Clustered charge-to-alanine mutagenesis of the vaccinia virus H5 gene: Isolation of a dominant, temperature-sensitive mutant with a profound defect in morphogenesis

The vaccinia virus H5 gene encodes a 22.3-kDa phosphoprotein that is expres sed during both the early and late phases of viral gene expression. It is a major component of virosomes and has been implicated in viral transcriptio n and, as a substrate of the B1 kinase, may participate in genome replicati on. To enable a genetic analysis of the role of H5 during the viral life cy cle, we:used clustered charge-to-alanine mutagenesis in an attempt to creat e a temperature-sensitive (ts) virus with a lesion in the H5 gene. Five mut ant viruses were isolated, with one of them, tsH5-4, having a strong fs phe notype as assayed by plaque formation and measurements of 24-h viral yield, Surprisingly, no defects in genome replication or viral gene expression we re detected at the nonpermissive temperature. By electron microscopy, we ob served a profound defect in the early stages of virion morphogenesis, with arrest occurring prior to the formation of crescent membranes or immature p articles. Nonfunctional, "curdled" virosomes were detected in tsH5-4 infect ions at the nonpermissive temperature. These structures appeared to revert to functional virosomes after a temperature shift to permissive conditions, We suggest an essential role for H5 in normal virosome formation and the i nitiation of virion morphogenesis. By constructing recombinant genomes cont aining two 115 alleles, wild type and H5-4, we determined that H5-4 exerted a dominant phenotype. tsH5-4 is the first example of a dominant ts mutant isolated and characterized in vaccinia virus.