Clustered charge-to-alanine mutagenesis of the vaccinia virus H5 gene: Isolation of a dominant, temperature-sensitive mutant with a profound defect in morphogenesis
J. Demasi et P. Traktman, Clustered charge-to-alanine mutagenesis of the vaccinia virus H5 gene: Isolation of a dominant, temperature-sensitive mutant with a profound defect in morphogenesis, J VIROLOGY, 74(5), 2000, pp. 2393-2405
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.