Effects of deletion or stringent repression of the H3L envelope gene on vaccinia virus replication

The C-terminal membrane anchor protein encoded by the H3L open reading fram e of vaccinia virus is located on the surfaces of intracellular mature viri ons. To investigate the role of the H3L protein, we constructed deletion (v H3 Delta) and inducible (vH3i) null mutants. The H3L protein was not detect ed in lysates of cells infected with vH3 Delta or vH3i in the absence of in ducer. Under these conditions, plaques were small and round instead of larg e and comet shaped, indicative of decreased virus replication or cell-to-ce ll spread. The mutant phenotype was correlated with reduced yields of infec tious intra- and extracellular virus in one-step growth experiments. The de fect in vH3i replication could not be attributed to a role of the H3L prote in in virus binding, internalization, or any event prior to late gene expre ssion. Electron microscopic examination of cells infected with vH3 Delta or vH3i in the absence of inducer revealed that virion assembly was impaired, resulting in a high ratio of immature to mature virus forms with an accumu lation of crescent membranes adjacent to granular material and DNA crystall oids, The absence of the H3L protein did not impair the membrane localizati on of virion surface proteins encoded by the A27L, D8L, and L1R genes. The wrapping of virions and actin tail formation were not specifically blocked, but there was an apparent defect in low-pN-mediated syncytium formation th at could be attributed to decreased virus particle production. The phenotyp es of the H3L deletion and repression mutants were identical to each other but differed from those produced by null mutations of genes encoding other vaccinia virus membrane components.