Mutation of a C-Terminal Motif Affects Kaposi's Sarcoma-Associated Herpesvirus ORF57 RNA Binding, Nuclear Trafficking, and Multimerization

ABSTRACT Terminal repeat (TR) elements of Kaposi's sarcoma-associated herpesvirus (KSHV), the potential origin sites of KSHV replication, have been demonstrated to play important roles in viral replication and transcription and are most likely also critical for the segregation of the KSHV genome to daughter cells. To search for the cellular proteins potentially involved in KSHV genome maintenance, we performed affinity chromatography analysis, using KSHV TR DNA as the affinity ligand. Proteomic analysis was then carried out to identify the TR-interacting proteins. We identified a total of 123 proteins from both KSHV-positive and -negative cells, among which most were identified exclusively from KSHV-positive cells. These proteins were categorized as proliferation/cell cycle regulatory proteins, proteins involved in spliceosome components, such as heterogeneous nuclear ribonuclear proteins, the DEAD/H family, the switch/sucrose nonfermenting protein family, splicing factors, RNA binding proteins, transcription regulation proteins, replication factors, modifying enzymes, and a number of proteins that could not be broadly categorized. To support the proteomic results, the presence of four candidate proteins, ATR, BRG1, NPM1 and PARP-1, in the elutions was further characterized in this study. The binding and colocalization of these proteins with the TR were verified using chromatin immunoprecipitation and immunofluorescence in situ hybridization analysis. These newly identified TR binding proteins provide a number of clues and potential links to understanding the mechanisms regulating the replication, transcription, and genome maintenance of KSHV. This study will facilitate the generation and testing of new hypotheses to further our understanding of the mechanisms involved in KSHV persistence and its associated pathogenesis.

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A posttranscriptional regulator of Kaposi's sarcoma-associated herpesvirus interacts with RNA-binding protein PCBP1 and controls gene expression through the IRES

Virology ◽

10.1016/j.virol.2004.04.041 ◽

2004 ◽

Vol 325 (2) ◽

pp. 364-378 ◽

Cited By ~ 15

Author(s):

Ken Nishimura ◽

Keiji Ueda ◽

Edhi Guwanan ◽

Shuhei Sakakibara ◽

Eunju Do ◽

...

Keyword(s):

Gene Expression ◽

Kaposi's Sarcoma ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Kaposi’S Sarcoma ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

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Binding of Cellular Export Factor REF/Aly by Kaposi's Sarcoma-Associated Herpesvirus (KSHV) ORF57 Protein Is Not Required for Efficient KSHV Lytic Replication

Journal of Virology ◽

10.1128/jvi.01190-12 ◽

2012 ◽

Vol 86 (18) ◽

pp. 9866-9874 ◽

Cited By ~ 14

Author(s):

Da-Jiang Li ◽

Dinesh Verma ◽

Sankar Swaminathan

Keyword(s):

Gene Expression ◽

Nuclear Export ◽

Kaposi's Sarcoma ◽

Rna Binding ◽

Virus Production ◽

Kaposi’S Sarcoma ◽

Lytic Replication ◽

Viral Mrnas ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

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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ORF57 Overcomes the Detrimental Sequence Bias of Kaposi's Sarcoma-Associated Herpesvirus Lytic Genes

Journal of Virology ◽

10.1128/jvi.03264-14 ◽

2015 ◽

Vol 89 (9) ◽

pp. 5097-5109 ◽

Cited By ~ 8

Author(s):

Carolin Vogt ◽

Christian Hackmann ◽

Alona Rabner ◽

Lars Koste ◽

Susann Santag ◽

...

Keyword(s):

Kaposi's Sarcoma ◽

Rna Binding ◽

Kaposi’S Sarcoma ◽

Target Sequence ◽

Nucleotide Bias ◽

Viral Rnas ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus ◽

Lytic Genes ◽

Sequence Bias

ABSTRACTKaposi'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 HIVgag, 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 HIVgagRNA 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.

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RNA Granules in Antiviral Innate Immunity: A Kaposi’s Sarcoma-Associated Herpesvirus Journey

Frontiers in Microbiology ◽

10.3389/fmicb.2021.794431 ◽

2022 ◽

Vol 12 ◽

Author(s):

Nishi R. Sharma ◽

Zhi-Ming Zheng

Keyword(s):

Innate Immunity ◽

Kaposi's Sarcoma ◽

Rna Binding ◽

Kaposi’S Sarcoma ◽

Productive Infection ◽

Dna Virus ◽

Rna Granules ◽

Infected Cells ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

RNA granules are cytoplasmic, non-membranous ribonucleoprotein compartments that form ubiquitously and are often referred to as foci for post-transcriptional gene regulation. Recent research on RNA processing bodies (PB) and stress granules (SG) has shown wide implications of these cytoplasmic RNA granules and their components in suppression of RNA translation as host intracellular innate immunity against infecting viruses. Many RNA viruses either counteract or co-opt these RNA granules; however, many fundamental questions about DNA viruses with respect to their interaction with these two RNA granules remain elusive. Kaposi’s sarcoma-associated herpesvirus (KSHV), a tumor-causing DNA virus, exhibits two distinct phases of infection and encodes ∼90 viral gene products during the lytic phase of infection compared to only a few (∼5) during the latent phase. Thus, productive KSHV infection relies heavily on the host cell translational machinery, which often links to the formation of PB and SG. One major question is how KSHV counteracts the hostile environment of RNA granules for its productive infection. Recent studies demonstrated that KSHV copes with the translational suppression by cellular RNA granules, PB and SG, by expressing ORF57, a viral RNA-binding protein, during KSHV lytic infection. ORF57 interacts with Ago2 and GW182, two major components of PB, and prevents the scaffolding activity of GW182 at the initial stage of PB formation in the infected cells. ORF57 also interacts with protein kinase R (PKR) and PKR-activating protein (PACT) to block PKR dimerization and kinase activation, and thus inhibits eIF2α phosphorylation and SG formation. The homologous immediate-early regulatory protein ICP27 of herpes simplex virus type 1 (HSV-1), but not the EB2 protein of Epstein-Barr virus (EBV), shares this conserved inhibitory function with KSHV ORF57 on PB and SG. Through KSHV ORF57 studies, we have learned much about how a DNA virus in the infected cells is equipped to evade host antiviral immunity for its replication and productive infection. KSHV ORF57 would be an excellent viral target for development of anti-KSHV-specific therapy.

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