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

Abstract

All sequenced poxviruses encode orthologs of the vaccinia virus L1 and F9 proteins, which are structurally similar and share about 20% amino acid identity. We found that F9 further resembles L1 as both proteins are membrane components of the mature virion with similar topologies and induce neutralizing antibodies. In addition, a recombinant vaccinia virus that inducibly expresses F9, like a previously described L1 mutant, had a conditional-lethal phenotype: plaque formation and replication of infectious virus were dependent on added inducer. However, only immature virus particles are made when L1 is repressed, whereas normal-looking intracellular and extracellular virions formed in the absence of F9. Except for the lack of F9, the polypeptide components of such virions were indistinguishable from those of wild-type virus. These F9-deficient virions bound to cells, but their cores did not penetrate into the cytoplasm. Furthermore, cells infected with F9-negative virions did not fuse after a brief low-pH treatment, as did cells infected with virus made in the presence of inducer. In these respects, the phenotype associated with F9 deficiency was identical to that produced by the lack of individual components of a previously described poxvirus entry/fusion complex. Moreover, F9 interacted with proteins of that complex, supporting a related role. Thus, despite the structural relationships of L1 and F9, the two proteins have distinct functions in assembly and entry, respectively.Vaccinia virus (VACV) is a member of the Poxviridae, a family of large enveloped double-stranded DNA viruses that replicate entirely in the cytoplasm (19). The nearly 200 genes of VACV encode enzymes and factors for transcription (5) and replication (21) of the genome, assembly of virus particles (8), cell entry (20), and modulation of the immune response (1). Assembly begins, in areas of the cytoplasm that are partially cleared of cellular organelles, with the creation of crescent membranes that enclose electron-dense material known as viroplasm. Membrane curvature, imposed by an external protein lattice, results in the formation of the spherical immature virion (IV) containing structural proteins, enzymes, and the viral genome. Further development, including the disassembly of the scaffold, proteolytic cleavages, and core condensation, results in the infectious barrel-shaped mature virion (MV). Most MVs remain in the cytoplasm until cell lysis enables their spread. A subpopulation of MVs undergoes wrapping by a double membrane derived from modified trans-Golgi or endosomal cisternae, allowing them to transit along microtubules to the periphery of the cell. There, the outer membrane fuses with the plasmalemma, resulting in exocytosis of the extracellular virion (EV), which is essentially an MV with an additional membrane. Prior to infecting another cell, the EV covering membrane is discarded (17), allowing the MV membrane to fuse with the cell membrane (4,6,7,15,39).Sequence-based predictions suggest that the Western Reserve (WR) strain of V...

Characterization of a Newly Identified 35-Amino-Acid Component of the Vaccinia Virus Entry/Fusion Complex Conserved in All Chordopoxviruses

“…A distinction between the integral components of the EFC and the so-called "EFC-associated" proteins is the exclusive role of the former in the assembly or stability of the complex (3,4,39). To determine the effect of repressing O3 expression, the A28R ORF in vO3-HAi was replaced with a copy containing tandem-affinity calmodulin and streptavidin binding peptide tags to form vO3-HAi-A28TAP.…”

Poxvirus Proteomics and Virus-Host Protein Interactions

“…These proteins are conserved in all poxviruses, which suggests that their functions are nonredundant and essential. Recently, coimmunoprecipitation studies confirmed that F9 and L1, which are also required for cell entry and membrane fusion, interact with the EFC (13,18). The poxvirus regulatory fusion proteins A56 and K2 have been shown to form a complex in the EV and at the cell surface by immunoprecipitation (166) ( Table 4).…”

Protein Primary Structure of the Vaccinia Virion at Increased Resolution

“…Of these proteins, A17L, A26L, and L1R are established membrane proteins, as are E8R (39) and F9L (40). G9R is presumed to be membrane associated, as a myristoylated member of the poxvirus entry-fusion complex (41).…”