scholarly journals Transcription of the murine gammaherpesvirus 68 ORF73 from promoters in the viral terminal repeats

2005 ◽  
Vol 86 (3) ◽  
pp. 561-574 ◽  
Author(s):  
Heather M. Coleman ◽  
Stacey Efstathiou ◽  
Philip G. Stevenson
Keyword(s):  
Transcription Initiation ◽  
Repeat Unit ◽  
Initiation Site ◽  
Reading Frame ◽  
Left Hand ◽  
Murine Gammaherpesvirus ◽  
Terminal Repeats ◽  
Gammaherpesvirus 68 ◽  
The Right ◽  
Murine Gammaherpesvirus 68

Gammaherpesviruses persist as latent episomes in a dynamic lymphocyte pool. The regulated production of an episome maintenance protein is therefore crucial to their survival. The transcription initiation site of the murine gammaherpesvirus 68 episome maintenance protein, ORF73, was mapped to the viral terminal repeats, more than 10 kb distant from the open reading frame (ORF) itself. A 5′ non-coding exon in the terminal repeats was spliced to the right end of the viral unique sequence, and then across ORFs 75a, 75b, 75c and 74 to ORF73. The right-hand portion of a single repeat unit was sufficient for constitutive promoter activity. The unique left end of the viral genome further enhanced ORF73 transcription. This, together with the large size of the predominant ORF73 mRNA, suggested that transcription initiates in distal repeat units and then splices between repeats to generate an extensive 5′ untranslated region. A second promoter in the left-hand portion of the proximal terminal repeat unit generated a transcript which overlapped that of ORF73, but failed to splice to the ORF73 coding exon and so transcribed ORF75a. In distal repeat copies, however, transcription from this promoter would enter the next repeat unit to become an ORF73 mRNA. There was a third promoter just upstream of ORF73 itself. These data indicate that ORF73 transcription is highly complex, and support the idea that the terminal repeats of gamma-2-herpesviruses constitute a vital component of episomal persistence.

scholarly journals Murine Gammaherpesvirus 68 Open Reading Frame 75c Tegument Protein Induces the Degradation of PML and Is Essential for Production of Infectious Virus

2008 ◽  
Vol 82 (16) ◽  
pp. 8000-8012 ◽  
Author(s):  
Paul D. Ling ◽  
Jie Tan ◽  
Jaturong Sewatanon ◽  
RongSheng Peng
Keyword(s):  
Infectious Virus ◽  
Nuclear Body ◽  
Tegument Protein ◽  
Suitable Model ◽  
Virus Infections ◽  
Reading Frame ◽  
Murine Gammaherpesvirus ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68

ABSTRACT Promyelocytic Leukemia nuclear body (PML NB) proteins mediate an intrinsic cellular host defense response against virus infections. Herpesviruses express proteins that modulate PML or PML-associated proteins by a variety of strategies, including degradation of PML or relocalization of PML NB proteins. The consequences of PML-herpesvirus interactions during infection in vivo have yet to be investigated in detail, largely because of the species-specific tropism of many human herpesviruses. Murine gammaherpesvirus 68 (γHV68) is emerging as a suitable model to study basic biological questions of virus-host interactions because it naturally infects mice. Therefore, we sought to determine whether γHV68 targets PML NBs as part of its natural life cycle. We found that γHV68 induces PML degradation through a proteasome-dependent mechanism and that loss of PML results in more robust virus replication in mouse fibroblasts. Surprisingly, γHV68-mediated PML degradation was mediated by the virion tegument protein ORF75c, which shares homology with the cellular formylglycinamide ribotide amidotransferase enzyme. In addition, we show that ORF75c is essential for production of infectious virus. ORF75 homologs are conserved in all rhadinoviruses but so far have no assigned functions. Our studies shed light on a potential role for this unusual protein in rhadinovirus biology and suggest that γHV68 will be a useful model for investigation of PML-herpesvirus interactions in vivo.

scholarly journals Identification of Closely Spaced but Distinct Transcription Initiation Sites for the Murine Gammaherpesvirus 68 Latency-Associated M2 Gene

2008 ◽  
Vol 82 (15) ◽  
pp. 7411-7421 ◽  
Author(s):  
Mark DeZalia ◽  
Samuel H. Speck
Keyword(s):  
Gene Product ◽  
Gene Transcription ◽  
Transcription Initiation ◽  
Splice Donor Site ◽  
Splice Acceptor ◽  
Splice Donor ◽  
Murine Gammaherpesvirus ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68

ABSTRACT Murine gammaherpesvirus 68 (MHV68) infection of mice provides a tractable small-animal system for assessing viral requirements for establishment of and reactivation from latency. The M2 gene product has no homology to any known proteins but has been shown to play a role in both the establishment of MHV68 latency and reactivation from latency. Furthermore, we have recently shown that M2 expression in primary murine B cells leads to enhanced proliferation, survival, and differentiation toward a preplasma memory B-cell phenotype (A. M. Siegel, J. H. Herskowitz, and S. H. Speck, PLoS Pathog. 4:e1000039, 2008). Previous studies have characterized the structure of the M2 transcript, but to date there has been no characterization of the M2 promoter, additional open reading frames (ORFs) in the M2 region, or identified splice acceptor and splice donor sites present in the previously characterized M2 gene transcript. Here we report (i) the identification and disruption of a novel transcript that encodes a short, previously unreported ORF (M2b) located in the intron between exon 1 and exon 2 of the M2 transcript; (ii) the identification of clustered but distinct M2 gene transcription initiation sites suggesting the presence of multiple promoters involved in regulating M2 gene transcription; (iii) the characterization in vivo of recombinant MHV68 harboring deletions within the identified M2 promoter region; and (iv) the in vivo analysis of recombinant MHV68 harboring mutations that ablate either the identified M2 splice acceptor or splice donor site. Finally, our 5′ rapid amplification of cDNA ends in conjunction with splice acceptor mutation analyses confirmed that all detected M2 gene transcripts expressed during MHV68 infection in mice splice into the M2 ORF downstream of the first AUG codon, providing strong evidence that initiation of the M2 gene product arises from the second AUG codon located at residue 8 in the M2 ORF. This initial detailed analysis of M2 gene transcription in vivo will aid future studies on regulation of M2 gene expression.

scholarly journals Identificationof Proteins Associated with Murine Gammaherpesvirus68Virions

2003 ◽  
Vol 77 (24) ◽  
pp. 13425-13432 ◽  
Author(s):  
Eric Bortz ◽  
Julian P. Whitelegge ◽  
Qingmei Jia ◽  
Z. Hong Zhou ◽  
James P. Stewart ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Cultured Cells ◽  
Gene Products ◽  
Reading Frame ◽  
Virion Assembly ◽  
Murine Gammaherpesvirus ◽  
Excellent Candidate ◽  
Associated Proteins ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68

ABSTRACT Murine gammaherpesvirus 68 (MHV68 [also known asγ HV-68]) is distinguished by its ability to replicate to high titers in cultured cells, making it an excellent candidate for studying gammaherpesvirus virion composition. Extracellular MHV68 virions were isolated, and abundant virion-associated proteins were identified by mass spectrometry. Five nucleocapsid protein homologues, the tegument protein homologue encoded by open reading frame (ORF) 75c, and envelope glycoproteins B and H were detected. In addition, gene products from MHV68 ORF20, ORF24, ORF28, ORF45, ORF48, and ORF52 were identified in association with virions, suggesting that these gammaherpesvirus genes are involved in the early phase of infection or virion assembly and egress.

scholarly journals Identification and Initial Characterization of the Murine Gammaherpesvirus 68 Gene M3, Encoding an Abundantly Secreted Protein

1999 ◽  
Vol 73 (5) ◽  
pp. 4524-4529 ◽  
Author(s):  
Victor van Berkel ◽  
Karen Preiter ◽  
Herbert W. Virgin ◽  
Samuel H. Speck
Keyword(s):  
Secreted Protein ◽  
S1 Nuclease ◽  
Reading Frame ◽  
Murine Gammaherpesvirus ◽  
Amino Terminal ◽  
Partial Cdna ◽  
Rapid Amplification ◽  
Gammaherpesvirus 68 ◽  
Partial Cdna Clone ◽  
Murine Gammaherpesvirus 68

ABSTRACT Several viruses, including members of the gammaherpesvirus family, encode proteins that are secreted into the extracellular environment. We have identified an abundant 44-kDa secreted protein that is present in the supernatant of fibroblasts infected with murine gammaherpesvirus 68 (γHV68; also referred to as MHV-68) but not in that of uninfected fibroblasts. Sequence analysis of the amino terminus and of internal peptides revealed that this protein is encoded by the γHV68 M3 open reading frame (ORF). The amino-terminal sequence of the secreted protein starts at residue 25 of the M3 ORF, consistent with the first 24 residues functioning as a signal peptide. Northern blot analysis revealed a single abundant ∼1.4-kb early-late lytic transcript encoded by the M3 ORF. Analysis of a partial cDNA clone and subsequent analyses of products of rapid amplification of cDNA ends coupled with S1 nuclease protection assays demonstrate that the M3 protein is encoded by an unspliced, polyadenylated mRNA initiating at bp 7294 and terminating at bp 6007 of the γHV68 genome. The 3′ end of the M3 transcript maps 9 bp downstream of a consensus polyadenylation signal. Thus, the predicted M3 ORF is a functional gene that encodes an abundant secreted protein which is a candidate for interacting with host cellular receptors or cytokines.

scholarly journals Murine Gammaherpesvirus 68 Contains Two Functional Lytic Origins of Replication

2007 ◽  
Vol 81 (13) ◽  
pp. 7300-7305 ◽  
Author(s):  
Heiko Adler ◽  
Beatrix Steer ◽  
Klaus Freimüller ◽  
Jürgen Haas
Keyword(s):  
Artificial Chromosome ◽  
Plasmid Replication ◽  
Origin Of Replication ◽  
Replication Assay ◽  
Murine Gammaherpesvirus ◽  
Origins Of Replication ◽  
Dna Fragment ◽  
Gammaherpesvirus 68 ◽  
The Right ◽  
Murine Gammaherpesvirus 68

ABSTRACT A 1.25-kbp DNA fragment from the right side of the genome containing the lytic origin of replication (oriLyt) of murine gammaherpesvirus 68 (MHV-68) has been identified by a plasmid replication assay. Here we show that a mutant MHV-68 with a deletion of an essential part of this oriLyt, generated by using an MHV-68 bacterial artificial chromosome, was only slightly attenuated and still able to replicate but that a mutant containing an additional deletion on the left side of the genome was replication deficient. The newly identified region was sufficient to support plasmid replication, thus providing evidence for a second oriLyt.

scholarly journals Murine Gammaherpesvirus 68 Open Reading Frame 45 Plays an Essential Role during the Immediate-Early Phase of Viral Replication

2005 ◽  
Vol 79 (8) ◽  
pp. 5129-5141 ◽  
Author(s):  
Qingmei Jia ◽  
Vasili Chernishof ◽  
Eric Bortz ◽  
Ian Mchardy ◽  
Ting-Ting Wu ◽  
...  
Keyword(s):  
Viral Replication ◽  
Translation Termination ◽  
Human Herpesvirus ◽  
Open Reading Frame ◽  
Viral Gene ◽  
Small Interfering Rnas ◽  
Reading Frame ◽  
Murine Gammaherpesvirus ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68

ABSTRACT Murine gammaherpesvirus 68 (MHV-68) has been developed as a model for the human gammaherpesviruses Epstein-Barr virus and human herpesvirus 8/Kaposi's sarcoma-associated herpesvirus (HHV-8/KSHV), which are associated with several types of human diseases. Open reading frame 45 (ORF45) is conserved among the members of the Gammaherpesvirinae subfamily and has been suggested to be a virion tegument protein. The repression of ORF45 expression by small interfering RNAs inhibits MHV-68 viral replication. However, the gene product of MHV-68 ORF45 and its function have not yet been well characterized. In this report, we show that MHV-68 ORF45 is a phosphorylated nuclear protein. We constructed an ORF45-null MHV-68 mutant virus (45STOP) by the insertion of translation termination codons into the portion of the gene encoding the N terminus of ORF45. We demonstrated that the ORF45 protein is essential for viral gene expression immediately after the viral genome enters the nucleus. These defects in viral replication were rescued by providing ORF45 in trans or in an ORF45-null revertant (45STOP.R) virus. Using a transcomplementation assay, we showed that the function of ORF45 in viral replication is conserved with that of its KSHV homologue. Finally, we found that the C-terminal 23 amino acids that are highly conserved among the Gammaherpesvirinae subfamily are critical for the function of ORF45 in viral replication.

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

2007 ◽  
Vol 81 (12) ◽  
pp. 6761-6764 ◽  
Author(s):  
Elaine Wong ◽  
Ting-Ting Wu ◽  
Nichole Reyes ◽  
Hongyu Deng ◽  
Ren Sun
Keyword(s):  
Gene Expression ◽  
Dna Replication ◽  
Viral Replication ◽  
Open Reading Frame ◽  
Late Gene ◽  
Late Gene Expression ◽  
Reading Frame ◽  
Murine Gammaherpesvirus ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68

ABSTRACT 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.

scholarly journals Murine Gammaherpesvirus 68 Open Reading Frame 11 Encodes a Nonessential Virion Component

2005 ◽  
Vol 79 (5) ◽  
pp. 3163-3168 ◽  
Author(s):  
Jessica M. Boname ◽  
Janet S. May ◽  
Philip G. Stevenson
Keyword(s):  
Lytic Replication ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Murine Gammaherpesvirus ◽  
Infected Cells ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68

ABSTRACT Open reading frame 11 (ORF11) is a conserved gammaherpesvirus gene that remains undefined. We identified the product of murine gammaherpesvirus 68 (MHV-68) ORF11, p43, as a virion component with a predominantly perinuclear distribution in infected cells. MHV-68 lacking p43 grew normally in vitro but showed reduced lytic replication in vivo and a delay in seeding to the spleen. Subsequent latency amplification was normal. Thus, MHV-68 ORF11 encoded a virion component that was important for in vivo lytic replication but dispensable for the establishment of latency.

scholarly journals Evidence for CDK-Dependent and CDK-Independent Functions of the Murine Gammaherpesvirus 68 v-Cyclin

2006 ◽  
Vol 80 (24) ◽  
pp. 11946-11959 ◽  
Author(s):  
Jason W. Upton ◽  
Samuel H. Speck
Keyword(s):  
Cell Cycle ◽  
Lytic Replication ◽  
Open Reading Frame ◽  
Virus Reactivation ◽  
Reading Frame ◽  
Murine Gammaherpesvirus ◽  
Cellular Environment ◽  
Independent Functions ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68

ABSTRACT Gamma-2 herpesviruses encode homologues of mammalian D-type cyclins (v-cyclins), which likely function to manipulate the cell cycle, thereby providing a cellular environment conducive to virus replication and/or reactivation from latency. We have previously shown that the v-cyclin of murine gammaherpesvirus 68 is an oncogene that binds and activates cellular cyclin-dependent kinases (CDKs) and is required for efficient reactivation from latency. To determine the contribution of v-cyclin-mediated cell cycle regulation to the viral life cycle, recombinant viruses in which specific point mutations (E133V or K104E) were introduced into the v-cyclin open reading frame were generated, resulting in the disruption of CDK binding and activation. While in vitro growth of these mutant viruses was unaffected, lytic replication in the lungs following low-dose intranasal inoculation was attenuated for both mutants deficient in CDK binding as well as virus in which the entire v-cyclin open reading frame was disrupted by the insertion of a translation termination codon. This replication defect was not apparent in spleens of mice following intraperitoneal inoculation, suggesting a cell type- and/or route-specific dependence on v-cyclin-CDK interactions during the acute phase of virus infection. Notably, although a v-cyclin-null virus was highly attenuated for reactivation from latency, the E133V v-cyclin CDK-binding mutant exhibited only a modest defect in virus reactivation from splenocytes, and neither the E133V nor K104E v-cyclin mutants were compromised in reactivation from peritoneal exudate cells. Taken together, these data suggest that lytic replication and reactivation in vivo are differentially regulated by CDK-dependent and CDK-independent functions of v-cyclin, respectively.

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