J5 is the 11th protein shown to be a functional component of the EFC. Determination of the role of J5 was more difficult than for other VACV entry proteins. Indeed, this study was stalled for more than 5 years because of an apparent contradiction: a deletion mutant could not be isolated, suggesting that J5 is essential, yet replication of a J5 mutant was incompletely inhibited in the absence of inducer even though the repression appeared complete based on Western blotting. We suspected that some J5 was made but the amount was below the detection threshold of the antibody and that the stringency of repression was insufficient for complete inhibition of virus replication. Furthermore, we considered it possible that there could be excess EFCs on the surfaces of VACV MVs and that entry into cultured cells could still occur with a greatly reduced number. Unfortunately, we cannot test this hypothesis, as methods to determine the EFC density on virions have not yet been devised. In this regard, however, it is worth noting that a single envelope trimer is sufficient for HIV entry (46
Several recent studies indicated that siRNAs can inhibit VACV replication, although they were not used to analyze the roles of the silenced genes (9
). In a screen of siRNAs to 12 monkeypox virus ORFs, knockdown of the J5 homolog reduced the virus yield by approximately 60% (2
). Although we did not expect siRNA alone to reduce the amount of J5 more efficiently than the inducible mutant, it occurred to us that the combination might work synergistically. Thus, if J5 mRNA could be greatly reduced by repressing synthesis, then the siRNA might effectively degrade the small remainder. We found that the yield of infectious virus was reduced 4- to 5-fold by repression alone, about 2-fold by siRNA alone, but by more than 60-fold together. The siRNAs were most effective when transfected prior to infection (C.L.W., unpublished data) probably because the RNA-induced silencing complex had additional time to form and was distributed throughout the cytoplasm before establishment of specialized DNA factories where VACV transcription occurs (18
). We are unsure why repression more efficiently prevents infectivity of other EFC mutants than the J5 mutant. However, the effectiveness of the inducible system appears to vary with gene location and the stringency needed to produce a functional defect, so that supplementation with siRNA may be a generally useful strategy.
The phenotype resulting from stringent reduction of J5 was similar to that occurring when expression of other EFC proteins is repressed, indicating that the J5 paralogs A16 and G9 cannot substitute for J5. When J5 was reduced, a normal yield of MVs formed, indicating the absence of effects on viral gene expression or morphogenesis. The protein composition of J5-deficient virions appeared similar to that of the wild type by staining SDS-polyacrylamide gels and Western blotting of representative core and membrane proteins, including components of the EFC. Nevertheless, the purified J5-deficient virions had very low infectivity. Further studies indicated that the block was at core entry rather than MV binding and that as a consequence early gene expression was severely inhibited.
Interestingly, repression of J5 was sufficient to prevent syncytium formation from within or without; the added stringency of siRNA was not required. This result suggests that more EFCs are needed to fuse cells together than to allow entry of virus particles.
We confirmed an earlier report that J5 is physically associated with the EFC (33
). The prior study used an affinity tag attached to the A28 EFC protein to capture the complex, and here we used a tag attached to the J5 protein itself to show association with other EFC proteins. An interesting feature of J5 is its homology with two other proteins of the EFC, A16 and G9. The three proteins are conserved in all poxviruses, suggesting that they arose from two gene duplications prior to the diversification of present poxvirus genera and subsequently evolved distinct functions in virion entry. The conservation of cysteine residues in the three proteins is striking. Evidence for intramolecular disulfide bonds was obtained for A16 (28
), and it is likely that these bonds are also formed in the related G9 and J5 proteins. The intramolecular disulfide bonds of A16 and other EFC proteins are formed by the VACV-encoded cytoplasmic redox system, which is also conserved in all poxviruses (35
), suggesting that the redox and entry systems arose together during evolution.
With 11 functional components of the EFC known, are there more to be identified? The small I2 protein is a good candidate, since the phenotype of a conditional lethal mutant is similar to that of EFC proteins with the exception that repression decreases the amounts of EFC components in MVs (26
). Because of its small size and hydrophobicity, the I2 protein, like the O3 EFC protein (30
), may have been missed by mass spectroscopy of the purified complex (33
). The overall architecture of the EFC has not been determined, although a few protein-protein interactions that resist destabilization of the complex have been identified (25