Clustered Charge-to-Alanine Mutagenesis of the Vaccinia Virus H5 Gene: Isolation of a Dominant, Temperature-Sensitive Mutant with a Profound Defect in Morphogenesis

Abstract

The vaccinia virus H5 gene encodes a 22.3-kDa phosphoprotein that is expressed during both the early and late phases of viral gene expression. It is a major component of virosomes and has been implicated in viral transcription and, as a substrate of the B1 kinase, may participate in genome replication. To enable a genetic analysis of the role of H5 during the viral life cycle, we used clustered charge-to-alanine mutagenesis in an attempt to create a temperature-sensitive (ts) virus with a lesion in the H5 gene. Five mutant viruses were isolated, with one of them, tsH5-4, having a strong ts phenotype as assayed by plaque formation and measurements of 24-h viral yield. Surprisingly, no defects in genome replication or viral gene expression were detected at the nonpermissive temperature. By electron microscopy, we observed a profound defect in the early stages of virion morphogenesis, with arrest occurring prior to the formation of crescent membranes or immature particles. Nonfunctional, "curdled" virosomes were detected in tsH5-4 infections 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 initiation of virion morphogenesis. By constructing recombinant genomes containing two H5 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.Vaccinia virus is the prototypical member of the Poxvirus family, members of which include the human pathogens variola virus, the causative agent of smallpox, and molluscum contagiosum virus, which causes benign but long-lived skin lesions. The 192-kb double-stranded DNA genome of vaccinia virus contains approximately 200 genes, enabling the virus to replicate quite autonomously within the cytoplasm of infected cells (17,31). The virus encodes a complex transcriptional machinery which directs the expression of three classes of temporally regulated genes: early, which encode the proteins required for replication of the viral genome, and intermediate and late, among whose products are those required for virion morphogenesis. Virion formation commences from cytoplasmic sites known as virosomes or viral factories, which contain viral DNA and viral proteins. Membrane crescents form, begin to enclose the virosome contents, and then enlarge and seal to form the visually distinguishable immature (IV) and mature (IMV) forms of the intracellular class of virions. A subset of these virions become wrapped in membranes derived from the transGolgi network and exit the cell. The genome also encodes a large number of effector proteins that enable the virus to evade host defenses.Protein phosphorylation is known to play a crucial role in the regulation of many cellular processes such as initiation of DNA replication, signal transduction, and cell cycle control. Similarly, vaccinia virus regulates its life cycle by ...

Inhibition of CD1d1‐mediated antigen presentation by the vaccinia virus B1R and H5R molecules

“…The former is the product of the VV H5R gene product, a transcription factor expressed both early and late in the virus life cycle, and is involved in the transcription of VV late genes [14,21]. A VV that contains a ts mutation in the gene encoding the H5R protein (tsH5R VV) was also included in the experiments with the B1R mutants [22]. As observed with the tsB1R VV, infection of L-CD1 cells for 4 h with the WT or tsH5R VV at the permissive 31 C resulted in a substantial decrease in CD1d1-dependent, NKT cell-produced IL-2 ( Fig.…”

“…Jonathan Yewdell and Jack Bennink (Laboratory of Viral Diseases, NIAID, NIH, Bethesda, MD). The VV containing temperature-sensitive B1R [17,18] or H5R [22] mutants or a mutant defective in viral DNA synthesis, tsVV-17.1 [17,18], were of the WR strain and obtained from R. Condit (B1R and 17.1; Univ. of Florida, Gainesville, FL) and P. Traktman (H5R; Medical College of Wisconsin, Milwaukee, WI).…”

Existence of an operative pathway from the endoplasmic reticulum to the immature poxvirus membrane

“…Consequently, the initial steps of viral membrane formation were not examined here. Previous studies had shown that several VACV proteins are needed to form recognizable viral membrane precursors; these include the F10 protein kinase (32) and the A11 (33), H5 (34), and G3 (35) proteins, none of which have TM domains. Repression of either the A17 (36,37) or A14 (38,39) membrane proteins leads to the accumulation of numerous vesicles and tubules.…”

Vaccinia Virus Uracil DNA Glycosylase Interacts with the A20 Protein to Form a Heterodimeric Processivity Factor for the Viral DNA Polymerase

“…For generation of vvfUDG, the overall strategy was to replace the endogenous D4 locus with one encoding a 3XFLAG-tagged allele; this was accomplished using the plasmid described above and wt virus. Generation of the virus was achieved by transient dominant selection as described (6,16,17).…”