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Control of Rta expression critically determines transcription of viral and cellular genes following gammaherpesvirus infection.

ABSTRACT: The replication and transcriptional activator (Rta), encoded by ORF50 of gammaherpesviruses, initiates the lytic cycle of gene expression; therefore understanding the impact of Rta on viral and cellular gene expression is key to elucidating the transcriptional events governing productive infection and reactivation from latency. To this end, the impact of altering Rta transcription on viral and cellular gene expression was studied in the context of a whole virus infection. Recombinant murine gammaherpesvirus (MHV)-68 engineered to overexpress Rta greatly accelerated expression of specific lytic cycle ORFs, but repressed transcription of the major latency gene, ORF73. Increased expression of Rta accelerated the dysregulation in transcription of specific cellular genes when compared with cells infected with wild-type and revertant viruses. A subset of cellular genes was dysregulated only in cells infected with Rta-overexpressing virus, and never in those infected with non-overexpressing viruses. These data highlight the critical role of Rta abundance in governing viral and cellular gene transcription, and demonstrate the importance of understanding how the relative expression of ORF50 during the virus life cycle impacts on these processes.

SUBMITTER: Hair JR

PROVIDER: S-EPMC2884955 | biostudies-literature | 2007 Jun

REPOSITORIES: biostudies-literature

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Control of Rta expression critically determines transcription of viral and cellular genes following gammaherpesvirus infection.

Hair James R JR Lyons Paul A PA Smith Kenneth G C KGC Efstathiou Stacey S

The Journal of general virology 20070601 Pt 6

The replication and transcriptional activator (Rta), encoded by ORF50 of gammaherpesviruses, initiates the lytic cycle of gene expression; therefore understanding the impact of Rta on viral and cellular gene expression is key to elucidating the transcriptional events governing productive infection and reactivation from latency. To this end, the impact of altering Rta transcription on viral and cellular gene expression was studied in the context of a whole virus infection. Recombinant murine gamm ...[more]

PMID: 17485528

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Project description:UnlabelledReplication and transcription activator (RTA) of gammaherpesvirus is an immediate early gene product and regulates the expression of many downstream viral lytic genes. ORF48 is also conserved among gammaherpesviruses; however, its expression regulation and function remained largely unknown. In this study, we characterized the transcription unit of ORF48 from murine gammaherpesvirus 68 (MHV-68) and analyzed its transcriptional regulation. We showed that RTA activates the ORF48 promoter via an RTA-responsive element (48pRRE). RTA binds to 48pRRE directly in vitro and also associates with ORF48 promoter in vivo. Mutagenesis of 48pRRE in the context of the viral genome demonstrated that the expression of ORF48 is activated by RTA through 48pRRE during de novo infection. Through site-specific mutagenesis, we generated an ORF48-null virus and examined the function of ORF48 in vitro and in vivo. The ORF48-null mutation remarkably reduced the viral replication efficiency in cell culture. Moreover, through intranasal or intraperitoneal infection of laboratory mice, we showed that ORF48 is important for viral lytic replication in the lung and establishment of latency in the spleen, as well as viral reactivation from latency. Collectively, our study identified ORF48 as an RTA-responsive gene and showed that ORF48 is important for MHV-68 replication both in vitro and in vivo.ImportanceThe replication and transcription activator (RTA), conserved among gammaherpesviruses, serves as a molecular switch for the virus life cycle. It works as a transcriptional regulator to activate the expression of many viral lytic genes. However, only a limited number of such downstream genes have been uncovered for MHV-68. In this study, we identified ORF48 as an RTA-responsive gene of MHV-68 and mapped the cis element involved. By constructing a mutant virus that is deficient in ORF48 expression and through infection of laboratory mice, we showed that ORF48 plays important roles in different stages of viral infection in vivo. Our study provides insights into the transcriptional regulation and protein function of MHV-68, a desired model for studying gammaherpesviruses.

Project description:UnlabelledThe essential immediate early transcriptional activator RTA, encoded by gene 50, is conserved among all characterized gammaherpesviruses. Analyses of a recombinant murine gammaherpesvirus 68 (MHV68) lacking both of the known gene 50 promoters (G50DblKo) revealed that this mutant retained the ability to replicate in the simian kidney epithelial cell line Vero but not in permissive murine fibroblasts following low-multiplicity infection. However, G50DblKo replication in permissive fibroblasts was partially rescued by high-multiplicity infection. In addition, replication of the G50DblKo virus was rescued by growth on mouse embryonic fibroblasts (MEFs) isolated from IFN-α/βR-/- mice, while growth on Vero cells was suppressed by the addition of alpha interferon (IFN-α). 5' rapid amplification of cDNA ends (RACE) analyses of RNAs prepared from G50DblKo and wild-type MHV68-infected murine macrophages identified three novel gene 50 transcripts initiating from 2 transcription initiation sites located upstream of the currently defined proximal and distal gene 50 promoters. In transient promoter assays, neither of the newly identified gene 50 promoters exhibited sensitivity to IFN-α treatment. Furthermore, in a single-step growth analysis RTA levels were higher at early times postinfection with the G50DblKo mutant than with wild-type virus but ultimately fell below the levels of RTA expressed by wild-type virus at later times in infection. Infection of mice with the MHV68 G50DblKo virus demonstrated that this mutant virus was able to establish latency in the spleen and peritoneal exudate cells (PECs) of C57BL/6 mice with about 1/10 the efficiency of wild-type virus or marker rescue virus. However, despite the ability to establish latency, the G50DblKo virus mutant was severely impaired in its ability to reactivate from either latently infected splenocytes or PECs. Consistent with the ability to rescue replication of the G50DblKo mutant by growth on type I interferon receptor null MEFs, infection of IFN-α/βR-/- mice with the G50DblKo mutant virus demonstrated partial rescue of (i) acute virus replication in the lungs, (ii) establishment of latency, and (iii) reactivation from latency. The identification of additional gene 50/RTA transcripts highlights the complex mechanisms involved in controlling expression of RTA, likely reflecting time-dependent and/or cell-specific roles of different gene 50 promoters in controlling virus replication. Furthermore, the newly identified gene 50 transcripts may also act as negative regulators that modulate RTA expression.ImportanceThe viral transcription factor RTA, encoded by open reading frame 50 (Orf50), is well conserved among all known gammaherpesviruses and is essential for both virus replication and reactivation from latently infected cells. Previous studies have shown that regulation of gene 50 transcription is complex. The studies reported here describe the presence of additional alternatively initiated, spliced transcripts that encode RTA. Understanding how expression of this essential viral gene product is regulated may identify new strategies for interfering with infection in the setting of gammaherpesvirus-induced diseases.

Project description:BackgroundEarly treatment of acute HIV infection with highly active antiretroviral therapy, followed by supervised treatment interruption (STI), has been associated with at least transient control of viremia. However, the durability of such control remains unclear. Here we present longitudinal follow-up of a single-arm, open-label study assessing the impact of STI in the setting of acute HIV-1 infection.Methods and findingsFourteen patients were treated during acute HIV-1 infection and subsequently subjected to an STI protocol that required retreatment if viral load exceeded 50,000 RNA copies/ml plasma or remained above 5,000 copies/ml for more than three consecutive weeks. Eleven of 14 (79%) patients were able to achieve viral loads of less than 5,000 RNA copies/ml for at least 90 d following one, two, or three interruptions of treatment. However, a gradual increase in viremia and decline in CD4+ T cell counts was observed in most individuals. By an intention-to-treat analysis, eight (57%), six (43%), and three (21%) of 14 patients achieved a maximal period of control of 180, 360, and 720 d, respectively, despite augmentation of HIV-specific CD4+ and CD8+ T cell responses. The magnitude of HIV-1-specific cellular immune responses before treatment interruption did not predict duration of viremia control. The small sample size and lack of concurrent untreated controls preclude assessment of possible clinical benefit despite failure to control viremia by study criteria.ConclusionsThese data indicate that despite initial control of viremia, durable viral control to less than 5,000 RNA copies/ml plasma in patients following treated acute HIV-1 infection occurs infrequently. Determination of whether early treatment leads to overall clinical benefit will require a larger and randomized clinical trial. These data may be relevant to current efforts to develop an HIV-1 vaccine designed to retard disease progression rather than prevent infection since they indicate that durable maintenance of low-level viremia may be difficult to achieve.

Project description:The immediate early viral protein replication and transcription activator (RTA) of Kaposi's sarcoma-associated herpesvirus (KSHV) is essential for activating the lytic cycle of KSHV. RTA induces the KSHV lytic cycle by several mechanisms, acting as a viral transcription factor that directly induces viral and host genes and acting as a viral E3 ubiquitin ligase by degrading host proteins that block viral lytic replication. Recently, we have characterized the global gene expression changes in primary effusion lymphoma (PEL) upon lytic reactivation of KSHV, which also led to the identification of rapidly downregulated genes such as ID2, an inhibitor of basic helix-loop-helix transcription factors. Here, we demonstrate that ID2 overexpression in PEL ablates KSHV lytic reactivation, indicating that ID2 inhibits the KSHV lytic cycle. Furthermore, we show that while ID2 is highly expressed during latency, its protein level is rapidly reduced by 4 h postinduction during lytic reactivation. Our results indicate that RTA binds to ID2 and induces its degradation during the KSHV lytic cycle by N-terminal ubiquitination through the ubiquitin-proteasome pathway. Importantly, we found that not only KSHV RTA but also its Epstein-Barr virus (EBV) and murine gammaherpesvirus 68 (MHV68) homologs interact with ID2, and they can induce the degradation of all four members of the ID protein family, suggesting an evolutionarily conserved interplay between gammaherpesvirus RTAs and ID proteins. Taken together, we propose that ID2 acts as a repressor of the KSHV lytic cycle, which is counteracted by its RTA-mediated degradation. We also predict that ID proteins may act as restriction factors of the lytic phase of the other gammaherpesviruses as well. IMPORTANCE In addition to its transcription regulatory role, RTA is also known to have an E3 ubiquitin ligase activity, which RTA utilizes for inducing protein degradation. However, it is still largely unknown what host factors are downregulated during KSHV lytic reactivation by RTA-mediated protein degradation and what the biological significance of the degradation of these host factors is. In this study, we discovered that RTA employs N-terminal ubiquitination to induce degradation of ID2, a potent transcription repressor of host genes, via the ubiquitin-proteasome pathway to promote KSHV lytic reactivation in PEL cells. Furthermore, we found that not only KSHV RTA but also RTA of EBV and MHV68 gammaherpesviruses can induce the degradation of all four human ID proteins, indicating that the interplay between gammaherpesvirus RTAs and ID proteins is evolutionarily conserved.

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Control of Rta expression critically determines transcription of viral and cellular genes following gammaherpesvirus infection.

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Control of Rta expression critically determines transcription of viral and cellular genes following gammaherpesvirus infection.

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