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

The original annotation of the vaccinia virus (VACV) genome was limited to open reading frames (ORFs) of at least 65 amino acids. Here, we characterized a 35-amino-acid ORF (O3L) located between ORFs O2L and I1L. ORFs similar in length to O3L were found at the same genetic locus in all vertebrate poxviruses. Although amino acid identities were low, the presence of a characteristic N-terminal hydrophobic domain strongly suggested that the other poxvirus genes were orthologs. Further studies demonstrated that the O3 protein was expressed at late times after infection and incorporated into the membrane of the mature virion. An O3L deletion mutant was barely viable, producing tiny plaques and a 3-log reduction in infectious progeny. A mutant VACV with a regulated O3L gene had a similar phenotype in the absence of inducer. There was no apparent defect in virus morphogenesis, though O3-deficient virus had low infectivity. The impairment was shown to be at the stage of virus entry, as cores were not detected in the cytoplasm after virus adsorption. Furthermore, O3-deficient virus did not induce fusion of infected cells when triggered by low pH. These characteristics are hallmarks of a group of proteins that form the entry/fusion complex (EFC). Affinity purification experiments demonstrated an association of O3 with EFC proteins. In addition, the assembly or stability of the EFC was impaired when expression of O3 was repressed. Thus, O3 is the newest recognized component of the EFC and the smallest VACV protein shown to have a function.Vaccinia virus (VACV), the best-studied member of the poxvirus family of cytoplasmic DNA viruses, encodes ∼200 genes, some of which are still uncharacterized (27). The focus of the present study is VACV O3L, a short 35-amino-acid open reading frame (ORF) that was recognized by homology to a 41-amino-acid ORF in molluscum contagiosum virus (37) but not previously investigated. Here, we show that O3L is conserved in all chordopoxviruses, expressed late in infection, and involved in cell entry.Considerable information regarding VACV entry has been obtained during the past several years (28). There are two related infectious forms of VACV: the mature virion (MV) and the enveloped virions (EV). The MV is comprised of a lipoprotein membrane enclosing a nucleoprotein core, whereas the EV has an additional outer membrane that must be disrupted before fusion can occur (24). The MV can enter cells either by fusion at the plasma membrane (7) or by a low-pH-mediated endosomal route involving macropinocytosis (20, 26, 44). Regardless of which route is used, the ability of VACV to enter cells depends on a large number of proteins in the MV membrane that form or are associated with the entry/fusion complex (EFC) (39). Using genetic and biochemical methods, 11 entry/fusion proteins have been identified: A16 (33), A21 (43), A28 (40), F9 (4), G3 (21), G9 (32), H2 (38), I2 (31), J5 (39), L1 (3), and L5 (42). Eight of these proteins (A16, A21, A28, G3, G9, H2, J5, and L5) comprise the EFC, which depends on multiple interactions for assembly or stability. Although the structure of the EFC remains to be elucidated, there is evidence for direct interactions between A28 and H2 (30) and between A16 and G9 (50). An additional role for A16 and G9 involves an interaction with the A56/K2 heterodimer, which is present on the surface of infected cells, to prevent spontaneous cell-cell fusion and superinfection by progeny virus (45, 46, 48-50). Binding of L1 to an unidentified cell receptor has been suggested (16). Roles in membrane fusion have also been considered for A17 and A27 (23).Here we provide physical and functional evidence that O3 (VACWR069.5) is an integral component of the EFC and participates in virus entry and membrane fusion. With just 35 amino acids, O3 is the smallest VACV protein with a defined function.