Intracellular Tat of human immunodeficiency virus type 1 activates lytic cycle replication of Kaposi's sarcoma-associated herpesvirus: role of JAK/STAT signaling.
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ABSTRACT:
SUBMITTER: Zeng Y
PROVIDER: S-EPMC1865948 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Herpesviruses exist in two states, latency and a lytic productive cycle. Here we identify an immediate-early gene encoded by Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus eight (HHV8) that activates lytic cycle gene expression from the latent viral genome. The gene is a homologue of Rta, a transcriptional activator encoded by Epstein-Barr virus (EBV). KSHV/Rta activated KSHV early lytic genes, including virus-encoded interleukin 6 and polyadenylated nuclear RNA, and a late gene, small viral capsid antigen. In cells dually infected with Epstein-Barr virus and KSHV, each Rta activated only autologous lytic cycle genes. Expression of viral cytokines under control of the KSHV/Rta gene is likely to contribute to the pathogenesis of KSHV-associated diseases.
| S-EPMC27987 | biostudies-literature
Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) is a human tumorigenic virus exhibiting two forms of infection, latent and lytic. Latent infection is abortive and allows the virus to establish lifelong infection, while lytic infection is productive, and is needed for virus dissemination within the host and between hosts. Latent infection may reactivate and switch towards the lytic cycle. This switch is a critical step in the maintenance of long-term infection and for the development of KSHV-related neoplasms. In this study, we examined the effect of nucleolar stress, manifested by failure in ribosome biogenesis or function and often coupled with p53 activation, on lytic reactivation of KSHV. To this end, we induced nucleolar stress by treatment with Actinomycin D, CX-5461 or BMH-21. Treatment with these compounds alone did not induce the lytic cycle. However, enhancement of the lytic cycle by these compounds was evident when combined with expression of the viral protein K-Rta. Further experiments employing combined treatments with Nutlin-3, knock-down of p53 and isogenic p53+/+ and p53-/- cells indicated that the enhancement of lytic reactivation by nucleolar stress does not depend on p53. Thus, our study identifies nucleolar stress as a novel regulator of KSHV infection, which synergizes with K-Rta expression to increase lytic reactivation. This suggests that certain therapeutic interventions, which induce nucleolar stress, may affect the outcome of KSHV infection.
| S-EPMC5862618 | biostudies-literature
Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) is an etiologic agent of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman disease. In studies of KSHV, efficient virus production and isolation are essential. Reactivation of KSHV can be initiated by treating latently infected cells with chemicals, such as 12-O-tetradecanoyl-phorbol-13-acetate and sodium butyrate. These chemicals have been used as tools to induce lytic replication and viral production in KSHV-producing cell lines. Dimethyl sulfoxide (DMSO) is an organosulfur compound that is frequently used as an aprotic solvent similar to water. In experiments exploring signaling pathways in KSHV-infected cells, DMSO treatment alone as a vehicle affected the lytic gene expression of KSHV. However, to the best of our knowledge, the effects of DMSO on KSHV-producing cells have not yet been reported. Therefore, in this study, we investigated whether DMSO could be used as a reagent to enhance viral production during lytic replication in KSHV-producing cells and assessed the underlying mechanisms. The effects of DMSO on KSHV production were analyzed in iSLK BAC16 cells, which have been widely used for recombinant KSHV production. We found that the production of KSHV virions was significantly increased by treatment with DMSO during the induction of lytic replication. Mechanistically, lytic genes of KSHV were enhanced by DMSO treatment, which was correlated with virion production. Additionally, DMSO induced the phosphorylation of JNK during lytic replication, and inhibition of JNK abolished the effects of DMSO on lytic replication and virion production. Our findings showed that additional treatment with DMSO during the induction of lytic replication significantly improved the yield of KSHV production.
| S-EPMC8716793 | biostudies-literature
Project description:Kaposi's sarcoma associated herpesvirus (KSHV) is associated with Kaposis's sarcoma (KS), primary effusion lymphoma and multicentric Castleman's disease. KSHV encodes at least 8 open reading frames (ORFs) that play important roles in its lytic DNA replication. Among which, ORF6 of KSHV encodes an ssDNA binding protein that has been proved to participate in origin-dependent DNA replication in transient assays. To define further the function of ORF6 in the virus life cycle, we constructed a recombinant virus genome with a large deletion within the ORF6 locus by using a bacterial artificial chromosome (BAC) system. Stable 293T cells carrying the BAC36 (wild type) and BAC?6 genomes were generated. When monolayers of 293T-BAC36 and 293T-BAC?6 cells were induced with 12-O-tetradecanoylphorbol-13-acetate (TPA) and sodium butyrate, infectious virus was detected from the 293T-BAC36 cell supernatants only and not from the 293T- BAC?6 cell supernatants. DNA synthesis was defective in 293T-BAC?6 cells. Expression of ORF6 in trans in BAC?6-containing cells was able to rescue both defects. Our results provide genetic evidence that ORF6 is essential for KSHV lytic replication. The stable 293T cells carrying the BAC36 and BAC?6 genomes could be used as tools to investigate the detailed functions of ORF6 in the lytic replication of KSHV.
| S-EPMC4050029 | biostudies-literature
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.
| S-EPMC5044832 | biostudies-literature
Project description:Lytic induction of latent Kaposi's sarcoma-associated herpesvirus (KSHV) has been considered as a therapeutic option for efficient treatment of several KSHV-associated malignancies. Here, we developed a robust high-throughput screening system that allows an easy and quantitative measurement of lytic induction of latent KSHV and discovered three anthracyclines as potent inducers from screen of FDA-approved drugs. Lytic induction of latent KSHV by three compounds was verified by the significant induction of lytic genes and subsequent production of infectious KSHV. Importantly, lytic induction by three compounds was much more efficient than that by sodium butyrate, a well-characterized inducer of KSHV lytic cycle. Mechanistically, the anthracyclines caused lytic induction of KSHV through apoptosis induced by their DNA intercalation rather than topoisomerase II inhibition. Consequently, our results clearly demonstrated a role of anthracyclines as effective lytic inducers of KSHV and also provided a molecular basis of their use for efficient treatment of diseases associated with KSHV infection.
| S-EPMC4226701 | biostudies-literature
Project description:UnlabelledThe K1 gene product of Kaposi's sarcoma-associated herpesvirus (KSHV) is encoded by the first open reading frame (ORF) of the viral genome. To investigate the role of the K1 gene during the KSHV life cycle, we constructed a set of recombinant viruses that contained either wild-type (WT) K1, a deleted K1 ORF (KSHVΔK1), stop codons within the K1 ORF (KSHV-K15×STOP), or a revertant K1 virus (KSHV-K1REV). We report that the recombinant viruses KSHVΔK1 and KSHV-K15×STOP displayed significantly reduced lytic replication compared to WT KSHV and KSHV-K1REV upon reactivation from latency. Additionally, cells infected with the recombinant viruses KSHVΔK1 and KSHV-K15×STOP also yielded smaller amounts of infectious progeny upon reactivation than did WT KSHV- and KSHV-K1REV-infected cells. Upon reactivation from latency, WT KSHV- and KSHV-K1REV-infected cells displayed activated Akt kinase, as evidenced by its phosphorylation, while cells infected with viruses deleted for K1 showed reduced phosphorylation and activation of Akt kinase. Overall, our results suggest that K1 plays an important role during the KSHV life cycle.ImportanceKaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of three human malignancies, and KSHV K1 is a signaling protein that has been shown to be involved in cellular transformation and to activate the phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR pathway. In order to investigate the role of the K1 protein in the life cycle of KSHV, we constructed recombinant viruses that were deficient for K1. We found that K1 deletion viruses displayed reduced lytic replication compared to the WT virus and also yielded smaller numbers of infectious progeny. We report that K1 plays an important role in the life cycle of KSHV.
| S-EPMC4988170 | biostudies-literature
Project description:The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated gene 9) system is an RNA-guided, DNA editing method that has been widely used for gene editing, including human viruses. Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8), following latent infection in human cells, can cause a variety of malignancies, such as Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman disease (MCD), with a high prevalence in immunocompromised patients. Of significant concern, the latent infection with KSHV has been shown to lead to increased resistance to antiviral therapies. MicroRNAs (miRNAs) are a set of non-coding, small RNA molecules that regulate protein-coding genes at the post-transcriptional and translational levels. KSHV has its miRNAs, most of which are expressed in latently infected cells and play a crucial role in maintaining KSHV latency. Notably, by regulating the expression of the downstream target genes in host cells, KSHV miRNAs can interact with the host environment to promote the development of KSHV-related diseases. Although CRISPR/Cas9 has been reported to edit KSHV protein-coding genes, there is no published literature on whether the CRISPR/Cas9 system can regulate the expression of KSHV miRNAs. In this study, we used CRISPR/Cas9 to inhibit the expression of KSHV miRNAs by directly editing the DNA sequences of individual KSHV miRNAs, or the promoter of clustered KHSV miRNAs, in latent KSHV-infected PEL cells. Our results show that CRISPR/Cas9 can ablate KSHV miRNAs expression, which in turn leads to the upregulation of viral lytic genes and alteration of host cellular gene expression. To the best of our knowledge, our study is the first reported demonstration of the CRISPR/Cas9 system editing KSHV miRNAs, further expanding the application of CRISPR/Cas9 as a novel antiviral strategy targeting KSHV latency.
| S-EPMC7813130 | 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:Many DNA virus replication-related proteins are associated with promyelocytic leukemia protein (PML), a component of nuclear domain 10 (ND10), which has been investigated for its potential involvement in viral replication. In the case of Kaposi's sarcoma-associated herpesvirus (KSHV) lytic gene products, K8 (K-bZIP), ORF59, and ORF75 have been shown to colocalize with PML, but its importance in KSHV lytic replication is still unclear. In this study, we analyzed the functional influence of PML on KSHV latency and lytic replication in KSHV-infected primary effusion lymphoma (PEL) cell lines. Stable PML-knockout (BC3-PMLKO) and PML-overexpressing BC3 cells (BC3PML) were successfully generated and the latency and reactivation status were analyzed. The results demonstrated that neither KSHV latency nor the episome copy number was affected in BC3-PMLKO cells. In the reactivation phase, the expression dynamics of KSHV immediate-early or early lytic proteins such as RTA, K9 (vIRF1), K5, K3, ORF59, and K8 (K-bZIP) were comparable between wild-type, control BC3, and BC3-PMLKO cells. Interestingly, KSHV lytic replication, virion production, and expression of late genes were downregulated in BC3-PMLKO cells and upregulated in BC3PML cells, compared to those in control or wild-type BC3 cells. Moreover, exogenous PML increased the size of the PML dots and recruited additional K8 (K-bZIP) to PML-NBs as dots. Therefore, PML would function as a positive regulator for KSHV lytic DNA replication by recruiting KSHV replication factors such as 8 (K-bZIP) or ORF59 to the PML-NBs.
| S-EPMC6186782 | biostudies-literature