Kaposi's sarcoma-associated herpesvirus ORF57 protein interacts with PYM to enhance translation of viral intronless mRNAs.
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ABSTRACT:
SUBMITTER: Boyne JR
PROVIDER: S-EPMC2885933 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 (MTA, mRNA transcript accumulation) is a multifunctional regulator of the expression of viral lytic genes. KSHV ORF57 is expressed during viral lytic infection and is essential for virus production. Like its homologues in the herpesvirus family, ORF57 promotes the accumulation (stabilization) and export of viral intronless RNA transcripts by a mechanism which remains to be defined. The ORF57-Aly/REF interaction plays only a small role in viral RNA export. Although other members of the family generally inhibit the splicing of cellular RNAs, KSHV ORF57 and EBV EB2, in sharp contrast, stimulate viral RNA splicing for the expression of viral intron-containing genes. The functions of KSHV ORF57 are independent of transcription and of other viral proteins; instead, these functions always rely on cellular components and occur in various protein-RNA complexes. ORF57 may synergize with KSHV ORF50 to transactivate a subset of viral promoters by an unknown mechanism. Thus, some functions of ORF57 have been conserved while others have diverged from its homologues as ORF57 adapted over evolution to KSHV biology and pathogenesis.
| S-EPMC2654597 | biostudies-literature
Project description:Kaposi's sarcoma-associated herpesvirus (KSHV [human herpesvirus 8; HHV-8]) open reading frame 57 (ORF57) is a viral early protein participating in posttranscriptional regulatory events, such as splicing, RNA stabilization, and protein expression. Recent data suggest that ORF57 recruits the transcription and export (TREX) complex to viral RNA and exports these transcripts to the cytoplasm. In this study, we show that although ORF57 promotes expression of a selection of KSHV viral intronless RNAs, it is not a bona fide export factor.
| S-EPMC3497679 | biostudies-literature
Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) encodes ORF57, which promotes the accumulation of specific KSHV mRNA targets, including ORF59 mRNA. We report that the cellular export NXF1 cofactors RBM15 and OTT3 participate in ORF57-enhanced expression of KSHV ORF59. We also found that ectopic expression of RBM15 or OTT3 augments ORF59 production in the absence of ORF57. While RBM15 promotes the accumulation of ORF59 RNA predominantly in the nucleus compared to the levels in the cytoplasm, we found that ORF57 shifted the nucleocytoplasmic balance by increasing ORF59 RNA accumulation in the cytoplasm more than in the nucleus. By promoting the accumulation of cytoplasmic ORF59 RNA, ORF57 offsets the nuclear RNA accumulation mediated by RBM15 by preventing nuclear ORF59 RNA from hyperpolyadenylation. ORF57 interacts directly with the RBM15 C-terminal portion containing the SPOC domain to reduce RBM15 binding to ORF59 RNA. Although ORF57 homologs Epstein-Barr virus (EBV) EB2, herpes simplex virus (HSV) ICP27, varicella-zoster virus (VZV) IE4/ORF4, and cytomegalovirus (CMV) UL69 also interact with RBM15 and OTT3, EBV EB2, which also promotes ORF59 expression, does not function like KSHV ORF57 to efficiently prevent RBM15-mediated nuclear accumulation of ORF59 RNA and RBM15's association with polyadenylated RNAs. Collectively, our data provide novel insight elucidating a molecular mechanism by which ORF57 promotes the expression of viral intronless genes.
| S-EPMC3028919 | biostudies-literature
Project description:UnlabelledKaposi's sarcoma-associated herpesvirus (KSHV) encodes ORF57, which enhances the expression of intronless KSHV genes on multiple posttranscriptional levels. However, it remains elusive how ORF57 recognizes viral RNAs. Here, we demonstrate that ORF57 also increases the expression of the multiple intron-containing K15 gene. The nucleotide bias of the K15 cDNA revealed an unusual high AT content. Thus, we optimized the K15 cDNA by raising the frequency of GC nucleotides, yielding an ORF57-independent version. To further prove the importance of the sequence bias of ORF57-dependent RNAs, we grouped KSHV mRNAs according to their AT content and found a correlation between AT-richness and ORF57 dependency. More importantly, latent genes, which have to be expressed in the absence of ORF57, have a low AT content and are indeed ORF57 independent. The nucleotide composition of K15 resembles that of HIV gag, which cannot be expressed unless RNA export is facilitated by the HIV Rev protein. Interestingly, ORF57 can partially rescue HIV Gag expression. Thus, the KSHV target RNAs of ORF57 and HIV gag RNA may share certain motifs based on the nucleotide bias. A bioinformatic comparison between wild-type and sequence-optimized K15 revealed a higher density for hnRNP-binding motifs in the former. We speculate that binding of particular hnRNPs to KSHV lytic transcripts is the prerequisite for ORF57 to enhance their expression.ImportanceThe mostly intronless genes of KSHV are only expressed in the presence of the viral regulator protein ORF57, but how ORF57 recognizes viral RNAs remains elusive. We focused on the multiple intron-containing KSHV gene K15 and revealed that its expression is also increased by ORF57. Moreover, sequences in the K15 cDNA mediate this enhancement. The quest for a target sequence or a response element for ORF57 in the lytic genes was not successful. Instead, we found the nucleotide bias to be the critical determinant of ORF57 dependency. Based on the fact that ORF57 has only a weak affinity for nucleic acids, we speculate that a cellular RNA-binding protein provides the sequence preference for ORF57. This study provides evidence that herpesviral RNA regulator proteins use the sequence bias of lytic genes and the resulting composition of the viral mRNP to distinguish between viral and cellular mRNAs.
| S-EPMC4403450 | biostudies-literature
Project description:Though similar to those of herpesvirus saimiri and Epstein-Barr virus (EBV), the Kaposi's sarcoma-associated herpesvirus (KSHV) genome features more splice genes and encodes many genes with bicistronic or polycistronic transcripts. In the present study, the gene structure and expression of KSHV ORF56 (primase), ORF57 (MTA), ORF58 (EBV BMRF2 homologue), and ORF59 (DNA polymerase processivity factor) were analyzed in butyrate-activated KSHV(+) JSC-1 cells. ORF56 was expressed at low abundance as a bicistronic ORF56/57 transcript that utilized the same intron, with two alternative branch points, as ORF57 for its RNA splicing. ORF56 was transcribed from two transcription start sites, nucleotides (nt) 78994 (minor) and 79075 (major), but selected the same poly(A) signal as ORF57 for RNA polyadenylation. The majority of ORF56 and ORF57 transcripts were cleaved at nt 83628, although other nearby cleavage sites were selectable. On the opposite strand of the viral genome, colinear ORF58 and ORF59 were transcribed from different transcription start sites, nt 95821 (major) or 95824 (minor) for ORF58 and nt 96790 (minor) or 96794 (major) for ORF59, but shared overlapping poly(A) signals at nt 94492 and 94488. Two cleavage sites, at nt 94477 and nt 94469, could be equally selected for ORF59 polyadenylation, but only the cleavage site at nt 94469 could be selected for ORF58 polyadenylation without disrupting the ORF58 stop codon immediately upstream. ORF58 was expressed in low abundance as a monocistronic transcript, with a long 5' untranslated region (UTR) but a short 3' UTR, whereas ORF59 was expressed in high abundance as a bicistronic transcript, with a short 5' UTR and a long 3' UTR similar to those of polycistronic ORF60 and ORF62. Both ORF56 and ORF59 are targets of ORF57 and were up-regulated significantly in the presence of ORF57, a posttranscriptional regulator.
| S-EPMC1676266 | biostudies-literature
Project description:The Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 protein is an essential multifunctional regulator of gene expression. ORF57 interaction with RNA is necessary for ORF57-mediated posttranscriptional functions, but little is known about the RNA elements that drive ORF57-RNA specificity. Here, we investigate the cis-acting factors on the KSHV PAN RNA that dictate ORF57 binding and activity. We show that ORF57 binds directly to the 5' end of PAN RNA in KSHV-infected cells. Furthermore, we employ in vitro and cell-based assays to define a 30-nucleotide (nt) core ORF57-responsive element (ORE) that is necessary and sufficient for ORF57 binding and activity. Mutational analysis of the core ORE further suggests that a 9-nt sequence is a specific binding site for ORF57. These studies provide insight into ORF57 specificity determinants and lay a foundation for future analyses of cellular and viral ORF57 targets.
| S-EPMC3177971 | biostudies-literature
Project description:UnlabelledKaposi's sarcoma-associated herpesvirus (KSHV) ORF57 plays an essential role in KSHV lytic infection by promoting viral gene expression at the posttranscriptional level. Using bioinformatic and biochemical approaches, we determined that ORF57 contains two structurally and functionally distinct domains: a disordered nonstructural N-terminal domain (amino acids [aa] 1 to 152) and a structured α-helix-rich C-terminal domain (aa 153 to 455). The N-terminal domain mediates ORF57 interaction with several RNA-protein complexes essential for ORF57 to function. The N-terminal phosphorylation by cellular casein kinase II (CKII) at S21, T32, and S43, and other cellular kinases at S95 and S97 residues in proximity of the caspase-7 cleavage site, 30-DETD-33, inhibits caspase-7 digestion of ORF57. The structured C-terminal domain mediates homodimerization of ORF57, and the critical region for this function was mapped carefully to α-helices 7 to 9. Introduction of point mutations into α-helix 7 at ORF57 aa 280 to 299, a region highly conserved among ORF57 homologues from other herpesviruses, inhibited ORF57 homodimerization and led to proteasome-mediated degradation of ORF57 protein. Thus, homodimerization of ORF57 via its C terminus prevents ORF57 from degrading and allows two structure-free N termini of the dimerized ORF57 to work coordinately for host factor interactions, leading to productive KSHV lytic infection and pathogenesis.ImportanceKSHV is a human oncogenic virus linked to the development of several malignancies. KSHV-mediated oncogenesis requires both latent and lytic infection. The KSHV ORF57 protein is essential for KSHV lytic replication, as it regulates the expression of viral lytic genes at the posttranscriptional level. This report provides evidence that the structural conformation of the ORF57 protein plays a critical role in regulation of ORF57 stability. Phosphorylation by CKII on the identified serine/threonine residues at the N-terminal unstructured domain of ORF57 prevents its digestion by caspase-7. The C-terminal domain of ORF57, which is rich in α-helices, contributes to homodimerization of ORF57 to prevent proteasome-mediated protein degradation. Elucidation of the ORF57 structure not only enables us to better understand ORF57 stability and functions but also provides an important tool for us to modulate ORF57's activity with the aim to inhibit KSHV lytic replication.
| S-EPMC4337553 | biostudies-literature
Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) is one of the few human oncogenic viruses, which causes a variety of malignancies, including Kaposi's sarcoma, multicentric Castleman disease, and primary effusion lymphoma, particularly in human immunodeficiency virus patients. The currently available treatment options cannot always prevent the invasion and dissemination of this virus. In recent times, siRNA-based therapeutics are gaining prominence over conventional medications as siRNA can be designed to target almost any gene of interest. The ORF57 is a crucial regulatory protein for lytic gene expression of KSHV. Disruption of this gene translation will inevitably inhibit the replication of the virus in the host cell. Therefore, the ORF57 of KSHV could be a potential target for designing siRNA-based therapeutics. Considering both sequence preferences and target site accessibility, several online tools (i-SCORE Designer, Sfold web server) had been utilized to predict the siRNA guide strand against the ORF57. Subsequently, off-target filtration (BLAST), conservancy test (fuzznuc), and thermodynamics analysis (RNAcofold, RNAalifold, and RNA Structure web server) were also performed to select the most suitable siRNA sequences. Finally, two siRNAs were identified that passed all of the filtration phases and fulfilled the thermodynamic criteria. We hope that the siRNAs predicted in this study would be helpful for the development of new effective therapeutics against KSHV.
| S-EPMC8752988 | biostudies-literature
Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) lytic infection increases the expression of viral and human interleukin-6 (vIL-6 and hIL-6, respectively), an important factor for cell growth and pathogenesis. Here, we report genome-wide analysis of viral RNA targets of KSHV ORF57 by a novel UV-cross-linking and immunoprecipitation (CLIP) assay. We identified 11 viral transcripts as putative ORF57 targets and demonstrate that vIL-6 mRNA is an authentic target of ORF57. Disrupting the ORF57 gene in the KSHV genome leads to inefficient expression of vIL-6. With transient transfection, the expression of vIL-6 could be enhanced greatly in the presence of ORF57 in a dose-dependent manner. We found that the open reading frame (ORF) region of vIL-6 RNA contains an MRE (MTA [ORF57]-responsive element) composed of two motifs, MRE-A and MRE-B, and binding of ORF57 to these two motifs stabilizes vIL-6 RNA and promotes vIL-6 translation. We demonstrate that vIL-6 MRE-B bears an miR-1293 binding site and that, mechanistically, ORF57 competes with miR-1293 for the same binding site to interact with vIL-6 RNA, thereby preventing vIL-6 RNA from association with the miR-1293-specified RNA-induced silencing complex (RISC). Consistent with this, ORF57 also interacts with an miR-608 binding site in the hIL-6 ORF and prevents miR-608 repression of hIL-6. Collectively, our results identify a novel function of ORF57 in being responsible for stabilization of viral and human IL-6 RNAs and the corresponding enhancement of RNA translation. In addition, our data provide the first evidence that a tumor virus may use a viral protein to interfere with microRNA (miRNA)-mediated repression of an miRNA target to induce cell proliferation and tumorigenesis during virus infection.
| S-EPMC3067933 | biostudies-literature
Project description:The Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 57 (ORF57)-encoded protein (Mta) is a multifunctional regulator of viral gene expression. ORF57 is essential for viral replication, so elucidation of its molecular mechanisms is important for understanding KSHV infection. ORF57 has been implicated in nearly every aspect of viral gene expression, including transcription, RNA stability, splicing, export, and translation. Here we demonstrate that ORF57 interacts with the KSHV K-bZIP protein in vitro and in cell extracts from lytically reactivated infected cells. To further test the biological relevance of the interaction, we performed a chromatin immunoprecipitation and microarray (ChIP-chip) analysis using anti-ORF57 antibodies and a KSHV tiling array. The results revealed four specific areas of enrichment, including the ORF4 and K8 (K-bZIP) promoters, as well as oriLyt, all of which interact with K-bZIP. In addition, ORF57 associated with DNA corresponding to the PAN RNA transcribed region, a known posttranscriptional target of ORF57. All of the peaks were RNase insensitive, demonstrating that ORF57 association with the viral genome is unlikely to be mediated exclusively by an RNA tether. Our data demonstrate that ORF57 associates with the viral genome by using at least two modes of recruitment, and they suggest that ORF57 and K-bZIP coregulate viral gene expression during lytic infection.
| S-EPMC3624208 | biostudies-literature