Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8 RTA Reactivates Murine Gammaherpesvirus 68 from Latency

Rickabaugh, Tammy M.; Brown, Helen; Wu, Ting; Song, Mee Hyun; Hwang, Seungmin; Deng, Hongyu; Mitsouras, Katherine; Sun, Ren

doi:10.1128/jvi.79.5.3217-3222.2005

Cited by 16 publications

(12 citation statements)

References 35 publications

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“…Real-time PCR using primers against MHV-68 M1 was performed (18). While pORF49 null transfection alone evidenced highly attenuated virus replication, cotransfection of pFLAG-ORF49 significantly increased the viral genome copy number of pORF49 null to a level comparable to that of the wild type (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Total genomic DNAs were extracted from the cells and digested with DpnI to remove the transfected BAC DNA. Real-time PCR using primers against MHV-68 M1 was performed to quantitate viral DNA, as described previously (18). (B) Increased viral gene expressions of the ORF49 null virus.…”

Section: Resultsmentioning

confidence: 99%

“…The whole genomic DNA (50 ng) was used for real-time PCR. Real-time PCR was performed using SYBR green PCR and specific primers in a 20-l reaction mixture for M1 (M1F, 5Ј-CCTGGCCATGGTTACATACTC-3Ј; M1R, 5Ј-GGAACATA ATCCATAAGCAGGGT-3Ј) (18). Real-time PCR was run at 50°C for 2 min and then 45 cycles at 95°C for 10 s, 58°C for 15 s, and 72°C for 20 s, followed by melting curve analysis.…”

Section: Methodsmentioning

confidence: 99%

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The ORF49 Protein of Murine Gammaherpesvirus 68 Cooperates with RTA in Regulating Virus Replication

Lee

Cho

Park

et al. 2007

J Virol

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Our functional mapping study of murine gammaherpesvirus 68 (MHV-68, or ␥HV-68) revealed that a mutant harboring a transposon at the ORF49 locus (ORF49 null ) evidenced a highly attenuated in vitro growth. ORF49 resides adjacent to and in an opposite direction from RTA, the primary switch of the gammaherpesvirus life cycle. A FLAG-tagged ORF49 protein was able to transcomplement ORF49 null , and a revertant of ORF49 null restored its attenuated growth to a level comparable to that of the wild type. The FLAG-tagged ORF49 protein promoted the ability of RTA to activate downstream target promoters and enhanced virus replication from the ORF50 null virus in the presence of RTA. Furthermore, ORF49 enhanced wild-type virus replication by increasing the RTA transcript levels. Our data indicate that ORF49 may perform an important function in MHV-68 replication in cooperation with RTA.Gammaherpesviruses, including Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), herpesvirus saimiri, and murine gammaherpesvirus 68 (MHV-68, or ␥HV-68), are important pathogens as they are associated with various malignancies (19). MHV-68 has been used as an animal model system for human gammaherpesviruses, based on its ability to establish both lytic and latent infections in vivo as well as in vitro (6,16,23,25).Although latent infection is critical for gammaherpesvirusassociated malignancies, reactivation from latency and lytic replication have been suggested to be important in the persistence and pathogenesis of gammaherpesviruses (13). An immediate-early protein, RTA (BRLF1 or ORF50), represents a sensitive switch system in the gammaherpesviruses (2-4, 15, 24, 27, 29, 30). ZTA (BZLF1 or ZEBRA) and RTA in EBV (gamma-1 herpesvirus) perform essential functions in the disruption of viral latency, thereby activating downstream genes in a cooperative fashion (2-4, 7, 17, 30). However, there is no functional ZTA homologue in gamma-2 herpesviruses and RTA alone is sufficient and necessary for the initiation of lytic replication (12,24,(27)(28)(29).Our functional mapping study using a genome-wide mutant library of MHV-68 revealed that a mutant harboring a transposon at the ORF49 locus (ORF49 null ) manifested attenuated growth, thereby suggesting the important functions of ORF49 in virus replication in vitro (22). As ORF49 is located adjacent to and in a reverse orientation relative to ORF50, the major coding region of RTA, it is conceivable that ORF49 may function in cooperation with RTA, the master switch gene of the virus life cycle.ORF49 is thought to be a positional homologue of the BRRF1 gene (Na) of EBV, which is located in an opposite orientation between the immediate-early genes ZTA and RTA and expressed under the control of ZTA (20). A recent study demonstrated that Na is a transcription factor that activates the ZTA IE promoter through its effects on c-Jun and suggested that Na enhances the RTA-mediated induction of lytic EBV infection in certain cell lines (9). In this report, we determined the function of ORF...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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The ORF49 Protein of Murine Gammaherpesvirus 68 Cooperates with RTA in Regulating Virus Replication

Lee

Cho

Park

et al. 2007

J Virol

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“…The pFLAG-CMV2 (Sigma)-based wild-type (WT) MHV-68 RTA mammalian expression vector was described previously (31). The alanine substitutions in the RTA coding region were introduced by two-step PCR.…”

Section: Methodsmentioning

confidence: 99%

“…7). Our previous studies have demonstrated that the transcomplementation assay is efficient for recapitulating the phenotypes of mutant viruses (2,17,31,39,40). For the transactivation assay, the cells were transfected with MHV-68 RTA promoter reporter plasmid (1-kb sequence upstream of the MHV-68 RTA exon 1 translational initiation codon driving a firefly luciferase gene) and vector plasmid or wild-type or mutant RTA expression plasmids.…”

Section: Generation Of the 15-bp Insertion Mutant Rta Locus Librarymentioning

confidence: 99%

High-Resolution Functional Profiling of a Gammaherpesvirus RTA Locus in the Context of the Viral Genome

Arumugaswami

Sitapara

Hwang

et al. 2009

J Virol

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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. Human gammaherpesviruses Epstein-Barr virus (EBV) andKaposi's sarcoma-associated herpesvirus (KSHV) are involved in the development of epithelial, mucosal, hematopoietic, and endothelial cell cancers (7, 28). Murine gammaherpesvirus 68 (MHV-68), a pathogen affecting wild rodents, has been used as an animal model to study the fundamental principles underlying replication of gammaherpesviruses (32,35,37). For gamma-2 herpesviruses, including KSHV and MHV-68, the balance between latent and lytic phases of the viral life cycle is controlled by the immediate-early viral protein, RTA (replication and transcription activator) (11,25,26,34,41); hence, RTA plays a pivotal role in the viral life cycle. RTA is a b-zipper protein consisting of N-terminal DNA binding (DBD) and dimerization domains and a C-terminal transactivation (TA) domain (9,10,13,15,36). Understanding both the regulation of RTA transcription and the mechanism of RTA function is important. The promoter of RTA contains many regulatory cis-elements that are bound by viral and cellular factors, resulting in transcriptional activation or repression of RTA. Protein kinase C, cyclic AMP/protein kinase A, AP-1, Ras/Raf/ MEK/ERK/Ets-1, and Notch signaling pathways have been demonstrated to induce KSHV reactivation through direct or indirect activation of RTA expression (8,23,38,43,45). NF-B and LANA negatively regulate the RTA expression (4,19,20).The promoter elements of RTA have been studied extensively using transient transfe...

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Inhibition of nasopharyngeal carcinoma growth by RTA‐expressing baculovirus vectors containing oriP

Wang

Shan

et al. 2008

The Journal of Gene Medicine

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Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8 RTA Reactivates Murine Gammaherpesvirus 68 from Latency

Cited by 16 publications

References 35 publications

The ORF49 Protein of Murine Gammaherpesvirus 68 Cooperates with RTA in Regulating Virus Replication

The ORF49 Protein of Murine Gammaherpesvirus 68 Cooperates with RTA in Regulating Virus Replication

High-Resolution Functional Profiling of a Gammaherpesvirus RTA Locus in the Context of the Viral Genome

Inhibition of nasopharyngeal carcinoma growth by RTA‐expressing baculovirus vectors containing oriP

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