During evolution, herpesviruses have developed numerous, and often very ingenious, strategies to counteract efficient host immunity. Specifically, Kaposi's sarcoma-associated herpesvirus (KSHV) eludes host immunity by undergoing a dormant stage, called latency wherein it expresses a minimal number of viral proteins to evade host immune activation. Here, we show that during latency, KSHV hijacks the complement pathway to promote cell survival. We detected strong deposition of complement membrane attack complex C5b-9 and the complement component C3 activated product C3b on Kaposi's sarcoma spindle tumor cells, and on human endothelial cells latently infected by KSHV, TIME-KSHV and TIVE-LTC, but not on their respective uninfected control cells, TIME and TIVE. We further showed that complement activation in latently KSHV-infected cells was mediated by the alternative complement pathway through down-regulation of cell surface complement regulatory proteins CD55 and CD59. Interestingly, complement activation caused minimal cell death but promoted the survival of latently KSHV-infected cells grown in medium depleted of growth factors. We found that complement activation increased STAT3 tyrosine phosphorylation (Y705) of KSHV-infected cells, which was required for the enhanced cell survival. Furthermore, overexpression of either CD55 or CD59 in latently KSHV-infected cells was sufficient to inhibit complement activation, prevent STAT3 Y705 phosphorylation and abolish the enhanced survival of cells cultured in growth factor-depleted condition. Together, these results demonstrate a novel mechanism by which an oncogenic virus subverts and exploits the host innate immune system to promote viral persistent infection.
The complement system is an important part of the innate immune system. Pathogens have evolved diverse strategies to evade host immune responses including attack of the complement system. Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with Kaposi's sarcoma (KS), primary effusion lymphoma and a subset of multicentric Castleman's disease. KSHV encodes a number of viral proteins to counter host immune responses during productive lytic replication. On the other hand, KSHV utilizes latency as a default replication program during which it expresses a minimal number of proteins to evade host immune detection. Thus, the complement system is expected to be silent during KSHV latency. In this study, we have found that the complement system is unexpectedly activated in latently KSHV-infected endothelial cells and in KS tumor cells wherein KSHV downregulates the expression of CD55 and CD59 complement regulatory proteins. More interestingly, most of latently KSHV-infected cells not only are resistant to complement-mediated cell killing, but also acquire survival advantage by inducing STAT3 tyrosine phosphorylation. These results demonstrate a novel mechanism by which an oncogenic virus exploits the host innate immune system to promote viral persistent infection.
The gammaherpesviruses, including Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), establish latency in memory B lymphocytes and promote lymphoproliferative disease in immunocompromised individuals. The precise immune mechanisms that prevent gammaherpesvirus reactivation and tumorigenesis are poorly defined. Murine gammaherpesvirus 68 (MHV68) is closely related to EBV and KSHV, and type I (alpha/beta) interferons (IFNαβ) regulate MHV68 reactivation from both B cells and macrophages by unknown mechanisms. Here we demonstrate that IFNβ is highly upregulated during latent infection, in the absence of detectable MHV68 replication. We identify an interferon-stimulated response element (ISRE) in the MHV68 M2 gene promoter that is bound by the IFNαβ-induced transcriptional repressor IRF2 during latency in vivo. The M2 protein regulates B cell signaling to promote establishment of latency and reactivation. Virus lacking the M2 ISRE (ISREΔ) overexpresses M2 mRNA and displays uncontrolled acute replication in vivo, higher latent viral load, and aberrantly high reactivation from latency. These phenotypes of the ISREΔ mutant are B-cell-specific, require IRF2, and correlate with a significant increase in virulence in a model of acute viral pneumonia. We therefore identify a mechanism by which a gammaherpesvirus subverts host IFNαβ signaling in a surprisingly cooperative manner, to directly repress viral replication and reactivation and enforce latency, thereby minimizing acute host disease. Since we find ISREs 5′ to the major lymphocyte latency genes of multiple rodent, primate, and human gammaherpesviruses, we propose that cooperative subversion of IFNαβ-induced IRFs to promote latent infection is an ancient strategy that ensures a stable, minimally-pathogenic virus-host relationship.
Herpesviruses establish life-long infection in a non-replicating state termed latency. During immune compromise, herpesviruses can reactivate and cause severe disease, including cancer. We investigated mechanisms by which interferons alpha/beta (IFNαβ), a family of antiviral immune genes, inhibit reactivation of murine gammaherpesvirus 68 (MHV68). MHV68 is related to Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, human gammaherpesviruses associated with multiple cancers. We made the surprising discovery that during latency, MHV68 cooperates with IFNαβ to inhibit its own replication. Specifically, a viral gene required for reactivation has evolved to be directly repressed by an IFNαβ-induced transcription factor, IRF2. Once virus replication has triggered sufficient IFNαβ production, expression of this viral gene is reduced and reactivation efficiency decreases. This strategy safeguards the health of the host, since a mutant virus that cannot respond to IRF2 replicates uncontrollably and is more virulent. Viral sensing of IFNαβ is also potentially subversive, since it allows MHV68 to detect periods of localized immune quiescence during which it can reactivate and spread to a new host. Thus, we highlight a novel path of virus-host coevolution, toward cooperative subversion of the antiviral immune response. These observations may illuminate new targets for drugs to inhibit herpesvirus reactivation or eliminate herpesvirus-associated tumors.
Kaposi's sarcoma-associated herpesvirus (KSHV) establishes a latent
infection in the host following an acute infection. Reactivation from latency
contributes to the development of KSHV-induced malignancies, which include
Kaposi's sarcoma (KS), the most common cancer in untreated AIDS patients,
primary effusion lymphoma and multicentric Castleman's disease. However,
the physiological cues that trigger KSHV reactivation remain unclear. Here, we
show that the reactive oxygen species (ROS) hydrogen peroxide
(H2O2) induces KSHV reactivation from latency through
both autocrine and paracrine signaling. Furthermore, KSHV spontaneous lytic
replication, and KSHV reactivation from latency induced by oxidative stress,
hypoxia, and proinflammatory and proangiogenic cytokines are mediated by
H2O2. Mechanistically, H2O2
induction of KSHV reactivation depends on the activation of mitogen-activated
protein kinase ERK1/2, JNK, and p38 pathways. Significantly,
H2O2 scavengers N-acetyl-L-cysteine (NAC), catalase
and glutathione inhibit KSHV lytic replication in culture. In a mouse model of
KSHV-induced lymphoma, NAC effectively inhibits KSHV lytic replication and
significantly prolongs the lifespan of the mice. These results directly relate
KSHV reactivation to oxidative stress and inflammation, which are physiological
hallmarks of KS patients. The discovery of this novel mechanism of KSHV
reactivation indicates that antioxidants and anti-inflammation drugs could be
promising preventive and therapeutic agents for effectively targeting KSHV
replication and KSHV-related malignancies.
Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of all
clinical forms of Kaposi's sarcoma (KS) and several other malignancies. The
life cycle of KSHV consists of latent and lytic phases. While establishment of
viral latency is essential for KSHV to evade host immune surveillances, viral
lytic replication promotes KSHV-induced malignancies. In this study, we show
that the reactive oxygen species (ROS) hydrogen peroxide
(H2O2) induces KSHV reactivation from latency.
Furthermore, induction of KSHV reactivation by oxidative stress, hypoxia, and
proinflammatory and proangiogenic cytokines, which are physiological hallmarks
in all clinical forms of KS patients, is mediated by H2O2.
Significantly, antioxidants inhibit H2O2-induced KSHV
lytic replication in culture and in a mouse model of KSHV-induced lymphoma.
These results show that ROS is likely an important physiological cue that
triggers KSHV replication. The discovery of this novel mechanism of KSHV
reactivation indicates that antioxidants and anti-inflammation drugs might be
promising preventive and therapeutic agents for effectively targeting KSHV
replication and KSHV-related malignancies.
Altered cell metabolism is inherently connected with pathological conditions including cancer and viral infections. Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma (KS). KS tumour cells display features of lymphatic endothelial differentiation and in their vast majority are latently infected with KSHV, while a small number are lytically infected, producing virions. Latently infected cells express only a subset of viral genes, mainly located within the latency-associated region, among them 12 microRNAs. Notably, the metabolic properties of KSHV-infected cells closely resemble the metabolic hallmarks of cancer cells. However, how and why KSHV alters host cell metabolism remains poorly understood. Here, we investigated the effect of KSHV infection on the metabolic profile of primary dermal microvascular lymphatic endothelial cells (LEC) and the functional relevance of this effect. We found that the KSHV microRNAs within the oncogenic cluster collaborate to decrease mitochondria biogenesis and to induce aerobic glycolysis in infected cells. KSHV microRNAs expression decreases oxygen consumption, increase lactate secretion and glucose uptake, stabilize HIF1α and decreases mitochondria copy number. Importantly this metabolic shift is important for latency maintenance and provides a growth advantage. Mechanistically we show that KSHV alters host cell energy metabolism through microRNA-mediated down regulation of EGLN2 and HSPA9. Our data suggest that the KSHV microRNAs induce a metabolic transformation by concurrent regulation of two independent pathways; transcriptional reprograming via HIF1 activation and reduction of mitochondria biogenesis through down regulation of the mitochondrial import machinery. These findings implicate viral microRNAs in the regulation of the cellular metabolism and highlight new potential avenues to inhibit viral latency.
Kaposi's sarcoma (KS) is the most common cancer in HIV-infected untreated individuals. Kaposi's sarcoma-associated herpesvirus (KSHV) is the infectious cause of this neoplasm. The discovery of KSHV and its oncogenic enigmas has enlightened many fields of tumor biology and viral oncogenesis. The metabolic properties of KS significantly differ from those of normal cells and resemble cancer cells in general, but the mechanisms employed by KSHV to alter host cell metabolism are poorly understood. Our work demonstrates that KSHV microRNAs can alter cell metabolism through coherent control of independent pathways, a key feature of microRNA-mediated control of cellular functions. This provides a fresh perspective for how microRNA-encoding pathogens shape a cell's metabolism to create an optimal environment for their survival and/or replication. Indeed, we show that, in the case of KSHV, viral microRNA-driven regulation of metabolism is important for viral latency. These findings will evoke new and exciting approaches to prevent KSHV from establishing latency and later on KS.
Autophagy is one of two major degradation systems in eukaryotic cells. The degradation mechanism of autophagy is required to maintain the balance between the biosynthetic and catabolic processes and also contributes to defense against invading pathogens. Recent studies suggest that a number of viruses can evade or subvert the host cell autophagic pathway to enhance their own replication. Here, we investigated the effect of autophagy on the KSHV (Kaposi's sarcoma-associated herpesvirus) life cycle. We found that the inhibition of autophagy reduces KSHV lytic reactivation from latency, and an enhancement of autophagy can be detected during KSHV lytic replication. In addition, RTA (replication and transcription activator), an essential viral protein for KSHV lytic reactivation, is able to enhance the autophagic process, leading to an increase in the number of autophagic vacuoles, an increase in the level of the lipidated LC3 protein, and the formation of autolysosomes. Moreover, the inhibition of autophagy affects RTA-mediated lytic gene expression and viral DNA replication. These results suggest that RTA increases autophagy activation to facilitate KSHV lytic replication. This is the first report demonstrating that autophagy is involved in the lytic reactivation of KSHV.
Kaposi's sarcoma-associated herpesvirus (KSHV) establishes persistent latent infection in immunocompetent hosts. Disruption of KSHV latency results in viral lytic replication, which promotes the development of KSHV-related malignancies in immunocompromised individuals. While inhibitors of classes I and II histone deacetylases (HDACs) potently reactivate KSHV from latency, the role of class III HDAC sirtuins (SIRTs) in KSHV latency remains unclear. Here, we examined the effects of inhibitors of SIRTs, nicotinamide (NAM) and sirtinol, on KSHV reactivation from latency. Treatment of latently KSHV-infected cells with NAM or sirtinol induced transcripts and proteins of the master lytic transactivator RTA (ORF50), early lytic genes ORF57 and ORF59, and late lytic gene ORF65 and increased the production of infectious virions. NAM increased the acetylation of histones H3 and H4 as well as the level of the active histone H3 trimethyl Lys4 (H3K4me3) mark but decreased the level of the repressive histone H3 trimethyl Lys27 (H3K27me3) mark in the RTA promoter. Consistent with these results, we detected SIRT1 binding to the RTA promoter. Importantly, knockdown of SIRT1 was sufficient to increase the expression of KSHV lytic genes. Accordingly, the level of the H3K4me3 mark in the RTA promoter was increased following SIRT1 knockdown, while that of the H3K27me3 mark was decreased. Furthermore, SIRT1 interacted with RTA and inhibited RTA transactivation of its own promoter and that of its downstream target, the viral interleukin-6 gene. These results indicate that SIRT1 regulates KSHV latency by inhibiting different stages of viral lytic replication and link the cellular metabolic state with the KSHV life cycle.
IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is the causal agent of several malignancies, including Kaposi's sarcoma, commonly found in immunocompromised patients. While latent infection is required for the development of KSHV-induced malignancies, viral lytic replication also promotes disease progression. However, the mechanism controlling KSHV latent versus lytic replication remains unclear. In this study, we found that class III histone deacetylases (HDACs), also known as SIRTs, whose activities are linked to the cellular metabolic state, mediate KSHV replication. Inhibitors of SIRTs can reactivate KSHV from latency. SIRTs mediate KSHV latency by epigenetically silencing a key KSHV lytic replication activator, RTA. We found that one of the SIRTs, SIRT1, binds to the RTA promoter to mediate KSHV latency. Knockdown of SIRT1 is sufficient to induce epigenetic remodeling and KSHV lytic replication. SIRT1 also interacts with RTA and inhibits RTA's transactivation function, preventing the expression of its downstream genes. Our results indicate that SIRTs regulate KSHV latency by inhibiting different stages of viral lytic replication and link the cellular metabolic state with the KSHV life cycle.
Autophagy is a highly conserved and regulated process in eukaryotic cells by which components of the cytoplasm, such as damaged organelles and foreign pathogens, become enveloped into double-membrane autophagosome vesicles that fuse with the lysosome for degradation. Viruses are adept at subverting host cellular pathways for their replication and survival. The human tumor viruses, Epstein-Barr virus (EBV), Kaposi’s Sarcoma-Associated Herpesvirus (KSHV), Hepatitis B virus (HBV), and Hepatitis C virus (HCV), have evolved novel ways of modulating autophagy during productive and latent stages of the virus life cycle. This review will discuss how the autophagy pathway becomes activated upon viral infection and the role of viral proteins in regulating the autophagy pathway. Specifically, we will examine how virus-encoded homologs of autophagy proteins evade autophagy-mediated degradation by blocking the induction, elongation, or maturation steps in the autophagy pathway. We will also discuss how certain viruses enhance autophagy induction or usurp autophagic machinery for their own replication. A comprehensive understanding of the autophagic response to tumor viruses may enable the discovery of novel antiviral and/or anticancer drug therapies.
autophagy; human tumor viruses; oncogene-induced senescence (OIS); unfolded protein response (UPR)
Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma (KS) and primary effusion B-cell lymphoma. KSHV induces reactive oxygen species (ROS) early during infection of human dermal microvascular endothelial (HMVEC-d) cells that are critical for virus entry. One of the downstream targets of ROS is nuclear factor E2-related factor 2 (Nrf2), a transcription factor with important anti-oxidative functions. Here, we show that KS skin lesions have high Nrf2 activity compared to healthy skin tissue. Within 30 minutes of de novo KSHV infection of HMVEC-d cells, we observed Nrf2 activation through ROS-mediated dissociation from its inhibitor Keap1, Ser-40 phosphorylation, and subsequent nuclear translocation. KSHV binding and consequent signaling through Src, PI3-K and PKC-ζ were also important for Nrf2 stability, phosphorylation and transcriptional activity. Although Nrf2 was dispensable for ROS homeostasis, it was essential for the induction of COX-2, VEGF-A, VEGF-D, Bcl-2, NQO1, GCS, HO1, TKT, TALDO and G6PD gene expression in KSHV-infected HMVEC-d cells. The COX-2 product PGE2 induced Nrf2 activity through paracrine and autocrine signaling, creating a feed-forward loop between COX-2 and Nrf2. vFLIP, a product of KSHV latent gene ORF71, induced Nrf2 and its target genes NQO1 and HO1. Activated Nrf2 colocalized with the KSHV genome as well as with the latency protein LANA-1. Nrf2 knockdown enhanced ORF73 expression while reducing ORF50 and other lytic gene expression without affecting KSHV entry or genome nuclear delivery. Collectively, these studies for the first time demonstrate that during de novo infection, KSHV induces Nrf2 through intricate mechanisms involving multiple signal molecules, which is important for its ability to manipulate host and viral genes, creating a microenvironment conducive to KSHV infection. Thus, Nrf2 is a potential attractive target to intervene in KSHV infection and the associated maladies.
KSHV infection of endothelial cells in vivo causes Kaposi's sarcoma and understanding the steps involved in de novo KSHV infection of these cells and the consequences is important to develop therapies to counter KSHV pathogenesis. Infection of endothelial cells in vitro is preceded by the induction of a network of host signaling agents that are necessary for virus entry, gene expression and establishment of latency. Our previous studies have implicated reactive oxygen species (ROS) as part of this network. In the current study, we show that ROS activate Nrf2, a master transcriptional regulator of genes involved in ROS homeostasis, apoptosis, glucose metabolism and angiogenesis. Besides ROS, KSHV utilizes additional aspects of host signaling to induce Nrf2 activity. We also observed that infection of endothelial cells deficient in Nrf2 resulted in downregulation of multiple genes important in KSHV pathogenesis, such as COX-2 and VEGF, and affected proper expression of two hallmark KSHV genes, lytic ORF50 and latent ORF73. Taken together, this study is the first to demonstrate the importance of Nrf2 during de novo KSHV infection of endothelial cells, and establishes Nrf2 as an attractive therapeutic target to control KSHV infection, establishment of latency and the associated cancers.
Like cancer cells, virally infected cells have dramatically altered metabolic requirements. We analyzed global metabolic changes induced by latent infection with an oncogenic virus, Kaposi's Sarcoma-associated herpesvirus (KSHV). KSHV is the etiologic agent of Kaposi's Sarcoma (KS), the most common tumor of AIDS patients. Approximately one-third of the nearly 200 measured metabolites were altered following latent infection of endothelial cells by KSHV, including many metabolites of anabolic pathways common to most cancer cells. KSHV induced pathways that are commonly altered in cancer cells including glycolysis, the pentose phosphate pathway, amino acid production and fatty acid synthesis. Interestingly, over half of the detectable long chain fatty acids detected in our screen were significantly increased by latent KSHV infection. KSHV infection leads to the elevation of metabolites involved in the synthesis of fatty acids, not degradation from phospholipids, and leads to increased lipid droplet organelle formation in the infected cells. Fatty acid synthesis is required for the survival of latently infected endothelial cells, as inhibition of key enzymes in this pathway led to apoptosis of infected cells. Addition of palmitic acid to latently infected cells treated with a fatty acid synthesis inhibitor protected the cells from death indicating that the products of this pathway are essential. Our metabolomic analysis of KSHV-infected cells provides insight as to how oncogenic viruses can induce metabolic alterations common to cancer cells. Furthermore, this analysis raises the possibility that metabolic pathways may provide novel therapeutic targets for the inhibition of latent KSHV infection and ultimately KS tumors.
In recent years there has been a resurgence in the study of metabolic changes in tumor cells. To determine if an oncogenic virus alters similar metabolic pathways as cancer cells, we measured the levels of a large number of metabolites in endothelial cells infected with Kaposi?s Sarcoma-associated herpesvirus (KSHV). KSHV is the etiologic agent of Kaposi's Sarcoma (KS), the most common tumor of AIDS patients world wide. Latent KSHV infection of endothelial cells altered a significant proportion of the host cell metabolites. Many metabolic pathways that are altered in most tumor cells were also altered by KSHV. In particular, KSHV upregulated fatty acid synthesis, a pathway that provides membrane material and metabolites critical for cell proliferation. Inhibitors of fatty acid synthesis kill many types of tumor cells and we found that these inhibitors led to death of cells latently infected with KSHV. In summary, we found that a directly oncogenic virus alters the same host metabolic pathways that are dysregulated in many cancer cells and that inhibition of these pathways can be used to kill off infected cells, thereby providing novel therapeutic targets for KSHV and ultimately KS tumors.
Activation-induced cytidine deaminase (AID) is specifically induced in germinal center B cells to carry out somatic hypermutation and class-switch recombination, two processes responsible for antibody diversification. Because of its mutagenic potential, AID expression and activity are tightly regulated to minimize unwanted DNA damage. Surprisingly, AID expression has been observed ectopically during pathogenic infections. However, the function of AID outside of the germinal centers remains largely uncharacterized. In this study, we demonstrate that infection of human primary naïve B cells with Kaposi's sarcoma-associated herpesvirus (KSHV) rapidly induces AID expression in a cell intrinsic manner. We find that infected cells are marked for elimination by Natural Killer cells through upregulation of NKG2D ligands via the DNA damage pathway, a pathway triggered by AID. Moreover, without having a measurable effect on KSHV latency, AID impinges directly on the viral fitness by inhibiting lytic reactivation and reducing infectivity of KSHV virions. Importantly, we uncover two KSHV-encoded microRNAs that directly regulate AID abundance, further reinforcing the role for AID in the antiviral response. Together our findings reveal additional functions for AID in innate immune defense against KSHV with implications for a broader involvement in innate immunity to other pathogens.
Immune responses to pathogens rely heavily on the ability of B cells to generate a unique set of antibodies that can bind and eliminate the pathogen. Activation-induced cytidine deaminase (AID) is the enzyme specifically upregulated in activated B cells to diversify the antibody repertoire by introducing mutations within the antibody coding genes. Curiously, AID expression has been observed outside of activated B cells upon infection with a number of viral and bacterial pathogens. However, in such cases AID function is poorly characterized and often deemed inappropriate since its mutagenic activity can put the cell at risk for oncogenic transformation. In this study, we investigate the expression of AID in response to infection with an oncogenic human pathogen Kaposi's sarcoma-associated herpesvirus (KSHV) and the antibody-independent immune defense it exerts. We show that AID marks infected cells for elimination by natural killer (NK) cells and directly impinges on viral fitness. Furthermore, we uncover novel viral immune evasion strategies employed by KSHV to counteract AID. Together, our findings demonstrate a protective role for AID in the response to infection with an oncogenic virus such as KSHV and suggest that AID may actually limit transformation rather than serve as its culprit.
Since Kaposi's sarcoma-associated herpesvirus (KSHV or human herpesvirus 8) was first identified in Kaposi's sarcoma (KS) lesions of HIV-infected individuals with AIDS, the basic biological understanding of KSHV has progressed remarkably. However, the absence of a proper animal model for KSHV continues to impede direct in vivo studies of viral replication, persistence, and pathogenesis. In response to this need for an animal model of KSHV infection, we have explored whether common marmosets can be experimentally infected with human KSHV. Here, we report the successful zoonotic transmission of KSHV into common marmosets (Callithrix jacchus, Cj), a New World primate. Marmosets infected with recombinant KSHV rapidly seroconverted and maintained a vigorous anti-KSHV antibody response. KSHV DNA and latent nuclear antigen (LANA) were readily detected in the peripheral blood mononuclear cells (PBMCs) and various tissues of infected marmosets. Remarkably, one orally infected marmoset developed a KS-like skin lesion with the characteristic infiltration of leukocytes by spindle cells positive for KSHV DNA and proteins. These results demonstrate that human KSHV infects common marmosets, establishes an efficient persistent infection, and occasionally leads to a KS-like skin lesion. This is the first animal model to significantly elaborate the important aspects of KSHV infection in humans and will aid in the future design of vaccines against KSHV and anti-viral therapies targeting KSHV coinfected tumor cells.
Kaposi's sarcoma-associated herpesvirus (KSHV or human herpesvirus 8), the most recently identified human tumor-inducing virus, has been linked to Kaposi's sarcoma, pleural effusion lymphomas and multicentric Castleman's disease. In fact, KSHV accounts for a large proportion of the cancer deaths in Africa. Further, the incidence of KSHV in the US and Europe has greatly increased due to the AIDS pandemic. Despite these pressing human health problems, studies of KSHV infection are greatly hampered by the lack of cell culture and animal models. To address this serious need, we set out to develop an animal model for KSHV infection. In this manuscript, we report the successful zoonotic transmission of KSHV into common marmosets (Callithrix jacchus, Cj), a New World primate. Our study demonstrates that experimental KSHV infection of the common marmoset is highly analogous to its infection of humans, including the means of infection, sustained serological responses, latent infection of PBMCs, virus persistence, and KS-like skin lesion development, although the latter was infrequent in experimental KSHV infections. This model thus provides a unique opportunity to dissect the molecular mechanisms of KSHV infection, persistence, and pathogenesis directly in primates.
Nucleophosmin (NPM) is a multifunctional nuclear phosphoprotein and a histone chaperone implicated in chromatin organization and transcription control. Oncogenic Kaposi's sarcoma herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD). In the infected host cell KSHV displays two modes of infection, the latency and productive viral replication phases, involving extensive viral DNA replication and gene expression. A sustained balance between latency and reactivation to the productive infection state is essential for viral persistence and KSHV pathogenesis. Our study demonstrates that the KSHV v-cyclin and cellular CDK6 kinase phosphorylate NPM on threonine 199 (Thr199) in de novo and naturally KSHV-infected cells and that NPM is phosphorylated to the same site in primary KS tumors. Furthermore, v-cyclin-mediated phosphorylation of NPM engages the interaction between NPM and the latency-associated nuclear antigen LANA, a KSHV-encoded repressor of viral lytic replication. Strikingly, depletion of NPM in PEL cells leads to viral reactivation, and production of new infectious virus particles. Moreover, the phosphorylation of NPM negatively correlates with the level of spontaneous viral reactivation in PEL cells. This work demonstrates that NPM is a critical regulator of KSHV latency via functional interactions with v-cyclin and LANA.
Latency is the predominant mode of viral persistence in KS and PEL tumors, and has a fundamental impact on KSHV tumorigenesis. Establishment and maintenance of latency involves a number of viral and cellular factors. This study provides a novel functional link between LANA and v-cyclin by showing that phosphorylation of nucleophosmin (NPM) by the v-cyclin-CDK6 kinase complex supports its interaction with LANA, and thus enables the transcriptional silencing of KSHV lytic genes needed for latency. These findings indicate that KSHV has evolved mechanisms to utilize host proteins for maintaining the latency, and underscores the role of NPM as a regulator of not only mammalian transcription but also of viral transcription. Taken together, our data suggests that a cellular protein, NPM, is a critical factor for the latency of this oncogenic human virus, and may thus represent an attractive novel target for intervention.
The DNA damage response (DDR) that evolved to repair host cell DNA damage also recognizes viral DNA entering the nucleus during infections. Here, we investigated the modulation of DDR signaling during de novo infection of primary endothelial cells by Kaposi's sarcoma-associated herpesvirus (KSHV). Phosphorylation of representative DDR-associated proteins, such as ataxia telangiectasia mutated (ATM) and H2AX, was induced as early as 30 min (0.5 h) postinfection and persisted during in vitro KSHV latency. Phosphorylated H2AX (γH2AX) colocalized at 30 min (0.5 h) with the KSHV genome entering the nuclei. Total H2AX protein levels also increased, and the increase was attributed to a decrease in degradative H2AX Lys48-linked polyubiquitination with a concomitant increase in Lys63-linked polyubiquitination that was shown to increase protein stability. ATM and H2AX phosphorylation and γH2AX nuclear foci were also induced by UV-inactivated KSHV, which ceased at later times of infection. Inhibition of ATM kinase activity by KU-55933 and H2AX knockdown by small interfering RNA significantly reduced the expression of the KSHV latency-associated nuclear antigen 1 (LANA-1; ORF73) and LANA-1 nuclear puncta. Knockdown of H2AX also resulted in a >80% reduction in the nuclear KSHV DNA copy numbers. Similar results were also observed in ATM-negative cells, although comparable levels of viral DNA entered ATM-negative and ATM-positive cell nuclei. In contrast, knockdown of CHK1 and CHK2 did not affect ORF73 expression. Collectively, these results demonstrate that KSHV induces ATM and H2AX, a selective arm of the DDR, for the establishment and maintenance of its latency during de novo infection of primary endothelial cells.
IMPORTANCE Eukaryotic cells mount a DNA damage response (DDR) to sense and repair different types of cellular DNA damage. In addition, DDR also recognizes exogenous genetic material, such as the viral DNA genome entering the nucleus during infections. The present study was undertaken to determine whether de novo Kaposi's sarcoma-associated herpesvirus (KSHV) infection modulates DDR. Our results demonstrate that early during de novo infection of primary endothelial cells, KSHV induces a selective arm of DDR signaling, such as the ATM kinase and its downstream target, H2AX, which are essential for KSHV's latent gene expression and the establishment of latency. These studies suggest that targeting ATM and H2AX could serve as an attractive strategy to block the establishment of KSHV latent infection and the associated malignancies.
Kaposi sarcoma is a tumor consisting of Kaposi sarcoma herpesvirus (KSHV)–infected tumor cells that express endothelial cell (EC) markers and viral genes like v-cyclin, vFLIP, and LANA. Despite a strong link between KSHV infection and certain neoplasms, de novo virus infection of human primary cells does not readily lead to cellular transformation. We have studied the consequences of expression of v-cyclin in primary and immortalized human dermal microvascular ECs. We show that v-cyclin, which is a homolog of cellular D-type cyclins, induces replicative stress in ECs, which leads to senescence and activation of the DNA damage response. We find that antiproliferative checkpoints are activated upon KSHV infection of ECs, and in early-stage but not late-stage lesions of clinical Kaposi sarcoma specimens. These are some of the first results suggesting that DNA damage checkpoint response also functions as an anticancer barrier in virally induced cancers.
Recent findings have indicated that DNA hyper-replication triggered by oncogenes can induce cellular senescence, which together with the oncogene-induced DNA damage checkpoint confers a barrier to tumorigenesis. Kaposi sarcoma herpesvirus (KSHV) can infect human dermal microvascular endothelial cells (ECs) in vitro, but KSHV infection does not seem to provide growth advantage to the cells, but rather leads to retarded growth. Moreover, the proliferative index has long been known to be low in KSHV-infected spindle cells in Kaposi sarcoma (KS) tumors. Our results provide an explanation for these observations by showing that activation of the DNA damage response, exerted by KSHV and a latent viral protein v-cyclin, functions as a barrier against transformation of KSHV-infected cells. Interestingly, the antiproliferative checkpoints are activated during the initial stages of KSHV infection and KS tumorigenesis. During the course of infection, the infected cells are imposed to overcome the checkpoint, and oncogenic stress elicited by the expression of v-cyclin may further contribute to the induction of genomic instability and malignant transformation.
The Kaposi's sarcoma-associated herpesvirus (KSHV) genome encodes a G protein-coupled receptor (vGPCR). vGPCR is a ligand-independent, constitutively active signaling molecule that promotes cell growth and proliferation; however, it is not clear how vGPCR is negatively regulated. We report here that the KSHV K7 small membrane protein interacts with vGPCR and induces its degradation, thereby dampening vGPCR signaling. K7 interaction with vGPCR is readily detected in transiently transfected human cells. Mutational analyses reveal that the K7 transmembrane domain is necessary and sufficient for this interaction. Biochemical and confocal microscopy studies indicate that K7 retains vGPCR in the endoplasmic reticulum (ER) and induces vGPCR proteasomeal degradation. Indeed, the knockdown of K7 by shRNA-mediated silencing increases vGPCR protein expression in BCBL-1 cells that are induced for KSHV lytic replication. Interestingly, K7 expression significantly reduces vGPCR tumorigenicity in nude mice. These findings define a viral factor that negatively regulates vGPCR protein expression and reveal a post-translational event that modulates GPCR-dependent transformation and tumorigenicity.
Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma. KSHV is also found in primary effusion lymphoma and multicentric Castleman's disease, rare lymphoproliferative diorders associated with immuno-suppression. The KSHV genome encodes a G protein-coupled receptor (vGPCR) that is believed to contribute to the KSHV-associated malignancies. vGPCR is a ligand-independent, constitutively active signaling molecule. It is not clear how vGPCR is negatively regulated. Here, we report that the KSHV small membrane K7 protein interacts with vGPCR through its putative transmembrane domain. Interaction with K7 retains vGPCR in the ER and facilitates its degradation by the proteasome, thereby reducing vGPCR protein expression. Consequently, K7 significantly reduces vGPCR-mediated transformation in vitro and tumor formation in nude mice. Our findings reveal that K7 functions as a viral factor to dampen vGPCR protein expression and negatively modulate the tumor-inducing capacity of vGPCR, implying that KSHV has evolved mechanisms to avoid deleterious effects and to permit persistent infection within its host.
Small Ubiquitin-related MOdifier (SUMO) modification was initially identified as a reversible post-translational modification that affects the regulation of diverse cellular processes, including signal transduction, protein trafficking, chromosome segregation, and DNA repair. Increasing evidence suggests that the SUMO system also plays an important role in regulating chromatin organization and transcription. It is thus not surprising that double-stranded DNA viruses, such as Kaposi’s sarcoma-associated herpesvirus (KSHV), have exploited SUMO modification as a means of modulating viral chromatin remodeling during the latent-lytic switch. In addition, SUMO regulation allows the disassembly and assembly of promyelocytic leukemia protein-nuclear bodies (PML-NBs), an intrinsic antiviral host defense, during the viral replication cycle. Overcoming PML-NB-mediated cellular intrinsic immunity is essential to allow the initial transcription and replication of the herpesvirus genome after de novo infection. As a consequence, KSHV has evolved a way as to produce multiple SUMO regulatory viral proteins to modulate the cellular SUMO environment in a dynamic way during its life cycle. Remarkably, KSHV encodes one gene product (K-bZIP) with SUMO-ligase activities and one gene product (K-Rta) that exhibits SUMO-targeting ubiquitin ligase (STUbL) activity. In addition, at least two viral products are sumoylated that have functional importance. Furthermore, sumoylation can be modulated by other viral gene products, such as the viral protein kinase Orf36. Interference with the sumoylation of specific viral targets represents a potential therapeutic strategy when treating KSHV, as well as other oncogenic herpesviruses. Here, we summarize the different ways KSHV exploits and manipulates the cellular SUMO system and explore the multi-faceted functions of SUMO during KSHV’s life cycle and pathogenesis.
KSHV; SUMO; epigenetic; PML-NB; interferon
Tetherin (CD317/BST2) is an interferon-induced membrane protein that inhibits the release of diverse enveloped viral particles. Several mammalian viruses have evolved countermeasures that inactivate tetherin, with the prototype being the HIV-1 Vpu protein. Here we show that the human herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) is sensitive to tetherin restriction and its activity is counteracted by the KSHV encoded RING-CH E3 ubiquitin ligase K5. Tetherin expression in KSHV-infected cells inhibits viral particle release, as does depletion of K5 protein using RNA interference. K5 induces a species-specific downregulation of human tetherin from the cell surface followed by its endosomal degradation. We show that K5 targets a single lysine (K18) in the cytoplasmic tail of tetherin for ubiquitination, leading to relocalization of tetherin to CD63-positive endosomal compartments. Tetherin degradation is dependent on ESCRT-mediated endosomal sorting, but does not require a tyrosine-based sorting signal in the tetherin cytoplasmic tail. Importantly, we also show that the ability of K5 to substitute for Vpu in HIV-1 release is entirely dependent on K18 and the RING-CH domain of K5. By contrast, while Vpu induces ubiquitination of tetherin cytoplasmic tail lysine residues, mutation of these positions has no effect on its antagonism of tetherin function, and residual tetherin is associated with the trans-Golgi network (TGN) in Vpu-expressing cells. Taken together our results demonstrate that K5 is a mechanistically distinct viral countermeasure to tetherin-mediated restriction, and that herpesvirus particle release is sensitive to this mode of antiviral inhibition.
To replicate efficiently in their hosts, viruses must avoid antiviral cellular defenses that comprise part of the innate immune system. Tetherin, an antiviral membrane protein that inhibits the release of several enveloped viruses from infected cells, is antagonized by the HIV-1 Vpu protein. The K5 protein of the human pathogen Kaposi's sarcoma-associated herpesvirus (KSHV) modulates the cell surface levels of several host proteins including tetherin. We show that KSHV release is sensitive to tetherin, and that K5 expression is required for efficient virus production in tetherin-expressing cells. K5 is also capable of rescuing Vpu-defective HIV-1 virus release from tetherin. K5 expression induces a down-regulation of cell-surface tetherin levels and degradation in late endosomes, which depends on a single lysine residue in the tetherin cytoplasmic tail. Finally, we show that the ESCRT pathway, which promotes the trafficking of cell surface receptors for degradation, is required for K5-mediated tetherin removal from the plasma membrane. Thus, we demonstrate that herpesviruses are sensitive to the antiviral effects of tetherin and that KSHV has evolved a mechanism for its destruction. These findings extend the list of viruses sensitive to tetherin, suggesting that tetherin counter-measures are widespread defense mechanisms amongst enveloped viruses.
Viral invasion of a host cell triggers immune responses with both innate and adaptive components. The innate immune response involving the induction of type I interferons (alpha and beta interferons [IFN-α and -β]) constitutes the first line of antiviral defenses. The type I IFNs signal the transcription of a group of antiviral effector proteins, the IFN-stimulated genes (ISGs), which target distinct viral components and distinct stages of the viral life cycle, aiming to eliminate invading viruses. In the case of Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent of Kaposi's sarcoma (KS), a sudden upsurge of type I IFN-mediated innate antiviral signals is seen immediately following both primary de novo infection and viral lytic reactivation from latency. Potent subversion of these responses thus becomes mandatory for the successful establishment of a primary infection following viral entry as well as for efficient viral assembly and egress. This review gives a concise overview of the induction of the type I IFN signaling pathways in response to viral infection and provides a comprehensive understanding of the antagonizing effects exerted by KSHV on type I IFN pathways wielded at various stages of the viral life cycle. Information garnered from this review should result in a better understanding of KSHV biology essential for the development of immunotherapeutic strategies targeted toward KSHV-associated malignancies.
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.
KSHV is a ubiquitous herpesvirus that establishes a life-long persistent infection in humans and is associated with Kaposi's sarcoma and several lymphoid malignancies. During latency, the KSHV genome persists as a multicopy circular DNA assembled into nucleosomal structures. While viral latency is characterized by restricted viral gene expression, reactivation induces the lytic replication program and the expression of viral genes in defined sequential and temporal order. Posttranslational modifications of the viral chromatin structure have been implicated to regulate viral gene expressions but the underlying gene regulatory mechanisms are still elusive. Here, we demonstrate that the latent and lytic chromatins of KSHV are associated with a distinctive pattern of activating and repressive histone modifications whose distribution changes upon reactivation in an organized manner in correlation with the temporally ordered expression of viral lytic genes. Furthermore, we demonstrate that the evolutionarily conserved Polycomb group proteins, that maintain the repression of genes involved in hematopoiesis, X-chromosome inactivation, cell proliferation and stem cell differentiation, also play a critical role in the regulation of KSHV latency by maintaining a repressive chromatin structure. Thus, the epigenetic program of KSHV is at the crux of restricting latent gene expression and the orderly expression of lytic genes.
Kaposi's sarcoma-associated herpesvirus (KSHV) is causally linked to several human cancers, including Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman's disease, malignancies commonly found in HIV-infected patients. While KSHV encodes diverse functional products, its mechanism of oncogenesis remains unknown. In this study, we determined the roles KSHV microRNAs (miRs) in cellular transformation and tumorigenesis using a recently developed KSHV-induced cellular transformation system of primary rat mesenchymal precursor cells. A mutant with a cluster of 10 precursor miRs (pre-miRs) deleted failed to transform primary cells, and instead, caused cell cycle arrest and apoptosis. Remarkably, the oncogenicity of the mutant virus was fully restored by genetic complementation with the miR cluster or several individual pre-miRs, which rescued cell cycle progression and inhibited apoptosis in part by redundantly targeting IκBα and the NF-κB pathway. Genomic analysis identified common targets of KSHV miRs in diverse pathways with several cancer-related pathways preferentially targeted. These works define for the first time an essential viral determinant for KSHV-induced oncogenesis and identify NF-κB as a critical pathway targeted by the viral miRs. Our results illustrate a common theme of shared functions with hierarchical order among the KSHV miRs.
Kaposi's sarcoma-associated herpesvirus (KSHV) is the causal agent of several human cancers. KSHV encodes over two dozen genes that regulate diverse cellular pathways. However, the molecular mechanism of KSHV-induced oncogenesis remains unknown. In this study, we determined the roles of KSHV microRNAs (miRs) in KSHV-induced oncogenesis using a recently developed KSHV cellular transformation system of primary rat mesenchymal precursor cells. A KSHV mutant with a cluster of 10 precursor miRs (pre-miRs) deleted failed to transform primary cells, and instead, caused cell cycle arrest and apoptosis. Expression of the miR cluster or several pre-miRs was sufficient to restore the oncogenicity of the mutant virus. KSHV miRs regulated cell cycle progression and inhibited apoptosis in part by redundantly targeting IκBα and the NF-κB pathway. By integrating gene expression profiling and target prediction, we identified common targets of KSHV miRs in diverse pathways. Importantly, several cancer-related pathways were preferentially targeted by KSHV miRs. These works have demonstrated for the first time the important roles of KSHV miRs in oncogenesis and identified NF-κB as a critical pathway targeted by the miRs. Our results reveal that shared function is a common theme of KSHV miRs, which manifest functional hierarchical order.
Kaposi's Sarcoma (KS), caused by Kaposi's Sarcoma Herpesvirus (KSHV), is a highly vascularised angiogenic tumor of endothelial cells, characterized by latently KSHV-infected spindle cells and a pronounced inflammatory infiltrate. Several KSHV proteins, including LANA-1 (ORF73), vCyclin (ORF72), vGPCR (ORF74), vIL6 (ORF-K2), vCCL-1 (ORF-K6), vCCL-2 (ORF-K4) and K1 have been shown to exert effects that can lead to the proliferation and atypical differentiation of endothelial cells and/or the secretion of cytokines with angiogenic and inflammatory properties (VEGF, bFGF, IL6, IL8, GROα, and TNFβ). To investigate a role of the KSHV K15 protein in KSHV-mediated angiogenesis, we carried out a genome wide gene expression analysis on primary endothelial cells infected with KSHV wildtype (KSHVwt) and a KSHV K15 deletion mutant (KSHVΔK15). We found RCAN1/DSCR1 (Regulator of Calcineurin 1/Down Syndrome critical region 1), a cellular gene involved in angiogenesis, to be differentially expressed in KSHVwt- vs KSHVΔK15-infected cells. During physiological angiogenesis, expression of RCAN1 in endothelial cells is regulated by VEGF (vascular endothelial growth factor) through a pathway involving the activation of PLCγ1, Calcineurin and NFAT1. We found that K15 directly recruits PLCγ1, and thereby activates Calcineurin/NFAT1-dependent RCAN1 expression which results in the formation of angiogenic tubes. Primary endothelial cells infected with KSHVwt form angiogenic tubes upon activation of the lytic replication cycle. This effect is abrogated when K15 is deleted (KSHVΔK15) or silenced by an siRNA targeting the K15 expression. Our study establishes K15 as one of the KSHV proteins that contribute to KSHV-induced angiogenesis.
Kaposi's Sarcoma Herpesvirus (KSHV) causes a multifocal angio-proliferative neoplasm, Kaposi's Sarcoma (KS), whose development involves angiogenic growth factors and cytokines. The K15 protein of KSHV upregulates the host factor RCAN1/DSCR1. RCAN1/DSCR1 has been implicated in angiogenesis but its role in KS has never been investigated. In this study we show that the increased expression of RCAN1/DSCR1 in KSHV-infected endothelial cells depends on K15 and that K15, by recruiting PLCγ1, activates PLCγ1, Calcineurin and NFAT1 to induce RCAN1/DSCR1 expression and capillary tube formation. Deleting the K15 gene from the viral genome, or silencing its expression with siRNA, reduces the ability of KSHV to induce angiogenesis in infected endothelial cells in tissue culture. These findings suggest that the K15 protein contributes to the angiogenic properties of this virus.
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.
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 infection.
The control of RNA stability is a key determinant in cellular gene expression. The stability of any transcript is modulated through the activity of cis- or trans-acting regulatory factors as well as cellular quality control systems that ensure the integrity of a transcript. As a result, invading viral pathogens must be able to subvert cellular RNA decay pathways capable of destroying viral transcripts. Here we report that the Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 protein binds to a unique KSHV polyadenylated nuclear RNA, called PAN RNA, and protects it from degradation by cellular factors. ORF57 increases PAN RNA levels and its effects are greatest on unstable alleles of PAN RNA. Kinetic analysis of transcription pulse assays shows that ORF57 protects PAN RNA from a rapid cellular RNA decay process, but ORF57 has little effect on transcription or PAN RNA localization based on chromatin immunoprecipitation and in situ hybridization experiments, respectively. Using a UV cross-linking technique, we further demonstrate that ORF57 binds PAN RNA directly in living cells and we show that binding correlates with function. In addition, we define an ORF57-responsive element (ORE) that is necessary for ORF57 binding to PAN RNA and sufficient to confer ORF57-response to a heterologous intronless β-globin mRNA, but not its spliced counterparts. We conclude that ORF57 binds to viral transcripts in the nucleus and protects them from a cellular RNA decay pathway. We propose that KSHV ORF57 protein functions to enhance the nuclear stability of intronless viral transcripts by protecting them from a cellular RNA quality control pathway.
In order to replicate efficiently, a virus must ensure that its genes are properly expressed in the context of an infected host cell. Recent work has demonstrated that eukaryotic cells have RNA quality control pathways that degrade improperly processed, aberrant RNAs. Our published findings using an unusual Kaposi's sarcoma-associated herpesvirus (KSHV) nuclear RNA, called PAN RNA, have suggested that intronless polyadenylated transcripts are subject to such a quality control system. Because most KSHV genes lack introns, we hypothesized that KSHV must have evolved mechanisms that bypass this quality control system. In support of this idea, we show that the ORF57 protein, a multifunctional enhancer of KSHV gene expression, binds to and stabilizes PAN RNA. We further define an element called the ORF57-responsive element (ORE) in PAN RNA that is necessary for ORF57-binding and activity on PAN RNA. In addition, we show that the ORE is sufficient to confer ORF57-responsiveness to a heterologous intronless mRNA, but not its spliced counterpart. These observations substantiate the model that ORF57 enhances KSHV gene expression by protecting viral transcripts from host RNA surveillance pathways. More broadly, these data suggest that viruses producing intronless nuclear RNAs require mechanisms to evade host quality control mechanisms.
Inhibition of host cell gene expression by the human herpesvirus KSHV occurs via a novel mechanism involving polyadenylation-linked RNA turnover.
Regulation of messenger RNA (mRNA) stability plays critical roles in controlling gene expression, ensuring transcript fidelity, and allowing cells to respond to environmental cues. Unregulated enhancement of mRNA turnover could therefore dampen cellular responses to such signals. Indeed, several herpesviruses instigate widespread destruction of cellular mRNAs to block host gene expression and evade immune detection. Kaposi's sarcoma-associated herpesvirus (KSHV) promotes this phenotype via the activity of its viral SOX protein, although the mechanism of SOX-induced mRNA turnover has remained unknown, given its apparent lack of intrinsic ribonuclease activity. Here, we report that KSHV SOX stimulates cellular transcriptome turnover via a unique mechanism involving aberrant polyadenylation. Transcripts in SOX-expressing cells exhibit extended poly(A) polymerase II-generated poly(A) tails and polyadenylation-linked mRNA turnover. SOX-induced polyadenylation changes correlate with its RNA turnover function, and inhibition of poly(A) tail formation blocks SOX activity. Both nuclear and cytoplasmic poly(A) binding proteins are critical cellular cofactors for SOX function, the latter of which undergoes striking nuclear relocalization by SOX. SOX-induced mRNA turnover therefore represents both a novel mechanism of host shutoff as well as a new model system to probe the regulation of poly(A) tail-stimulated mRNA turnover in mammalian cells.
During viral infection, many essential cellular functions are targets for viral manipulation, yet aside from RNA interference, surprisingly few examples of viruses disrupting RNA turnover have been documented. Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus that induces widespread cellular messenger RNA destabilization during lytic infection. The viral protein SOX is a critical effector of this phenotype, yet it lacks ribonuclease activity, so presumably it targets cellular factors governing RNA stability. Here, we show that SOX stimulates host mRNA destruction via a unique mechanism involving polyadenylation. During SOX expression, newly formed messages have longer than normal poly(A) tails, leading to their retention in the nucleus. Coincident with this hyperadenylation, poly(A) binding protein (PABPC) is relocalized from the cytoplasm to the nucleus. PABPC has prominent roles in translation, messenger RNA stabilization, and quality control in the cytoplasm; we find its nuclear relocalization by SOX correlates with enhanced mRNA turnover in the cytoplasm. Thus, KSHV appears to have evolved distinct polyadenylation-linked mechanisms to target both new messages in the nucleus and preexisting cytoplasmic messages for destruction, thereby effectively inhibiting cellular gene expression.
Kaposi's sarcoma-associated herpesvirus (KSHV) is a human herpesvirus that causes Kaposi's sarcoma and is associated with the development of lymphoproliferative diseases. KSHV reactivation from latency and virion production is dependent on efficient transcription of over eighty lytic cycle genes and viral DNA replication. CTCF and cohesin, cellular proteins that cooperatively regulate gene expression and mediate long-range DNA interactions, have been shown to bind at specific sites in herpesvirus genomes. CTCF and cohesin regulate KSHV gene expression during latency and may also control lytic reactivation, although their role in lytic gene expression remains incompletely characterized. Here, we analyze the dynamic changes in CTCF and cohesin binding that occur during the process of KSHV viral reactivation and virion production by high resolution chromatin immunoprecipitation and deep sequencing (ChIP-Seq) and show that both proteins dissociate from viral genomes in kinetically and spatially distinct patterns. By utilizing siRNAs to specifically deplete CTCF and Rad21, a cohesin component, we demonstrate that both proteins are potent restriction factors for KSHV replication, with cohesin knockdown leading to hundred-fold increases in viral yield. High-throughput RNA sequencing was used to characterize the transcriptional effects of CTCF and cohesin depletion, and demonstrated that both proteins have complex and global effects on KSHV lytic transcription. Specifically, both proteins act as positive factors for viral transcription initially but subsequently inhibit KSHV lytic transcription, such that their net effect is to limit KSHV RNA accumulation. Cohesin is a more potent inhibitor of KSHV transcription than CTCF but both proteins are also required for efficient transcription of a subset of KSHV genes. These data reveal novel effects of CTCF and cohesin on transcription from a relatively small genome that resemble their effects on the cellular genome by acting as gene-specific activators of some promoters, but differ in acting as global negative regulators of transcription.
Kaposi's sarcoma-associated herpesvirus (KSHV) is a human virus that causes Kaposi's sarcoma and lymphoma. KSHV establishes a lifelong infection in B lymphocytes, and persists in a latent form as circular DNA molecules. Reactivation and replication yield infectious virions, allowing transmission and maintenance of latent infection. The cellular mechanisms controlling reactivation remain incompletely characterized. Host proteins that regulate RNA transcription play an important role in controlling viral reactivation. In this study, we used high-throughput techniques to analyze the binding of two cellular proteins, CTCF and Rad21, to the KSHV genome as the virus reactivated to produce infectious virions. We found that these proteins dissociate from the latent genome when reactivation occurs. We also found that depleting cells of these proteins increases virus production as much as a hundredfold. Depleting the cell of CTCF or Rad21 caused complex changes in the synthesis of RNAs by KSHV, with the amounts of most KSHV RNAs increasing greatly. We also showed that Rad21 and CTCF are needed for the virus to synthesize RNAs efficiently. Our study provides new insights into how the cell uses CTCF and Rad21 to limit KSHV's ability to synthesize RNA and reactivate from latency to produce infectious virus.