Transcriptional repression and RNA silencing act synergistically to demonstrate the function of the eleventh component of the vaccinia virus entry-fusion complex.

Poxviruses have an elaborate system for infecting cells comprising several proteins for attachment and a larger number dedicated to membrane fusion and entry. Thus far, 11 proteins have been identified as components of the vaccinia virus (VACV) entry-fusion complex (EFC), and 10 of these proteins have been shown to be required for entry. J5, the remaining functionally uncharacterized component of the complex, is conserved in all poxviruses, has a predicted C-terminal transmembrane domain, and is an N-terminally truncated paralog of two other EFC proteins. To determine the role of J5, we constructed a mutant that inducibly regulates J5 transcription. Although the virus yield was reduced only about 80% without inducer, the inability to isolate a J5 deletion mutant suggested an essential function. To enhance stringency, we employed RNA silencing alone and together with transcriptional repression of the inducible mutant. The yield of infectious virus was reduced 4- to 5-fold by repression, 2-fold by silencing, and 60-fold by the combination of the two. Virus particles made under the latter conditions appeared to contain a full complement of proteins excluding J5 but had very low infectivity. Further studies indicated that after binding to cells, J5-deficient virions had a defect in core entry and an inability to induce syncytium formation. In addition, we confirmed that J5 is associated with the EFC by affinity purification. These data indicate that J5 is a functional component of the EFC and highlights the advantage of combining transcriptional repression and RNA silencing for stringent reduction of gene expression.