9A and B). of the transmembrane domain, their small size, and their sequence diversity. Nevertheless,trans-complementation experiments demonstrated the ability of a representative O3-like protein from each chordopoxvirus genus to rescue the infectivity of a VACV mutant that was unable to express endogenous O3. Moreover, recombinant viruses expressing O3 homologs in place of O3 replicated and formed plaques as well or nearly as well as wild-type VACV. The O3 homologs expressed by the recombinant VACVs were incorporated into the membranes of mature virions and, with one exception, remained stably associated with the detergent-extracted and affinity-purified EFC. The ability of the sequence-divergent O3 homologs to coordinate function with VACV entry proteins suggests the conservation of structural motifs. Analysis of chimeras formed by swapping domains of O3 with those of other proteins indicated that the N-terminal transmembrane segment was responsible for EFC interactions and for the complementation of infectivity. == INTRODUCTION == Vaccinia virus (VACV), the best-studied member of thePoxviridae, is a large DNA virus that encodes about 200 proteins and replicates entirely in the cytoplasm of infected cells (17). The mature virion (MV) enters cells via the plasma membrane at a neutral pH or by a low-pH-dependent endosomal route following macropinocytosis or fluid-phase uptake (1,11,16,18,27). Four proteins participate in cell attachment via glycosaminoglycans and laminin (6,7,10,15), but no specific cell receptor has been identified. At least 12 MV proteins participate in membrane fusion and Methyl Hesperidin core entry; with one possible exception, these proteins are physically associated to form the entry-fusion complex (EFC) (3,4,13,1926). The roles of the entry proteins have been determined mainly by analysis of inducible conditional lethal mutants. Under nonpermissive conditions, normal-appearing but noninfectious virions form. These defective virions can bind to cells but are unable to mediate membrane fusion and core entry. In addition, the absence of individual EFC proteins destabilizes the entire complex. The protein requirements for the entry of poxviruses into cells have been studied exclusively with VACV. However, homologs of 11 of the 12 EFC proteins are highly conserved in all chordopoxviruses, making it likely that they use the same entry mechanism. A possible exception is the VACV O3 protein. The O3L open reading frame (ORF), encoding the O3 protein, was not annotated originally Rabbit Polyclonal to ITGA5 (L chain, Cleaved-Glu895) because of its small size (35 amino acids). Nevertheless, we demonstrated that the protein was expressed and that the phenotype of an inducible mutant under nonpermissive conditions was similar to that of other EFC mutants (22). Although a VACV O3L deletion mutant was constructed, it replicated poorly, and entry was very inefficient. A BLAST search against the translated nucleotide database (tblastn) revealed homologous proteins with 100% sequence identity in other orthopoxviruses; the exception was the monkeypox virus homolog, with 2 to 3 3 amino acid substitutions. However, BLAST hits were not found in all chordopoxvirus genera. Moreover, the statistical significance of the hits was weakened because of the small size of the proteins and the bias caused by the contribution of the transmembrane (TM) domain (30). Nevertheless, an ORF predicting a small hydrophobic protein is located in the same relative position and orientation in all chordopoxvirus genomes (22). Since the O3 protein is required to stabilize the VACV EFC, presumably through multiple protein interactions (22), it seemed possible that the sequence-divergent O3-like Methyl Hesperidin proteins might exhibit genus specificity. Remarkably, we found that the related proteins of other chordopoxviruses can complement a VACV O3 deficiency despite Methyl Hesperidin extensive sequence diversity. Furthermore, we demonstrate the importance of the N-terminal hydrophobic domain in EFC interactions and the function of the O3 homologs. == MATERIALS AND METHODS == == Cells and viruses. == BS-C-1 cells.