Genetic and molecular biological characterization of a vaccinia virus gene which renders the virus dependent on isatin-β-thiosemicarbazone (IBT)

Meis, Ronald J.; Condit, Richard C.

doi:10.1016/0042-6822(91)90585-y

Cited by 43 publications

(34 citation statements)

References 46 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The vaccinia G3L gene encodes a conserved polypeptide of 111 amino acids with a predicted molecular mass of 12.8 kDa (10). The G3L transcript was previously detected at the late phase of virus infection, indicating that G3L belongs to the viral late gene family (23). Hydropathy analysis (18) predicted that G3L has two hydrophobic domains at the N and C termini (Fig.…”

Section: Resultsmentioning

confidence: 92%

“…To be sure that insertion of the inducible G3L gene cassette did not interrupt expression of the neighboring G2R ORF, we included 50-nucleotide upstream sequences that contain putative early promoters for G2R ORF in the flanking region. That viG3L virus was successfully isolated and did not exhibit the G2R mutant phenotype (23) suggested that G2R expression is intact in viG3L virus and that early promoter for G2R resides within the upstream 50 nucleotides as expected. On the other hand, Meis and Condit determined the G2 early transcript (23) and concluded that the G2R promoter is 600 nucleotides upstream of the G2R ORF, which means that G2R transcription is initiated in G1L and transcribed through the entire G3L ORF and into G2R.…”

mentioning

confidence: 69%

See 1 more Smart Citation

The Envelope G3L Protein Is Essential for Entry of Vaccinia Virus into Host Cells

Izmailyan

Huang

Mohammad

et al. 2006

J Virol

View full text Add to dashboard Cite

The vaccinia virus G3L/WR079 gene encodes a conserved protein with a predicted transmembrane domain. Our proteomic analyses of vaccinia virus revealed that G3L protein is incorporated into intracellular mature virus; however, the function of G3L protein in the vaccinia virus life cycle has not been investigated. In this study, a recombinant vaccinia virus, viG3L, expressing G3L protein under IPTG (isopropyl-␤-D-thiogalactopyranoside) regulation was constructed. Under permissive conditions when G3L protein was expressed, the vaccinia virus life cycle proceeded normally, resulting in plaque formation in BSC40 cells. In contrast, under nonpermissive conditions when G3L protein expression was repressed, no plaques were formed, showing that G3L protein is essential for vaccinia virus growth in cell cultures. In infected cells when G3L protein was not expressed, the formation of intracellular mature virus (IMV) and cell-associated enveloped virus occurred normally, showing that G3L protein is not required for virion morphogenesis. IMV particles containing (G3L ؉ ) or lacking (G3L ؊ ) G3L protein were purified and were found to be indistinguishable on microscopic examination. Both G3L ؉ and G3L ؊ IMV bound to HeLa cells; however, G3L ؊ IMV failed to enter the cells, showing that G3L protein is required for IMV penetration into cells. Finally, G3L protein was required for fusion of the infected cells under low-pH treatment. Thus, our results provide direct evidence that G3L is an essential component of the vaccinia virus fusion complex, in addition to the previously reported A28, H2, L5, A21, and A16 proteins.Vaccinia virus, the prototype of the orthopoxvirus genus of the family Poxviridae, infects many cell lines and animals (9) and produces several forms of infectious particles, namely, intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV), and extracellular enveloped virus (EEV) (25). The membrane structure of IMV is different from that of IEV, which, again, is different from that of CEV and EEV (40). Although IMV and EEV were originally shown to enter cells through different mechanisms (22,35,39), recent studies have revealed that a conserved viral fusion complex is required for the penetration of both viruses (26,27,31,32,37,38). Although no cellular coreceptor has been identified for vaccinia virus, some data suggest that cell-bound IMV initiates plasma membrane fusion to deliver viral cores into cells (1,2,8,22,39). In addition, IMV entry has been shown to trigger cell signaling pathways that involve Rac, MEK, extracellular signal-regulated kinase, protein kinase A, and protein kinase C activation, but the molecular mechanism is unknown (7,20,22).IMV represents the majority of the infectious progeny produced in cells, and our recent proteomic study revealed that it contains 75 viral proteins (4). Of these, nine have been shown to play a role in vaccinia virus entry (see below). The viral envelope proteins, H3L and A27L, bind to heparan sulfates, whereas D8L b...

show abstract

Section: Resultsmentioning

confidence: 92%

mentioning

confidence: 69%

The Envelope G3L Protein Is Essential for Entry of Vaccinia Virus into Host Cells

Izmailyan

Huang

Mohammad

et al. 2006

J Virol

View full text Add to dashboard Cite

show abstract

“…To identify viral proteins involved in transcriptional processivity, Condit and colleagues isolated viral mutants resistant to isatin-␤-thiosemicarbazone (IBT) (55)(56)(57)(58)(59)(60). IBT leads to the synthesis of run-on transcripts, which in turn generate excessive dsRNA that leads to the arrest of infection by the antiviral response.…”

Section: Discussionmentioning

confidence: 99%

Genetic Confirmation that the H5 Protein Is Required for Vaccinia Virus DNA Replication

Boyle

Greseth

Traktman

2015

J Virol

View full text Add to dashboard Cite

The duplication of the poxvirus double-stranded DNA genome occurs in cytoplasmic membrane-delimited factories. This physical autonomy from the host nucleus suggests that poxvirus genomes encode the full repertoire of proteins committed for genome replication. Biochemical and genetic analyses have confirmed that six viral proteins are required for efficient DNA synthesis; indirect evidence has suggested that the multifunctional H5 protein may also have a role. Here we show that H5 localizes to replication factories, as visualized by immunofluorescence and immunoelectron microscopy, and can be retrieved upon purification of the viral polymerase holoenzyme complex. The temperature-sensitive (ts) mutant Dts57, which was generated by chemical mutagenesis and has a lesion in H5, exhibits defects in DNA replication and morphogenesis under nonpermissive conditions, depending upon the experimental protocol. The H5 variant encoded by the genome of this mutant is ts for function but not stability. For a more precise investigation of how H5 contributes to DNA synthesis, we placed the ts57 H5 allele in an otherwise wild-type viral background and also performed small interfering RNA-mediated depletion of H5. Finally, we generated a complementing cell line, CV-1-H5, which allowed us to generate a viral recombinant in which the H5 open reading frame was deleted and replaced with mCherry (v⌬H5). Analysis of v⌬H5 allowed us to demonstrate conclusively that viral DNA replication is abrogated in the absence of H5. The loss of H5 does not compromise the accumulation of other early viral replication proteins or the uncoating of the virion core, suggesting that H5 plays a direct and essential role in facilitating DNA synthesis. IMPORTANCEVariola virus, the causative agent of smallpox, is the most notorious member of the Poxviridae family. Poxviruses are unique among DNA viruses that infect mammalian cells, in that their replication is restricted to the cytoplasm of the cell. This physical autonomy from the nucleus has both cell biological and genetic ramifications. Poxviruses must establish cytoplasmic niches that support replication, and the genomes must encode the repertoire of proteins necessary for genome synthesis. Here we focus on H5, a multifunctional and abundant viral protein. We confirm that H5 associates with the DNA polymerase holoenzyme and localizes to the sites of DNA synthesis. By generating an H5-expressing cell line, we were able to isolate a deletion virus that lacks the H5 gene and show definitively that genome synthesis does not occur in the absence of H5. These data support the hypothesis that H5 is a crucial participant in cytoplasmic poxvirus genome replication. S mallpox has plagued humans throughout history. The etiological agent of this deadly disease is variola virus, a member of the Poxviridae family of viruses. Smallpox was declared eradicated as a natural pathogen in 1980 after a global vaccination campaign that utilized a closely related poxvirus, vaccinia virus. Vaccinia virus is now the protot...

show abstract

“…A role for the vaccinia virus G2R protein in transcription termination was initially revealed by the mapping of mutations conferring dependence on the anti-poxvirus drug isatin b-thiosemicarbazone (IBT) (Meis & Condit, 1991). G2R mutants display a phenotype in which intermediate and late transcripts are shorter than normal (Black & Condit, 1996), just the opposite of that of A18R mutants.…”

Section: Termination Of Intermediate and Late Transcriptionmentioning

confidence: 99%

Vaccinia virus transcription

Broyles

2003

Journal of General Virology

193

168

View full text Add to dashboard Cite

Genetic and molecular biological characterization of a vaccinia virus gene which renders the virus dependent on isatin-β-thiosemicarbazone (IBT)

Cited by 43 publications

References 46 publications

The Envelope G3L Protein Is Essential for Entry of Vaccinia Virus into Host Cells

The Envelope G3L Protein Is Essential for Entry of Vaccinia Virus into Host Cells

Genetic Confirmation that the H5 Protein Is Required for Vaccinia Virus DNA Replication

Vaccinia virus transcription

Contact Info

Product

Resources

About