Identification of viral genes essential for replication of murine γ-herpesvirus 68 using signature-tagged mutagenesis

The herpesvirus glycoprotein H (gH) and gL associate to form a heterodimer that plays a central role in virus-driven membrane fusion. When archetypal alpha-or betaherpesviruses lack gL, gH misfolds and progeny virions are noninfectious. In order to define the role that gL plays in gamma-2 herpesvirus infections, we disrupted its coding sequence in murine gammaherpesvirus-68 (MHV-68). MHV-68 lacking gL folded gH into a conformation antigenically distinct from the form that normally predominates on infected cells. gL-deficient virions bound less well than the wild type to epithelial cells and fibroblasts. However, they still incorporated gH and remained infectious. The cell-to-cell spread of gL-deficient viruses was remarkably normal, as was infection, dissemination, and latency establishment in vivo. Viral membrane fusion was therefore gL independent. The major function of gL appeared to be allowing gH to participate in cell binding prior to membrane fusion. This function was most important for the entry of MHV-68 virions into fibroblasts and epithelial cells.

Section: Vol 81 2007contrasting

confidence: 46%

Glycoprotein L Disruption Reveals Two Functional Forms of the Murine Gammaherpesvirus 68 Glycoprotein H

Gillet

May

Colaco

et al. 2007

“…ORF64 is a tegument protein that is essential for MHV-68 replication (22). Because of technical difficulties in expressing fulllength ORF64, we analyzed fragments of the ORF64 coding sequence and identified two ZAP-responsive elements (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Expression of RTA is necessary and sufficient to trigger reactivation into lytic replication in latently infected cell lines (16)(17)(18)(19). RTA activates transcription of multiple genes expressed in the lytic phase (16,17,20,21), some of which are essential for lytic replication (22).…”

mentioning

confidence: 99%

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ZAP Inhibits Murine Gammaherpesvirus 68 ORF64 Expression and Is Antagonized by RTA

Xuan

Gong

et al. 2013

bZinc finger antiviral protein (ZAP) is an interferon-inducible host antiviral factor that specifically inhibits the replication of certain viruses, including HIV-1 and Ebola virus. ZAP functions as a dimer formed through intermolecular interactions of its N-terminal tails. ZAP binds directly to specific viral mRNAs and inhibits their expression by repressing translation and/or promoting degradation of the target mRNA. ZAP is not a universal antiviral factor, since some viruses grow normally in ZAP-expressing cells. It is not fully understood what determines whether a virus is susceptible to ZAP. We explored the interaction between ZAP and murine gammaherpesvirus 68 (MHV-68), whose life cycle has latent and lytic phases. We previously reported that ZAP inhibits the expression of M2, which is expressed mainly in the latent phase, and regulates MHV-68 latency in cultured cells. Here, we report that ZAP inhibits the expression of ORF64, a tegument protein that is expressed in the lytic phase and is essential for lytic replication. MHV-68 infection induced ZAP expression. However, ZAP did not inhibit lytic replication of MHV-68. We provide evidence showing that the antiviral activity of ZAP is antagonized by MHV-68 RTA, a critical viral transactivator expressed in the lytic phase. We further show that RTA inhibits the antiviral activity of ZAP by disrupting the N-terminal intermolecular interaction of ZAP. Our results provide an example of how a virus can escape ZAP-mediated immunity.

“…Open reading frame 24 (ORF24) is conserved among all beta-and gammaherpesviruses. Both ORF24 of MHV-68 and its human cytomegalovirus homolog (UL87) have previously been identified as being essential for lytic replication by genome-wide mutagenesis (7,19,25). ORF24 of MHV-68 is 39% and 26% identical to the Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus homologs, respectively (23).…”

mentioning

confidence: 99%

Murine Gammaherpesvirus 68 Open Reading Frame 24 Is Required for Late Gene Expression after DNA Replication

Wong¹,

Wu²,

Reyes³

et al. 2007

Self Cite

Open reading frame 24 (ORF24) of murine gammaherpesvirus 68 (MHV-68) is conserved among beta-and gammaherpesviruses; however, its function in viral replication has not been defined. Using MHV-68 as a model, we have identified ORF24 as being essential for viral replication. An ORF24-null virus was generated and shown to be defective in late gene expression. Expression of early genes, as well as viral genome replication, was not affected. Furthermore, the defect in late gene expression was likely due to a deficiency in transcription. Thus, we have identified an MHV-68 protein, ORF24, that is essential for the expression of viral late proteins yet dispensable for viral DNA replication.Murine gammaherpesvirus 68 (MHV-68) has been used to study the replication cycle of gammaherpesvirus due to its ability to lytically infect various cell lines, including those of human and murine origins (6,17,(20)(21)(22)(23). Open reading frame 24 (ORF24) is conserved among all beta-and gammaherpesviruses. Both ORF24 of MHV-68 and its human cytomegalovirus homolog (UL87) have previously been identified as being essential for lytic replication by genome-wide mutagenesis (7,19,25). ORF24 of MHV-68 is 39% and 26% identical to the Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus homologs, respectively (23). However, it does not have any significant homology to any cellular proteins. The gene product of ORF24 was found to be associated with MHV-68 virions, but its function is unknown (4). Previously, array studies did not provide conclusive information regarding the kinetic class of ORF24 due to the lack of sensitivity of the arrays for less abundant transcripts (8, 13). However, one group has classified ORF24 as being an early gene (1).To characterize this viral gene, an ORF24-null virus, 24S, was generated by the insertion of a triple stop codon with a PstI restriction site into the N-terminal region (nucleotide [nt] 40056) of the ORF24 coding sequence on the wild-type (WT) MHV-68 bacterial artificial chromosome (BAC) by allelic exchange as described previously (3, 10, 18). A revertant virus (24R) was subsequently generated using allelic exchange of the 24S BAC plasmid with a FLAG-tagged WT ORF24 shuttle plasmid. Digestion with three restriction enzymes confirmed the correct genomic structure of the three viruses (Fig. 1A).Transfection of 24S BAC DNA into BHK-21 cells did not result in any detectable productive viral infection when surveyed for 7 days. However, when a FLAG-tagged ORF24 expression plasmid was cotransfected along with the 24S BAC DNA, a virally induced cytopathic effect was observed by 5 days posttransfection, similar to transfection with WT or 24R BAC DNA. Viral replication was further confirmed by examining the expression of capsid protein ORF65 in the transfected cells (Fig. 1B). No capsid protein was detected in the transfection of 24S DNA alone. However, capsid protein expression was restored in the 24S transfection by complementation with an ORF24 expression plasmid. To reconstitute the 24S virus, 2...