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High-resolution functional profiling of a gammaherpesvirus RTA locus in the context of the viral genome.

ABSTRACT: Gammaherpesviruses Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus are associated with multiple human cancers. Our goal was to develop a quantitative, high-throughput functional profiling system to identify viral cis-elements and protein subdomains critical for virus replication in the context of the herpesvirus genome. In gamma-2 herpesviruses, the transactivating factor RTA is essential for initiation of lytic gene expression and viral reactivation. We used the RTA locus as a model to develop the functional profiling approach. The mutant murine gammaherpesvirus 68 viral library, containing 15-bp random insertions in the RTA locus, was passaged in murine fibroblast cells for multiple rounds of selection. The effect of each 15-bp insertion was characterized using fluorescent-PCR profiling. We identified 1,229 insertions in the 3,845-bp RTA locus, of which 393, 282, and 554 were critically impaired, attenuated, and tolerated, respectively, for viral growth. The functional profiling phenotypes were verified by examining several individual RTA mutant clones for transactivating function of the RTA promoter and transcomplementing function of the RTA-null virus. Thus, the profiling approach enabled us to identify several novel functional domains in the RTA locus in the context of the herpesvirus genome. Importantly, our study has demonstrated a novel system to conduct high-density functional genetic mapping. The genome-scale expansion of the genetic profiling approach will expedite the functional genomics research on herpesvirus.

SUBMITTER: Arumugaswami V

PROVIDER: S-EPMC2643748 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature

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High-resolution functional profiling of a gammaherpesvirus RTA locus in the context of the viral genome.

Arumugaswami Vaithilingaraja V Sitapara Ronika R Hwang Seungmin S Song Moon Jung MJ Ho Tuyet Ngoc TN Su Nancy Qi NQ Sue Eric Y EY Kanagavel Vidhya V Xing Fangfang F Zhang Xiaolin X Zhao Minglei M Deng Hongyu H Wu Ting-Ting TT Kanagavel Sudhakar S Zhang LuLu L Dandekar Sugandha S Papp Jeanette J Sun Ren R

Journal of virology 20081210 4

Gammaherpesviruses Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus are associated with multiple human cancers. Our goal was to develop a quantitative, high-throughput functional profiling system to identify viral cis-elements and protein subdomains critical for virus replication in the context of the herpesvirus genome. In gamma-2 herpesviruses, the transactivating factor RTA is essential for initiation of lytic gene expression and viral reactivation. We used the RTA locus as a mo ...[more]

PMID: 19073723

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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: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:In the process of characterizing the requirements for expression of the essential immediate-early transcriptional activator (RTA) encoded by gene 50 of murine gammaherpesvirus 68 (MHV68), a recombinant virus was generated in which the known gene 50 promoter was deleted (G50pKO). Surprisingly, the G50pKO mutant retained the ability to replicate in permissive murine fibroblasts, albeit with slower kinetics than wild-type MHV68. 5'-rapid amplification of cDNA ends analyses of RNA prepared from G50pKO-infected fibroblasts revealed a novel upstream transcription initiation site, which was also utilized during wild-type MHV68 infection of permissive cells. Furthermore, the region upstream of the distal gene 50/RTA transcription initiation site exhibited promoter activity in both permissive NIH 3T12 fibroblasts as well as in the murine macrophage cell line RAW 264.7. In addition, in RAW 264.7 cells the activity of the distal gene 50/RTA promoter was strongly upregulated (>20-fold) by treatment of the cells with lipopolysaccharide. Reverse transcriptase PCR analyses of RNA prepared from Kaposi's sarcoma-associated herpesvirus- and Epstein-Barr virus-infected B-cell lines, following induction of virus reactivation, also revealed the presence of gene 50/RTA transcripts initiating upstream of the known transcription initiation site. The latter argues that alternative initiation of gene 50/RTA transcription is a strategy conserved among murine and human gammaherpesviruses. Infection of mice with the MHV68 G50pKO demonstrated the ability of this mutant virus to establish latency in the spleen and peritoneal exudate cells (PECs). However, the G50pKO mutant was unable to reactivate from latently infected splenocytes and also exhibited a significant reactivation defect from latently infected PECs, arguing in favor of a model where the proximal gene 50/RTA promoter plays a critical role in virus reactivation from latency, particularly from B cells. Finally, analyses of viral genome methylation in the regions upstream of the proximal and distal gene 50/RTA transcription initiation sites revealed that the distal promoter is partially methylated in vivo and heavily methylated in MHV68 latently infected B-cell lines, suggesting that DNA methylation may serve to silence the activity of this promoter during virus latency.

Dataset Information

High-resolution functional profiling of a gammaherpesvirus RTA locus in the context of the viral genome.

Publications

High-resolution functional profiling of a gammaherpesvirus RTA locus in the context of the viral genome.

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