia Virus F9 Virion Membrane Protein Is Required for Entry but Not Virus Assembly, in Contrast to the Related L1 Protein

“…Initial experiments indicated that the protein was associated with MVs purified by sucrose density gradient centrifugation. Compared to L1, F9 was poorly soluble upon extraction with NP-40 or NP-40 plus DTT (data not shown) as found for some components of the entry/fusion complex (22,23). Evidence for the location of F9 in the MV membrane was obtained by surface biotinylation of purified MVs.…”

“…G. Cohen and R. Eisenberg (University of Pennsylvania) provided R192 and R180 rabbit antibodies raised against secreted baculovirus-expressed F9 and L1 recombinant proteins, respectively. Rabbit polyclonal antibodies against the following VACV peptides or proteins were used: A10 (R. Doms and B. Moss, unpublished), A4 (10), A21 (37), L5 (36), A16 (23), and A28 (G. Nelson and B. Moss, unpublished). The anti-L1 monoclonal antibody 7D11 (42) was prepared from a hybridoma kindly provided by A. Schmalljohn (United States Army Medical Research Institute for Infectious Diseases).…”

“…Those proteins associated with the MV membrane can be placed into at least four functional groups involved in membrane crescent formation, e.g., A14 and A17 (25,27,38,41); virion maturation, e.g., A9 and L1 (24,43); virus attachment to glycosaminoglycans, e.g., H3 and D8 (14,18); and virus entry, e.g., A16, A21, A28, G9, H2, and L5 (22,23,30,32,36). When expression of any one of the aforementioned entry proteins is repressed, typical-looking MVs and EVs form but they are defective in entry and induction of syncytia after brief low-pH treatment.…”