ORF18 Is a Transfactor That Is Essential for Late Gene Transcription of a Gammaherpesvirus

In this study, we adopted a conditional protein genetic approach to characterize the role of the human cytomegalovirus (HCMV) gene UL79 during virus infection. We constructed ADddUL79, a recombinant HCMV in which the annotated UL79 open reading frame (ORF) was tagged with the destabilization domain of a highly unstable variant of the human FKBP12 protein (ddFKBP). The ddFKBP domain targets the tagged protein for rapid proteasomal degradation, but the synthetic ligand Shield-1 can stabilize ddFKBP, allowing accumulation of the tagged protein. ADddUL79 failed to replicate without Shield-1, but it grew at wild-type levels with Shield-1 or in human foreskin fibroblasts overexpressing hemagglutinin (HA)-tagged UL79 (HF-UL79HA cells), indicating an essential role of UL79 and the effectiveness of this approach. Without Shield-1, representative immediate-early and early viral proteins as well as viral DNA accumulated normally, but late transcripts and proteins were markedly reduced. UL79 was transcribed with early-late kinetics, which was also regulated via a positive-feedback loop. Using HF-UL79HA cells, we found that the UL79 protein localized to viral replication compartments during HCMV infection. Finally, we created a second UL79 mutant virus (ADinUL79 stop ) in which the UL79 ORF was disrupted by a stop codon mutation and found that ADinUL79 stop phenocopied ADddUL79 under the destabilizing condition. Taking these results together, we conclude that UL79 acts after viral DNA replication to promote the accumulation of late viral transcripts. Importantly, the comparative analysis of ADddUL79 and ADinUL79 stop viruses provide additional proof for the power of the protein stability-based conditional approach to dissect the role of viral factors in HCMV biology.

Section: Discussionmentioning

confidence: 99%

“…8). However, UL79 shares only 28% amino acid identity to ORF18 (2) and is transcribed with early-late kinetics (Fig. 6), whereas ORF18 was identified as an early gene (33).…”

Section: Discussionmentioning

confidence: 99%

The Human Cytomegalovirus Gene UL79 Is Required for the Accumulation of Late Viral Transcripts

Perng

Zhou

Fehr

et al. 2011

“…These MHV-68 ORFs are expressed early but are not required for viral DNA replication. Since these MHV-68 proteins activate late viral promoters driving expression of an indicator gene, they are considered late viral transactivators (3,54,55). How late viral transactivators specifically activate a late viral promoter remains unclear.…”

Section: Discussionmentioning

confidence: 99%

“…However, the HCMV IE genes alone are not sufficient for activation of the true late viral genes. Which HCMV early genes are required for late gene expression is not known.ORFs 18, 24, 30, and 34 of murine gammaherpesvirus 68 (MHV-68) are essential for late gene transcription (3,54,55) and have homology to HCMV ORFs UL79, -87, and -95. The MHV-68 gene products function specifically to stimulate the promoters of late genes, since their absence has little effect on early viral gene expression or DNA replication.…”

mentioning

confidence: 99%

The Human Cytomegalovirus Gene Products Essential for Late Viral Gene Expression Assemble into Prereplication Complexes before Viral DNA Replication

Isomura

Stinski

Murata

et al. 2011

The regulation of human cytomegalovirus (HCMV) late gene expression by viral proteins is poorly understood, and these viral proteins could be targets for novel antivirals. HCMV open reading frames (ORFs) UL79, -87, and -95 encode proteins with homology to late gene transcription factors of murine gammaherpesvirus 68 ORFs 18, 24, and 34, respectively. To determine whether these HCMV proteins are also essential for late gene transcription of a betaherpesvirus, we mutated HCMV ORFs UL79, -87, and -95. Cells were infected with the recombinant viruses at high and low multiplicities of infection (MOIs). While viral DNA was detected with the recombinant viruses, infectious virus was not detected unless the wild-type viral proteins were expressed in trans. At a high MOI, mutation of ORF UL79, -87, or -95 had no effect on the level of major immediate-early ( Human cytomegalovirus (HCMV) is a member of the betaherpesvirus family. The viral genome is 240,000 bp long and has at least 150 predicted open reading frames (ORFs) (9, 57). Although infection by HCMV occurs in most individuals, it is usually asymptomatic. The virus is reactivated under immunosuppressive conditions and causes pneumonitis, hepatitis, retinitis, and gastrointestinal diseases. The virus replicates productively in terminally differentiated cells such as fibroblasts, epithelial and endothelial cells, and monocyte-derived macrophages (11,12,18,28,41,42,48).During productive infection, HCMV genes are expressed in a temporal cascade, with temporal phases designated immediate-early (IE), early, and late. The major IE genes (MIE) UL123 and UL122 (IE1/IE2) play a critical role in subsequent viral gene expression and the efficiency of viral replication (19,20,24,(33)(34)(35). The early viral genes encode proteins necessary for viral DNA replication (36). Following viral DNA replication, delayed early and late viral genes are expressed which encode structural proteins for the virion. Expression of true late (gamma 2 class) viral genes is prevented by inhibition of viral DNA synthesis. UL75 (gH), UL99 (pp28), and the middle transcription start site of UL44 are examples of HCMV genes that are true late transcripts (26,29,32,53). While the regulation of HCMV immediate-early and early gene expression has been studied extensively, little is known about the specific mechanisms of regulating late gene expression.With HCMV, the IE2 protein can be found in microfoci early after infection and is associated with parental viral genomes by direct DNA contact. Some of the microfoci enlarge to form replication compartments (RCs) at late times after infection (43). However, the HCMV IE genes alone are not sufficient for activation of the true late viral genes. Which HCMV early genes are required for late gene expression is not known.ORFs 18, 24, 30, and 34 of murine gammaherpesvirus 68 (MHV-68) are essential for late gene transcription (3,54,55) and have homology to HCMV ORFs UL79, -87, and -95. The MHV-68 gene products function specifically to stimulate the promoters of late g...

“…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.…”

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...