The K1 Protein of Kaposi's Sarcoma-Associated Herpesvirus Augments Viral Lytic Replication

ABSTRACT The 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-K1 5×STOP ), or a revertant K1 virus (KSHV-K1 REV ). We report that the recombinant viruses KSHVΔK1 and KSHV-K1 5×STOP displayed significantly reduced lytic replication compared to WT KSHV and KSHV-K1 REV upon reactivation from latency. Additionally, cells infected with the recombinant viruses KSHVΔK1 and KSHV-K1 5×STOP also yielded smaller amounts of infectious progeny upon reactivation than did WT KSHV- and KSHV-K1 REV -infected cells. Upon reactivation from latency, WT KSHV- and KSHV-K1 REV -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. IMPORTANCE Kaposi'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

Epigenetic Analysis of KSHV Latent and Lytic Genomes

Epigenetic modifications of the herpesviral genome play a key role in the transcriptional control of latent and lytic genes during a productive viral lifecycle. In this study, we describe for the first time a comprehensive genome-wide ChIP-on-Chip analysis of the chromatin associated with the Kaposi's sarcoma-associated herpesvirus (KSHV) genome during latency and lytic reactivation. Depending on the gene expression class, different combinations of activating [acetylated H3 (AcH3) and H3K4me3] and repressive [H3K9me3 and H3K27me3] histone modifications are associated with the viral latent genome, which changes upon reactivation in a manner that is correlated with their expression. Specifically, both the activating marks co-localize on the KSHV latent genome, as do the repressive marks. However, the activating and repressive histone modifications are mutually exclusive of each other on the bulk of the latent KSHV genome. The genomic region encoding the IE genes ORF50 and ORF48 possesses the features of a bivalent chromatin structure characterized by the concomitant presence of the activating H3K4me3 and the repressive H3K27me3 marks during latency, which rapidly changes upon reactivation with increasing AcH3 and H3K4me3 marks and decreasing H3K27me3. Furthermore, EZH2, the H3K27me3 histone methyltransferase of the Polycomb group proteins (PcG), colocalizes with the H3K27me3 mark on the entire KSHV genome during latency, whereas RTA-mediated reactivation induces EZH2 dissociation from the genomic regions encoding IE and E genes concurrent with decreasing H3K27me3 level and increasing IE/E lytic gene expression. Moreover, either the inhibition of EZH2 expression by a small molecule inhibitor DZNep and RNAi knockdown, or the expression of H3K27me3-specific histone demethylases apparently induced the KSHV lytic gene expression cascade. These data indicate that histone modifications associated with the KSHV latent genome are involved in the regulation of latency and ultimately in the control of the temporal and sequential expression of the lytic gene cascade. In addition, the PcG proteins play a critical role in the control of KSHV latency by maintaining a reversible heterochromatin on the KSHV lytic genes. Thus, the regulation of the spatial and temporal association of the PcG proteins with the KSHV genome may be crucial for propagating the KSHV lifecycle

The K1 Protein of Kaposi's Sarcoma-Associated Herpesvirus Augments Viral Lytic Replication

The 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-K1(5×STOP)), or a revertant K1 virus (KSHV-K1(REV)). We report that the recombinant viruses KSHVΔK1 and KSHV-K1(5×STOP) displayed significantly reduced lytic replication compared to WT KSHV and KSHV-K1(REV) upon reactivation from latency. Additionally, cells infected with the recombinant viruses KSHVΔK1 and KSHV-K1(5×STOP) also yielded smaller amounts of infectious progeny upon reactivation than did WT KSHV- and KSHV-K1(REV)-infected cells. Upon reactivation from latency, WT KSHV- and KSHV-K1(REV)-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. IMPORTANCE Kaposi'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

Activation of p90 Ribosomal S6 Kinases (RSKs) by ORF45 of Kaposi Sarcoma-Associated Herpesvirus is Critical for Optimal Production of Infectious Viruses

We have previously shown that ORF45, an immediate-early and tegument protein of Kaposi's sarcoma associated herpesvirus (KSHV), causes sustained activation of p90 ribosomal S6 kinases (RSKs) and ERK. We have now identified the critical region of ORF45 that is involved in RSK interaction and activation. Alanine scanning mutagenesis of this region revealed that a single F66A point mutation abolished binding of ORF45 to RSK or ERK, and consequently its ability to activate the kinases. We introduced the F66A mutation into BAC16 (a bacterial artificial chromosome clone containing the entire infectious KSHV genome), producing BAC16-45F66A. In parallel, we also repaired the mutation and obtained a revertant BAC16-45A66F. Reconstitution of these mutants in iSLK cells demonstrated that the ORF45-F66A mutant failed to cause sustained ERK and RSK activation during lytic reactivation, resulting in dramatic differences in the phosphoproteomic profile between the wild-type virus-infected cells and the mutant virus-infected cells. ORF45 mutation or deletion was also accompanied by noticeably decreased viral gene expression during lytic reactivation. The ORF45-F66A mutant consequently produced significantly fewer infectious progeny virions than the wild type or the revertant. These results suggest a critical role for ORF45-mediated RSK activation in KSHV lytic replication. IMPORTANCE STATEMENT: Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of three human malignancies. KSHV pathogenesis is intimately linked to its ability to modulate the host cell microenvironment, and facilitate efficient production of progeny viral particles. We have previously described the mechanism by which the KSHV lytic protein ORF45 activates the cellular kinases ERK and RSK. We now have mapped the critical region of ORF45 responsible for binding and activation of ERK/RSK to a single residue, F66. We mutated this amino acid of ORF45 (F66A) and introduced the mutation into a newly developed bacterial artificial chromosome containing the KSHV genome (BAC16). This system has provided us with a useful tool to characterize the functions of ORF45-activated RSK upon KSHV lytic reactivation. We show that viral gene expression and virion production are significantly reduced by F66A mutation, indicating a critical role for ORF45-activated RSK during KSHV lytic replicatio

Reinitiation after Translation of Two Upstream Open Reading Frames (ORF) Governs Expression of the ORF35-37 Kaposi's Sarcoma-Associated Herpesvirus Polycistronic mRNA

The Kaposi's sarcoma-associated herpesvirus (KSHV) ORF36 protein kinase is translated as a downstream gene from the ORF35-37 polycistronic mRNA via a unique mechanism involving short upstream open reading frames (uORFs) located in the 5′ untranslated region. Here, we confirm that ORF35-37 is functionally dicistronic during infection and demonstrate that mutation of the dominant uORF restricts KSHV replication. Leaky scanning past the uORFs facilitates ORF35 expression, while a reinitiation mechanism after translation of the uORFs enables ORF36 expression

Kaposi's Sarcoma Associated Herpesvirus Tegument Protein ORF75 Is Essential for Viral Lytic Replication and Plays a Critical Role in the Antagonization of ND10Instituted Intrinsic Immunity - art. no. e1003863

Nuclear domain 10 (ND10) components are restriction factors that inhibit herpesviral replication. Effector proteins of different herpesviruses can antagonize this restriction by a variety of strategies, including degradation or relocalization of ND10 proteins. We investigated the interplay of Kaposi's Sarcoma-Associated Herpesvirus (KSHV) infection and cellular defense by nuclear domain 10 (ND10) components. Knock-down experiments in primary human cells show that KSHV-infection is restricted by the ND10 components PML and Sp100, but not by ATRX. After KSHV infection, ATRX is efficiently depleted and Daxx is dispersed from ND10, indicating that these two ND10 components can be antagonized by KSHV. We then identified the ORF75 tegument protein of KSHV as the viral factor that induces the disappearance of ATRX and relocalization of Daxx. ORF75 belongs to a viral protein family (viral FGARATs) that has homologous proteins in all gamma-herpesviruses. Isolated expression of ORF75 in primary cells induces a relocalization of PML and dispersal of Sp100, indicating that this viral effector protein is able to influence multiple ND10 components. Moreover, by constructing a KSHV mutant harboring a stop codon at the beginning of ORF75, we could demonstrate that ORF75 is absolutely essential for viral replication and the initiation of viral immediate-early gene expression. Using recombinant viruses either carrying Flag- or YFP-tagged variants of ORF75, we could further corroborate the role of ORF75 in the antagonization of ND10-mediated intrinsic immunity, and show that it is independent of the PML antagonist vIRF3. Members of the viral FGARAT family target different ND10 components, suggesting that the ND10 targets of viral FGARAT proteins have diversified during evolution. We assume that overcoming ND10 intrinsic defense constitutes a critical event in the replication of all herpesviruses; on the other hand, restriction of herpesviral replication by ND10 components may also promote latency as the default outcome of infection.Author Summary Kaposi's Sarcoma-Associated Herpesvirus (KSHV) establishes a lifelong persistent infection in humans and is associated with tumors and lymphoproliferative disease, particularly upon immunosuppression. The virus has to overcome cellular intrinsic immunity in order to initiate viral protein expression and genome replication in primary infection. We demonstrated that KSHV is restricted by a cellular intrinsic immunity complex called nuclear domain 10 (ND10) and identified a critical role of the KSHV ORF75 protein, which is part of the viral particle, in this process. We found that ORF75 is essential for viral replication and that ORF75 leads to disappearance of the ND10 protein ATRX. Furthermore, it induces the relocalization of several other ND10 components. Noteworthy, all herpesviruses studied so far have evolved mechanisms for ND10 counteraction, indicating the importance of this step for herpesviral replication. The individual mechanisms, however, including the extent of ND10-antagonization, are of considerable variation between different herpesviruses. We speculate that, in contrast to efficient lytic replication of alphaherpesviruses, less effective ND10 counteraction may represent a doorway for gammaherpesviruses to latent infectio