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Kaposi's sarcoma-associated herpesvirus processivity factor (PF-8) recruits cellular E3 ubiquitin ligase CHFR to promote PARP1 degradation and lytic replication.

ABSTRACT: Kaposi's sarcoma-associated herpesvirus (KSHV), which belongs to the gammaherpesvirus subfamily, is associated with the pathogenesis of various tumors. Nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP1) catalyzes the polymerization of ADP-ribose units on target proteins. In KSHV-infected cells, PARP1 inhibits replication and transcription activator (RTA), a molecular switch that initiates lytic replication, through direct interaction. Thus, for efficient replication, KSHV has to overcome the molecular barrier in the form of PARP1. Previously, we have demonstrated that KSHV downregulates the expression of PARP1 through PF-8, a viral processivity factor. PF-8 induces ubiquitin-proteasome system-mediated degradation of PARP1 via direct physical association and enhances RTA transactivation activity. Here, we showed that dimerization domains of PF-8 are crucial not only for PARP1 interaction and degradation but also for enhancement of the RTA transactivation activity. PF-8 recruited CHFR for the PARP1 degradation. A knockdown of CHFR attenuated the PF-8-induced PARP1 degradation and enhancement of the RTA transactivation activity, leading to reduced KSHV lytic replication. These findings reveal a mechanism by which KSHV PF-8 recruits a cellular E3 ligase to curtail the inhibitory effect of PARP1 on KSHV lytic replication.

SUBMITTER: Chung WC

PROVIDER: S-EPMC7872283 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Kaposi's sarcoma-associated herpesvirus processivity factor (PF-8) recruits cellular E3 ubiquitin ligase CHFR to promote PARP1 degradation and lytic replication.

Chung Woo-Chang WC Lee Seungrae S Kim Yejin Y Seo Jong Bok JB Song Moon Jung MJ

PLoS pathogens 20210128 1

Kaposi's sarcoma-associated herpesvirus (KSHV), which belongs to the gammaherpesvirus subfamily, is associated with the pathogenesis of various tumors. Nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP1) catalyzes the polymerization of ADP-ribose units on target proteins. In KSHV-infected cells, PARP1 inhibits replication and transcription activator (RTA), a molecular switch that initiates lytic replication, through direct interaction. Thus, for efficient replication, KSHV has to overcome the m ...[more]

PMID: 33508027

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Project description:The downregulation of immune synapse components such as major histocompatibility complex class I (MHC-I) and ICAM-1 is a common viral immune evasion strategy that protects infected cells from targeted elimination by cytolytic effector functions of the immune system. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes two membrane-bound ubiquitin E3 ligases, called K3 and K5, which share the ability to induce internalization and degradation of MHC-I molecules. Although individual functions of K3 and K5 outside the viral genome are well characterized, their roles during the KSHV life cycle are still unclear. In this study, we individually introduced the amino acid-coding sequences of K3 or K5 into a ?K3 ?K5 recombinant virus, at either original or interchanged genomic positions. Recombinants harboring coding sequences within the K5 locus showed higher K3 and K5 protein expression levels and more rapid surface receptor downregulation than cognate recombinants in which coding sequences were introduced into the K3 locus. To identify infected cells undergoing K3-mediated downregulation of MHC-I, we employed a novel reporter virus, called red-green-blue-BAC16 (RGB-BAC16), which was engineered to harbor three fluorescent protein expression cassettes: EF1?-monomeric red fluorescent protein 1 (mRFP1), polyadenylated nuclear RNA promoter (pPAN)-enhanced green fluorescent protein (EGFP), and pK8.1-monomeric blue fluorescent protein (tagBFP), marking latent, immediate early, and late viral gene expression, respectively. Analysis of RGB-derived K3 and K5 deletion mutants showed that while the K5-mediated downregulation of MHC-I was concomitant with pPAN induction, the reduction of MHC-I surface expression by K3 was evident in cells that were enriched for pPAN-driven EGFP(high) and pK8.1-driven blue fluorescent protein-positive (BFP(+)) populations. These data support the notion that immunoreceptor downregulation occurs by a sequential process wherein K5 is critical during the immediately early phase and K3 plays a significant role during later stages.Although the roles of K3 and K5 outside the viral genome are well characterized, the function of these proteins in the context of the KSHV life cycle has remained unclear, particularly in the case of K3. This study examined the relative contributions of K3 and K5 to the downregulation of MHC-I during the lytic replication of KSHV. We show that while K5 acts immediately upon entry into the lytic phase, K3-mediated downregulation of MHC-I was evident during later stages of lytic replication. The identification of distinctly timed K3 and K5 activities significantly advances our understanding of KSHV-mediated immune evasion. Crucial to this study was the development of a novel recombinant KSHV, called RGB-BAC16, which facilitated the delineation of stage-specific phenotypes.

Project description:UnlabelledKaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of commonly fatal malignancies of immunocompromised individuals, including primary effusion lymphoma (PEL) and Kaposi's sarcoma (KS). A hallmark of all herpesviruses is their biphasic life cycle-viral latency and the productive lytic cycle-and it is well established that reactivation of the KSHV lytic cycle is associated with KS pathogenesis. Therefore, a thorough appreciation of the mechanisms that govern reactivation is required to better understand disease progression. The viral protein replication and transcription activator (RTA) is the KSHV lytic switch protein due to its ability to drive the expression of various lytic genes, leading to reactivation of the entire lytic cycle. While the mechanisms for activating lytic gene expression have received much attention, how RTA impacts cellular function is less well understood. To address this, we developed a cell line with doxycycline-inducible RTA expression and applied stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative proteomics. Using this methodology, we have identified a novel cellular protein (AT-rich interacting domain containing 3B [ARID3B]) whose expression was enhanced by RTA and that relocalized to replication compartments upon lytic reactivation. We also show that small interfering RNA (siRNA) knockdown or overexpression of ARID3B led to an enhancement or inhibition of lytic reactivation, respectively. Furthermore, DNA affinity and chromatin immunoprecipitation assays demonstrated that ARID3B specifically interacts with A/T-rich elements in the KSHV origin of lytic replication (oriLyt), and this was dependent on lytic cycle reactivation. Therefore, we have identified a novel cellular protein whose expression is enhanced by KSHV RTA with the ability to inhibit KSHV reactivation.ImportanceKaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of fatal malignancies of immunocompromised individuals, including Kaposi's sarcoma (KS). Herpesviruses are able to establish a latent infection, in which they escape immune detection by restricting viral gene expression. Importantly, however, reactivation of productive viral replication (the lytic cycle) is necessary for the pathogenesis of KS. Therefore, it is important that we comprehensively understand the mechanisms that govern lytic reactivation, to better understand disease progression. In this study, we have identified a novel cellular protein (AT-rich interacting domain protein 3B [ARID3B]) that we show is able to temper lytic reactivation. We showed that the master lytic switch protein, RTA, enhanced ARID3B levels, which then interacted with viral DNA in a lytic cycle-dependent manner. Therefore, we have added a new factor to the list of cellular proteins that regulate the KSHV lytic cycle, which has implications for our understanding of KSHV biology.

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.

Dataset Information

Kaposi's sarcoma-associated herpesvirus processivity factor (PF-8) recruits cellular E3 ubiquitin ligase CHFR to promote PARP1 degradation and lytic replication.

Publications

Kaposi's sarcoma-associated herpesvirus processivity factor (PF-8) recruits cellular E3 ubiquitin ligase CHFR to promote PARP1 degradation and lytic replication.

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