Nucleolar disruption impairs Kaposi's sarcoma-associated herpesvirus ORF57-mediated nuclear export of intronless viral mRNAs

Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 protein is expressed early during lytic KSHV replication, enhances expression of many KSHV genes, and is essential for virus production. ORF57 is a member of a family of proteins conserved among all human and many animal herpesviruses that are multifunctional regulators of gene expression and act posttranscriptionally to increase accumulation of their target mRNAs. The mechanism of ORF57 action is complex and may involve effects on mRNA transcription, stability, and export. ORF57 directly binds to REF/Aly, a cellular RNA-binding protein component of the TREX complex that mediates RNA transcription and export. We analyzed the effects of an ORF57 mutation known to abrogate REF/Aly binding and demonstrate that the REF-binding mutant is impaired in activation of viral mRNAs and noncoding RNAs confined to the nucleus. Although the inability to bind REF leads to decreased ORF57 activity in enhancing gene expression, there is no demonstrable effect on nuclear export of viral mRNA or the ability of ORF57 to support KSHV replication and virus production. These data indicate that REF/Aly-ORF57 interaction is not essential for KSHV lytic replication but may contribute to target RNA stability independent of effects on RNA export, suggesting a novel role for REF/Aly in viral RNA metabolism.

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Identification of the Nuclear Export and Adjacent Nuclear Localization Signals for ORF45 of Kaposi's Sarcoma-Associated Herpesvirus

Journal of Virology ◽

10.1128/jvi.02209-08 ◽

2008 ◽

Vol 83 (6) ◽

pp. 2531-2539 ◽

Cited By ~ 22

Author(s):

Xiaojuan Li ◽

Fanxiu Zhu

Keyword(s):

Subcellular Localization ◽

Nuclear Localization ◽

Nuclear Export ◽

Kaposi's Sarcoma ◽

Nuclear Export Signal ◽

Kaposi’S Sarcoma ◽

Lytic Replication ◽

Wild Type ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Open reading frame 45 (ORF45) of Kaposi's sarcoma-associated herpesvirus 8 (KSHV) is an immediate-early phosphorylated tegument protein and has been shown to play important roles at both early and late stages of viral infection. Homologues of ORF45 exist only in gammaherpesviruses, and their homology is limited. These homologues differ in their protein lengths and subcellular localizations. We and others have reported that KSHV ORF45 is localized predominantly in the cytoplasm, whereas its homologue in murine herpesvirus 68 is localized exclusively in the nucleus. We observed that ORF45s of rhesus rhadinovirus and herpesvirus saimiri are found exclusively in the nucleus. As a first step toward understanding the mechanism underlying the distinct intracellular distribution of KSHV ORF45, we identified the signals that control its subcellular localization. We found that KSHV ORF45 accumulated rapidly in the nucleus in the presence of leptomycin B, an inhibitor of CRM1 (exportin 1)-dependent nuclear export, suggesting that it could shuttle between the nucleus and cytoplasm. Mutational analysis revealed that KSHV ORF45 contains a CRM1-dependent, leucine-rich-like nuclear export signal and an adjacent nuclear localization signal. Replacement of the key residues with alanines in these motifs of ORF45 disrupts its shuttling between the cytoplasm and nucleus. The resulting ORF45 mutants have restricted subcellular localizations, being found exclusively either in the cytoplasm or in the nucleus. Recombinant viruses were reconstituted by introduction of these mutations into KSHV bacterial artificial chromosome BAC36. The resultant viruses have distinct phenotypes. A mutant virus in which ORF45 is restricted to the cytoplasm behaves as an ORF45-null mutant and produces 5- to 10-fold fewer progeny viruses than the wild type. In contrast, mutants in which the ORF45 protein is mostly restricted to the nucleus produce numbers of progeny viruses similar to those produced by the wild type. These data suggest that the subcellular localization signals of ORF45 have important functional roles in KSHV lytic replication.

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Recruitment of the Complete hTREX Complex Is Required for Kaposi's Sarcoma–Associated Herpesvirus Intronless mRNA Nuclear Export and Virus Replication

PLoS Pathogens ◽

10.1371/journal.ppat.1000194 ◽

2008 ◽

Vol 4 (10) ◽

pp. e1000194 ◽

Cited By ~ 71

Author(s):

James R. Boyne ◽

Kevin J. Colgan ◽

Adrian Whitehouse

Keyword(s):

Virus Replication ◽

Nuclear Export ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

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Restricted Kaposi’s Sarcoma (KS) Herpesvirus Transcription in KS Lesions from Patients on Successful Antiretroviral Therapy

mBio ◽

10.1128/mbio.00138-11 ◽

2011 ◽

Vol 2 (6) ◽

Cited By ~ 16

Author(s):

Dirk P. Dittmer

Keyword(s):

Antiretroviral Therapy ◽

Kaposi's Sarcoma ◽

Treatment Options ◽

Human Herpesvirus ◽

Kaposi’S Sarcoma ◽

Molecular Signature ◽

Viral Mrnas ◽

Cd4 Counts ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACTKaposi’s sarcoma (KS) is caused by Kaposi’s sarcoma-associated herpesvirus (KSHV; human herpesvirus 8). KS is an AIDS-defining cancer, and it is changing in the post-antiretroviral therapy (post-ART) era. In countries with ready access to ART, approximately one-third of KS cases present in patients with undetectable HIV loads and CD4 counts of ≥200 cells/µl. This is in contrast to pre-ART era KS, which was associated with systemic HIV replication and CD4 counts of ≤200 cells/µl. Using primary patient biopsy specimens, we identified a novel molecular signature that characterizes AIDS KS lesions that develop in HIV-suppressed patients on ART: KSHV transcription is limited in HIV-suppressed patients. With one exception, only the canonical viral latency mRNAs were detectable. In contrast, early AIDS KS lesions expressed many more viral mRNAs, including, for instance, the viral G protein-coupled receptor (vGPCR).IMPORTANCEThis is the first genomewide study of Kaposi’s sarcoma-associated herpesvirus (KSHV) transcription in KS lesions in the post-antiretroviral (post-ART) era. It shows that the gene expression of KSHV is altered in patients on ART, and it provides clinical evidence for active AIDS (as characterized by high HIV load and low CD4 counts) being a potential modulator of KSHV transcription. This implies a novel mode of pathogenesis (tightly latent KS), which may inform KS cancer treatment options in the post-ART era.

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The Kaposi's sarcoma-associated herpesvirus ORF57 protein: a pleurotropic regulator of gene expression

Biochemical Society Transactions ◽

10.1042/bst0340705 ◽

2006 ◽

Vol 34 (5) ◽

pp. 705-710 ◽

Cited By ~ 10

Author(s):

P. Malik ◽

E.C. Schirmer

Keyword(s):

Gene Expression ◽

Host Cell ◽

Nuclear Export ◽

Kaposi's Sarcoma ◽

Immune Cell ◽

Kaposi’S Sarcoma ◽

Nucleocytoplasmic Transport ◽

Wide Range ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

Herpesviridae comprises over 120 viruses infecting a wide range of vertebrates including humans and livestock. Herpesvirus infections typically produce dermal lesions or immune cell destruction, but can also lead to oncogenesis, especially with KSHV (Kaposi's sarcoma-associated herpesvirus). All herpesviruses are nuclear replicating viruses that subvert cellular processes such as nucleocytoplasmic transport for their advantage. For virus replication to take over the cell and produce lytic infection requires that virus gene expression outpace that of the host cell. KSHV ORF57 (open reading frame 57) appears to play a major role in this by (i) serving as a nuclear export receptor to carry intronless viral mRNAs out of the nucleus and (ii) inhibiting expression of intron-containing host mRNAs. As most of the virally encoded mRNAs are intronless compared with host cell mRNAs, these two mechanisms are critical to overcoming host gene expression.

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Identification of the Physiological Gene Targets of the Essential Lytic Replicative Kaposi's Sarcoma-Associated Herpesvirus ORF57 Protein

Journal of Virology ◽

10.1128/jvi.02663-14 ◽

2014 ◽

Vol 89 (3) ◽

pp. 1688-1702 ◽

Cited By ~ 17

Author(s):

Dinesh Verma ◽

Da-Jiang Li ◽

Brian Krueger ◽

Rolf Renne ◽

Sankar Swaminathan

Keyword(s):

Nuclear Export ◽

Kaposi's Sarcoma ◽

Human Herpesvirus ◽

Kaposi’S Sarcoma ◽

Lytic Replication ◽

Lytic Cycle ◽

Wild Type ◽

Mrna Stabilization ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACTThe Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 gene product is essential for lytic KSHV replication and virion production. Recombinant ORF57-null mutants fail to accumulate several lytic cycle mRNAs at wild-type levels, leading to decreased production of lytic proteins necessary for efficient replication. Several mechanisms by which ORF57 may enhance expression of lytic KSHV mRNAs have been proposed, including mRNA stabilization, mRNA nuclear export, increased polyadenylation, and transcriptional activation. ORF57 activity is also gene specific, with some genes being highly dependent on ORF57, whereas others are relatively independent. Most experiments have utilized transfection models for ORF57 and have not systematically examined the gene specificity and potential mechanisms of action of ORF57 in the context of KSHV-infected cells. In this study, the KSHV genes that are most highly upregulated by ORF57 during KSHV lytic replication were identified by a combination of high-throughput deep RNA sequencing, quantitative PCR, Northern blotting, and rapid amplification of cDNA ends methods. Comparison of gene expression from a ΔORF57 KSHV recombinant, a rescued ΔORF57 KSHV recombinant, and wild-type KSHV revealed that two clusters of lytic genes are most highly dependent on ORF57 for efficient expression. Despite contiguous location in the genome and shared polyadenylation of several of the ORF57-dependent genes, ORF57 regulation was promoter and polyadenylation signal independent, suggesting that the mRNAs are stabilized by ORF57. The eight genes identified to critically require ORF57 belong to both early and late lytic temporal classes, and seven are involved in DNA replication, virion assembly, or viral infectivity, explaining the essential role of ORF57 in infectious KSHV production.IMPORTANCEKaposi's sarcoma-associated herpesvirus (KSHV) is a human herpesvirus involved in the causation of several human cancers. The KSHV ORF57 protein is required for KSHV to replicate and produce infectious virus. We have identified several KSHV genes whose expression is highly dependent on ORF57 and shown that ORF57 increases expression of these genes specifically. These genes code for proteins that are required for the virus to replicate its DNA and to infect other cells. Identifying the targets and mechanism of action of ORF57 provides further approaches to discover antiviral therapy.

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