Kaposi's Sarcoma-associated Herpesvirus Genome Research Articles

KSHV Genome Replication and Maintenance.

Kaposi's sarcoma associated herpesvirus (KSHV) or human herpesvirus 8 (HHV8) is a major etiological agent for multiple severe malignancies in immune-compromised patients. KSHV establishes lifetime persistence in the infected individuals and displays two distinct life cycles, generally a prolonged passive latent, and a short productive or lytic cycle. During latent phase, the viral episome is tethered to the host chromosome and replicates once during every cell division. Latency-associated nuclear antigen (LANA) is a predominant multifunctional nuclear protein expressed during latency, which plays a central role in episome tethering, replication and perpetual segregation of the episomes during cell division. LANA binds cooperatively to LANA binding sites (LBS) within the terminal repeat (TR) region of the viral episome as well as to the cellular nucleosomal proteins to tether viral episome to the host chromosome. LANA has been shown to modulate multiple cellular signaling pathways and recruits various cellular proteins such as chromatin modifying enzymes, replication factors, transcription factors, and cellular mitotic framework to maintain a successful latent infection. Although, many other regions within the KSHV genome can initiate replication, KSHV TR is important for latent DNA replication and possible segregation of the replicated episomes. Binding of LANA to LBS favors the recruitment of various replication factors to initiate LANA dependent DNA replication. In this review, we discuss the molecular mechanisms relevant to KSHV genome replication, segregation, and maintenance of latency.

Triptolide decreases expression of latency-associated nuclear antigen 1 and reduces viral titers in Kaposi's sarcoma-associated and herpesvirus-related primary effusion lymphoma cells.

Kaposi's sarcoma-associated herpesvirus (KSHV) can establish a life-long persistence in the host after primary infection and is associated with certain malignancies, which are resistant to conventional chemotherapeutic agents with a poor prognosis. Latency-associated nuclear antigen 1 (LANA1) encoded by KSHV is essential for segregation, replication and maintenance of viral genome. In addition, LANA1 upregulates the transcriptional activity of signal transducer and activator of transcription 3 (STAT3), which plays an important role in promoting survival of KSHV-associated primary effusion lymphoma (PEL) cells. Furthermore, LANA1 mediates transcriptional modulation of KSHV and host genome in host cells. In the present study, the antitumor effect of triptolide was assessed. CCK-8 assays were performed to demonstrate that the proliferations of PEL cells were efficiently inhibited by triptolide in a dose- and time-dependent manner. Flow cytometric results indicated that triptolide induced cell cycle arrest and apoptosis. Western blot results suggested that triptolide downregulated LANA1 expression and reduced half-life of LANA1 in the KSHV-infected malignant cells. Viral titer experiments indicated that triptolide treatment impaired the number of viral DNA copies and the production of virions in BCBL-1 cells. Triptolide also suppressed STAT3 activity and inhibited secretion of IL-6 in PEL cells. In a mouse xenograft model of primary effusion lymphoma by BCBL-1 cells, triptolide treatment significantly inhibited ascites formation and diffused organ infiltration. These results indicate that triptolide impairs the expression of LANA1 and shows antitumor activity against PEL in vitro and in vivo. Triptolide may be a potential agent for treatment of PEL.

A KSHV microRNA Directly Targets G Protein-Coupled Receptor Kinase 2 to Promote the Migration and Invasion of Endothelial Cells by Inducing CXCR2 and Activating AKT Signaling.

Kaposi's sarcoma (KS) is a highly disseminated angiogenic tumor of endothelial cells linked to infection by Kaposi's sarcoma-associated herpesvirus (KSHV). KSHV encodes more than two dozens of miRNAs but their roles in KSHV-induced tumor dissemination and metastasis remain unknown. Here, we found that ectopic expression of miR-K12-3 (miR-K3) promoted endothelial cell migration and invasion. Bioinformatics and luciferase reporter analyses showed that miR-K3 directly targeted G protein-coupled receptor (GPCR) kinase 2 (GRK2, official gene symbol ADRBK1). Importantly, overexpression of GRK2 reversed miR-K3 induction of cell migration and invasion. Furthermore, the chemokine receptor CXCR2, which was negatively regulated by GRK2, was upregulated in miR-K3-transduced endothelial cells. Knock down of CXCR2 abolished miR-K3-induced cell migration and invasion. Moreover, miR-K3 downregulation of GRK2 relieved its direct inhibitory effect on AKT. Both CXCR2 induction and the release of AKT from GRK2 were required for miR-K3 maximum activation of AKT and induction of cell migration and invasion. Finally, deletion of miR-K3 from the KSHV genome abrogated its effect on the GRK2/CXCR2/AKT pathway and KSHV-induced migration and invasion. Our data provide the first-line evidence that, by repressing GRK2, miR-K3 facilitates cell migration and invasion via activation of CXCR2/AKT signaling, which likely contribute to the dissemination of KSHV-induced tumors.

Bub1 in Complex with LANA Recruits PCNA To Regulate Kaposi's Sarcoma-Associated Herpesvirus Latent Replication and DNA Translesion Synthesis.

Latent DNA replication of Kaposi's sarcoma-associated herpesvirus (KSHV) initiates at the terminal repeat (TR) element and requires trans-acting elements, both viral and cellular, such as ORCs, MCMs, and latency-associated nuclear antigen (LANA). However, how cellular proteins are recruited to the viral genome is not very clear. Here, we demonstrated that the host cellular protein, Bub1, is involved in KSHV latent DNA replication. We show that Bub1 constitutively interacts with proliferating cell nuclear antigen (PCNA) via a highly conserved PIP box motif within the kinase domain. Furthermore, we demonstrated that Bub1 can form a complex with LANA and PCNA in KSHV-positive cells. This strongly indicated that Bub1 serves as a scaffold or molecular bridge between LANA and PCNA. LANA recruited PCNA to the KSHV genome via Bub1 to initiate viral replication in S phase and interacted with PCNA to promote its monoubiquitination in response to UV-induced damage for translesion DNA synthesis. This resulted in increased survival of KSHV-infected cells. During latency in KSHV-infected cells, the viral episomal DNA replicates once each cell cycle. KSHV does not express DNA replication proteins during latency. Instead, KSHV LANA recruits the host cell DNA replication machinery to the replication origin. However, the mechanism by which LANA mediates replication is uncertain. Here, we show that LANA is able to form a complex with PCNA, a critical protein for viral DNA replication. Furthermore, our findings suggest that Bub1, a spindle checkpoint protein, serves as a scaffold or molecular bridge between LANA and PCNA. Our data further support a role for Bub1 and LANA in PCNA-mediated cellular DNA replication processes as well as monoubiquitination of PCNA in response to UV damage. These data reveal a therapeutic target for inhibition of KSHV persistence in malignant cells.

New insights into the expression and functions of the Kaposi's sarcoma-associated herpesvirus long noncoding PAN RNA

The Kaposi's sarcoma-associated herpesvirus (KSHV) is a clinically relevant pathogen associated with several human diseases that primarily affect immunocompromised individuals. KSHV encodes a noncoding polyadenylated nuclear (PAN) RNA that is essential for viral propagation and viral gene expression. PAN RNA is the most abundant viral transcript produced during lytic replication. The accumulation of PAN RNA depends on high levels of transcription driven by the Rta protein, a KSHV transcription factor necessary and sufficient for latent-to-lytic phase transition. In addition, KSHV uses several posttranscriptional mechanisms to stabilize PAN RNA. A cis-acting element, called the ENE, prevents PAN RNA decay by forming a triple helix with its poly(A) tail. The viral ORF57 and the cellular PABPC1 proteins further contribute to PAN RNA stability during lytic phase. PAN RNA functions are only beginning to be uncovered, but PAN RNA has been proposed to control gene expression by several different mechanisms. PAN RNA associates with the KSHV genome and may regulate gene expression by recruiting chromatin-modifying factors. Moreover, PAN RNA binds the viral latency-associated nuclear antigen (LANA) protein and decreases its repressive activity by sequestering it from the viral genome. Surprisingly, PAN RNA was found to associate with translating ribosomes, so this noncoding RNA may be additionally used to produce viral peptides. In this review, I highlight the mechanisms of PAN RNA accumulation and describe recent insights into potential functions of PAN RNA.

Inefficient Codon Usage Impairs mRNA Accumulation: the Case of the v-FLIP Gene of Kaposi's Sarcoma-Associated Herpesvirus.

Latent Kaposi's sarcoma-associated herpesvirus (KSHV) genomes encode a homolog of cellular FLICE-inhibitory proteins (termed v-FLIP) that activates NF-κB and can trigger important proinflammatory and antiapoptotic changes in latently infected cells. The protein is present at very low levels in infection and has generally been difficult to efficiently express in recombinant vectors. Here we show that codon usage in the v-FLIP gene is strikingly suboptimal. Optimization of codon use in expression vectors, as expected, restores efficient protein expression. Surprisingly, however, it also dramatically increases the steady-state level of v-FLIP mRNA, at least in part by increasing mRNA stability. When codon-optimized v-FLIP sequences are reintroduced into intact KSHV genomes, the resulting virus expresses readily detectable monocistronic v-FLIP mRNAs that are undetectable in wild-type (WT) infection by blot hybridization, suggesting that such RNAs are in fact transcribed in WT infection but fail to accumulate. The overexpression of v-FLIP by codon-optimized latent genomes results in a 5- to 7-fold decrement in virus production following lytic induction, indicating that maximizing NF-κB signaling is deleterious to induction. These studies provide a clear explanation for the evolution of inefficient codon usage in this gene and point to a strong connection between translational efficiency and RNA accumulation in mammalian cells. This study reports that inefficient codon usage in a herpesviral gene is strikingly correlated with the inability of its mRNA to accumulate in cells; correction of efficient translatability restores RNA abundance. A similar correlation has been reported in yeast species, but the mechanisms operating in mammalian cells appear substantially different.

Identification of the Essential Role of Viral Bcl-2 for Kaposi's Sarcoma-Associated Herpesvirus Lytic Replication.

Kaposi's sarcoma-associated herpesvirus (KSHV) evades host defenses through tight suppression of autophagy by targeting each step of its signal transduction: by viral Bcl-2 (vBcl-2) in vesicle nucleation, by viral FLIP (vFLIP) in vesicle elongation, and by K7 in vesicle maturation. By exploring the roles of KSHV autophagy-modulating genes, we found, surprisingly, that vBcl-2 is essential for KSHV lytic replication, whereas vFLIP and K7 are dispensable. Knocking out vBcl-2 from the KSHV genome resulted in decreased lytic gene expression at the mRNA and protein levels, a lower viral DNA copy number, and, consequently, a dramatic reduction in the amount of progeny infectious viruses, as also described in the accompanying article (A. Gelgor, I. Kalt, S. Bergson, K. F. Brulois, J. U. Jung, and R. Sarid, J Virol 89:5298-5307, 2015). More importantly, the antiapoptotic and antiautophagic functions of vBcl-2 were not required for KSHV lytic replication. Using a comprehensive mutagenesis analysis, we identified that glutamic acid 14 (E14) of vBcl-2 is critical for KSHV lytic replication. Mutating E14 to alanine totally blocked KSHV lytic replication but showed little or no effect on the antiapoptotic and antiautophagic functions of vBcl-2. Our study indicates that vBcl-2 harbors at least three important and genetically separable functions to modulate both cellular signaling and the virus life cycle. The present study shows for the first time that vBcl-2 is essential for KSHV lytic replication. Removal of the vBcl-2 gene results in a lower level of KSHV lytic gene expression, impaired viral DNA replication, and consequently, a dramatic reduction in the level of progeny production. More importantly, the role of vBcl-2 in KSHV lytic replication is genetically separated from its antiapoptotic and antiautophagic functions, suggesting that the KSHV Bcl-2 carries a novel function in viral lytic replication.

Viral Bcl-2 Encoded by the Kaposi's Sarcoma-Associated Herpesvirus Is Vital for Virus Reactivation.

The Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 16 (orf16) encodes a viral Bcl-2 (vBcl-2) protein which shares sequence and functional homology with the Bcl-2 family. Like its cellular homologs, vBcl-2 protects various cell types from apoptosis and can also negatively regulate autophagy. vBcl-2 is transcribed during lytic infection; however, its exact function has not been determined to date. By using bacterial artificial chromosome 16 (BAC16) clone carrying the full-length KSHV genome, we have generated recombinant KSHV mutants that fail to express vBcl-2 or express mCherry-tagged vBcl-2. We show that the vBcl-2 protein is expressed at relatively low levels during lytic induction and that a lack of vBcl-2 largely reduces the efficiency of KSHV reactivation in terms of lytic gene expression, viral DNA replication, and production of infectious particles. In contrast, the establishment of latency was not affected by the absence of vBcl-2. Our findings suggest an important role for vBcl-2 during initial phases of lytic reactivation and/or during subsequent viral propagation. Given the known functions of vBcl-2 in regulating apoptosis and autophagy, which involve its direct interaction with cellular proteins and thus require high levels of protein expression, it appears that vBcl-2 may have additional regulatory functions that do not depend on high levels of protein expression. The present study shows for the first time the expression of endogenous vBcl-2 protein in KSHV-infected cell lines and demonstrates the importance of vBcl-2 during the initial phases of lytic reactivation and/or during its subsequent propagation. It is suggested that vBcl-2 has additional regulatory functions beyond apoptosis and autophagy repression that do not depend on high levels of protein expression.

Generation of high-titre virus stocks using BrK.219, a B-cell line infected stably with recombinant Kaposi's sarcoma-associated herpesvirus

Kaposi's sarcoma-associated herpesvirus (KSHV) is a gamma-2-lymphotropic human oncogenic herpesvirus associated with Kaposi's sarcoma (KS) and two B-cell lymphoproliferative diseases, primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). KSHV establishes latency soon after infection in vivo and in vitro. Consequently, it is technically difficult to generate high-titre virus stocks required for infection experiments in tissue culture. Currently used methods of KSHV stock production involve induction of the lytic/productive cycle in PEL cell lines or in adherent cell lines harbouring recombinant KSHV genomes. In this study, the BJAB-derived B-cell line BrK.219, which is infected latently with a recombinant KSHV (rKSHV.219), is used to produce high-titre virus stocks. BrK.219 cells enter the lytic KSHV replication cycle upon cross-linking of B-cell receptors (BCRs) with anti-IgM antibodies without the need for additional, potentially toxic chemical inducers. High cell concentrations can be cultured and induced easily in spinner flasks, saving time and resources. The established protocol allows the generation of KSHV virus stocks with titres of up to 106IU/ml in unconcentrated culture supernatants, representing a 103–104-fold improvement compared to conventional methods.

HITS-CLIP Analysis Uncovers a Link between the Kaposi’s Sarcoma-Associated Herpesvirus ORF57 Protein and Host Pre-mRNA Metabolism

The Kaposi’s sarcoma associated herpesvirus (KSHV) is an oncogenic virus that causes Kaposi’s sarcoma, primary effusion lymphoma (PEL), and some forms of multicentric Castleman’s disease. The KSHV ORF57 protein is a conserved posttranscriptional regulator of gene expression that is essential for virus replication. ORF57 is multifunctional, but most of its activities are directly linked to its ability to bind RNA. We globally identified virus and host RNAs bound by ORF57 during lytic reactivation in PEL cells using high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP). As expected, ORF57-bound RNA fragments mapped throughout the KSHV genome, including the known ORF57 ligand PAN RNA. In agreement with previously published ChIP results, we observed that ORF57 bound RNAs near the oriLyt regions of the genome. Examination of the host RNA fragments revealed that a subset of the ORF57-bound RNAs was derived from transcript 5´ ends. The position of these 5´-bound fragments correlated closely with the 5´-most exon-intron junction of the pre-mRNA. We selected four candidates (BTG1, EGR1, ZFP36, and TNFSF9) and analyzed their pre-mRNA and mRNA levels during lytic phase. Analysis of both steady-state and newly made RNAs revealed that these candidate ORF57-bound pre-mRNAs persisted for longer periods of time throughout infection than control RNAs, consistent with a role for ORF57 in pre-mRNA metabolism. In addition, exogenous expression of ORF57 was sufficient to increase the pre-mRNA levels and, in one case, the mRNA levels of the putative ORF57 targets. These results demonstrate that ORF57 interacts with specific host pre-mRNAs during lytic reactivation and alters their processing, likely by stabilizing pre-mRNAs. These data suggest that ORF57 is involved in modulating host gene expression in addition to KSHV gene expression during lytic reactivation.

A Survey of the Interactome of Kaposi's Sarcoma-Associated Herpesvirus ORF45 Revealed Its Binding to Viral ORF33 and Cellular USP7, Resulting in Stabilization of ORF33 That Is Required for Production of Progeny Viruses

The ORF45 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus-specific immediate-early tegument protein. Our previous studies have revealed its crucial roles in both early and late stages of KSHV infection. In this study, we surveyed the interactome of ORF45 using a panel of monoclonal antibodies. In addition to the previously identified extracellular regulated kinase (ERK) and p90 ribosomal S6 kinase (RSK) proteins, we found several other copurified proteins, including prominent ones of ∼38 kDa and ∼130 kDa. Mass spectrometry revealed that the 38-kDa protein is viral ORF33 and the 130-kDa protein is cellular USP7 (ubiquitin-specific protease 7). We mapped the ORF33-binding domain to the highly conserved carboxyl-terminal 19 amino acids (aa) of ORF45 and the USP7-binding domain to the reported consensus motif in the central region of ORF45. Using immunofluorescence staining, we observed colocalization of ORF45 with ORF33 or USP7 both under transfected conditions and in KSHV-infected cells. Moreover, we noticed ORF45-dependent relocalization of a portion of ORF33/USP7 from the nucleus to the cytoplasm. We found that ORF45 caused an increase in ORF33 protein accumulation that was abolished if either the ORF33- or USP7-binding domain in ORF45 was deleted. Furthermore, deletion of the conserved carboxyl terminus of ORF45 in the KSHV genome drastically reduced the level of ORF33 protein in KSHV-infected cells and abolished production of progeny virions. Collectively, our results not only reveal new components of the ORF45 interactome, but also demonstrate that the interactions among these proteins are crucial for KSHV lytic replication. Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of several human cancers. KSHV ORF45 is a multifunctional protein that is required for KSHV lytic replication, but the exact mechanisms by which ORF45 performs its critical functions are unclear. Our previous studies revealed that all ORF45 protein in cells exists in high-molecular-weight complexes. We therefore sought to characterize the interactome of ORF45 to provide insights into its roles during lytic replication. Using a panel of monoclonal antibodies, we surveyed the ORF45 interactome in KSHV-infected cells. We identified two new binding partners of ORF45: the viral protein ORF33 and cellular ubiquitin-specific protease 7 (USP7). We further demonstrate that the interaction between ORF45 and ORF33 is crucial for the efficient production of KSHV viral particles, suggesting that the targeted interference with this interaction may represent a novel strategy to inhibit KSHV lytic replication.

Association between malaria exposure and Kaposi's sarcoma-associated herpes virus seropositivity in Uganda.

ObjectiveUnlike other herpes viruses, Kaposi's sarcoma-associated herpes virus (KSHV) is not ubiquitous worldwide and is most prevalent in sub-Saharan Africa. The reasons for this are unclear. As part of a wider investigation of factors that facilitate transmission in Uganda, a high prevalence country, we examined the association between antimalaria antibodies and seropositivity against KSHV.MethodsAntibodies against P. falciparum merozoite surface protein (PfMSP)-1, P. falciparum apical membrane antigen (PfAMA)-1 and KSHV antigens (ORF73 and K8.1) were measured in samples from 1164 mothers and 1227 children.ResultsKaposi's sarcoma-associated herpes virus seroprevalence was 69% among mothers and 15% children. Among mothers, KSHV seroprevalence increased with malaria antibody titres: from 60% to 82% and from 54% to 77%, comparing those with the lowest and highest titres for PfMSP-1 and PfAMA-1, respectively (P < 0.0001). Among children, only antibodies to PfAMA-1 were significantly associated with KSHV seropositivity, (P < 0.0001). In both mothers and children, anti-ORF73 antibodies were more strongly associated with malaria antibodies than anti-K8.1 antibodies.ConclusionThe association between malaria exposure and KSHV seropositivity suggests that malaria is a cofactor for KSHV infection or reactivation.

Chromatinization of the KSHV Genome During the KSHV Life Cycle.

Kaposi’s sarcoma-associated herpesvirus (KSHV) belongs to the gamma herpesvirus family and is the causative agent of various lymphoproliferative diseases in humans. KSHV, like other herpesviruses, establishes life-long latent infection with the expression of a limited number of viral genes. Expression of these genes is tightly regulated by both the viral and cellular factors. Recent advancements in identifying the expression profiles of viral transcripts, using tilling arrays and next generation sequencing have identified additional coding and non-coding transcripts in the KSHV genome. Determining the functions of these transcripts will provide a better understanding of the mechanisms utilized by KSHV in altering cellular pathways involved in promoting cell growth and tumorigenesis. Replication of the viral genome is critical in maintaining the existing copies of the viral episomes during both latent and lytic phases of the viral life cycle. The replication of the viral episome is facilitated by viral components responsible for recruiting chromatin modifying enzymes and replication factors for altering the chromatin complexity and replication initiation functions, respectively. Importantly, chromatin modification of the viral genome plays a crucial role in determining whether the viral genome will persist as latent episome or undergo lytic reactivation. Additionally, chromatinization of the incoming virion DNA, which lacks chromatin structure, in the target cells during primary infection, helps in establishing latent infection. Here, we discuss the recent advancements on our understating of KSHV genome chromatinization and the consequences of chromatin modifications on viral life cycle.

Molecular biology of KSHV lytic reactivation.

Kaposi’s sarcoma-associated herpesvirus (KSHV) primarily persists as a latent episome in infected cells. During latent infection, only a limited number of viral genes are expressed that help to maintain the viral episome and prevent lytic reactivation. The latent KSHV genome persists as a highly ordered chromatin structure with bivalent chromatin marks at the promoter-regulatory region of the major immediate-early gene promoter. Various stimuli can induce chromatin modifications to an active euchromatic epigenetic mark, leading to the expression of genes required for the transition from the latent to the lytic phase of KSHV life cycle. Enhanced replication and transcription activator (RTA) gene expression triggers a cascade of events, resulting in the modulation of various cellular pathways to support viral DNA synthesis. RTA also binds to the origin of lytic DNA replication to recruit viral, as well as cellular, proteins for the initiation of the lytic DNA replication of KSHV. In this review we will discuss some of the pivotal genetic and epigenetic factors that control KSHV reactivation from the transcriptionally restricted latent program.

Kaposi's sarcoma-associated herpesvirus genome programming during the early stages of primary infection of peripheral blood mononuclear cells.

ABSTRACTThe early period of Kaposi’s sarcoma-associated herpesvirus (KSHV) infection involves the dynamic expression of viral genes, which are temporally and epigenetically regulated. KSHV can effectively infect and persist in endothelial as well as human B cells with different gene expression patterns. To understand the temporal epigenetic changes which occur when KSHV infects the lymphocytic compartment, we infected human peripheral blood mononuclear cells (PBMCs) and comprehensively analyzed the changes which occurred at the binding sites of virally encoded lytic as well as latent proteins along with epigenetic modifications across the KSHV genome during early primary infection. Using chromatin immunoprecipitation (ChIP) assays, we showed that the KSHV genome acquires a uniquely distinct histone modification pattern of methylation (H3K4me3, H3K9me3, and H3K27me3) and acetylation (H3Ac) during de novo infection of human PBMCs. This pattern showed that the epigenetic changes were temporally controlled. The binding profiles of KSHV latent protein LANA and the immediate early proteins RTA and K8 showed specific patterns at different times postinfection, which reflects the gene expression program. Further analysis demonstrated that KSHV can concurrently express lytic and latent genes which were associated with histone modifications at these specific regions on the viral genome. We identified three KSHV genes, K3, ORF49, and ORF64, which exhibited different profiles of histone modifications during the early stages of PBMC infection. These studies established a distinct pattern of epigenetic modification which correlates with viral gene expression temporally regulated during the first 7 days of PBMC infection and provides clues to the regulatory program required for successful infection by KSHV of human PBMCs.

Kaposi's Sarcoma-Associated Herpesvirus Induces Nrf2 during De Novo Infection of Endothelial Cells to Create a Microenvironment Conducive to Infection

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.

Activation of p90 ribosomal S6 kinases by ORF45 of Kaposi's 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 extracellular regulated kinase (ERK) (E. Kuang, Q. Tang, G. G. Maul, and F. Zhu, J Virol 82:1838-1850, 2008, http://dx.doi.org/10.1128/JVI.02119-07). We now have 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. The 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 also was accompanied by a noticeable decreased in viral gene expression during lytic reactivation. Consequently, the ORF45-F66A mutant 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. KSHV is the causative agent of three human malignancies. KSHV pathogenesis is intimately linked to its ability to modulate the host cell microenvironment and to facilitate efficient production of progeny viral particles. We 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 replication.

Lymphoid hyperplasia and lymphoma in KSHV K1 transgenic mice.

Growing evidence supports the involvement of human herpervirus 8, Kaposi's sarcoma associated herpesvirus (KSHV), in the pathology of primary effusion lymphoma, multicentric Castleman's disease, and Kaposi's sarcoma, but the exact mechanism of KSHV contribution to the oncogenic process remains elusive. We studied transgenic mice expressing the ORF K1 of KSHV, whose position in the KSHV genome corresponds to known lymphoproliferative genes of other herpesviruses. K1 protein was previously shown to contain a constitutively active ITAM domain, involved in activation of Akt and pro-survival signaling, and to inhibit Fas-mediated apoptosis by interfering with binding of FasL. All this pointed to a possible role of K1 in the pathogenesis of KSHV-associated cancers. K1 transgenic mice (80-90%) developed lymphoid hyperplasia and splenomegaly at 8 and 10 months of age, 25% had confirmed diagnosis of lymphoma, and 50% developed abdominal and/or hepatic tumors by 18 months of age. Histological examination showed loss of splenic architecture and increased cellularity. Lymph nodes showed disrupted architecture with effaced follicles and other pathological changes, including signs of angiofollicular lymphoid hyperplasia. One of the livers showed signs of angiosarcoma. In summary, our histology results revealed pathological changes in K1 transgenic mice similar to lymphoma, Castleman's disease, and angiosarcoma, suggesting that K1 may contribute to the development of KSHV-associated cancers.

P130Cas scaffolds the signalosome to direct adaptor-effector cross talk during Kaposi's sarcoma-associated herpesvirus trafficking in human microvascular dermal endothelial cells.

Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with cell surface receptors, such as heparan sulfate, integrins (α3β1, αVβ3, and αVβ5), and EphrinA2 (EphA2), and activates focal adhesion kinase (FAK), Src, phosphoinositol 3-kinase (PI3-K), c-Cbl, and RhoA GTPase signal molecules early during lipid raft (LR)-dependent productive macropinocytic entry into human dermal microvascular endothelial cells. Our recent studies have identified CIB1 as a signal amplifier facilitating EphA2 phosphorylation and subsequent cytoskeletal cross talk during KSHV macropinocytosis. Although CIB1 lacks an enzymatic activity and traditional adaptor domain or known interacting sequence, it associated with the KSHV entry signal complex and the CIB1-KSHV association was sustained over 30 min postinfection. To identify factors scaffolding the EphA2-CIB1 signal axis, the role of major cellular scaffold protein p130Cas (Crk-associated substrate of Src) was investigated. Inhibitor and small interfering RNA (siRNA) studies demonstrated that KSHV induced p130Cas in an EphA2-, CIB1-, and Src-dependent manner. p130Cas and Crk were associated with KSHV, LRs, EphA2, and CIB1 early during infection. Live-cell microscopy and biochemical studies demonstrated that p130Cas knockdown did not affect KSHV entry but significantly reduced productive nuclear trafficking of viral DNA and routed KSHV to lysosomal degradation. p130Cas aided in scaffolding adaptor Crk to downstream guanine nucleotide exchange factor phospho-C3G possibly to coordinate GTPase signaling during KSHV trafficking. Collectively, these studies demonstrate that p130Cas acts as a bridging molecule between the KSHV-induced entry signal complex and the downstream trafficking signalosome in endothelial cells and suggest that simultaneous targeting of KSHV entry receptors with p130Cas would be an attractive potential avenue for therapeutic intervention in KSHV infection. Eukaryotic cell adaptor molecules, without any intrinsic enzymatic activity, are well known to allow a great diversity of specific and coordinated protein-protein interactions imparting signal amplification to different networks for physiological and pathological signaling. They are involved in integrating signals from growth factors, extracellular matrix molecules, bacterial pathogens, and apoptotic cells. The present study identifies human microvascular dermal endothelial (HMVEC-d) cellular scaffold protein p130Cas (Crk-associated substrate) as a platform to promote Kaposi's sarcoma-associated herpesvirus (KSHV) trafficking. Early during KSHV de novo infection, p130Cas associates with lipid rafts and scaffolds EphrinA2 (EphA2)-associated critical adaptor members to downstream effector molecules, promoting successful nuclear delivery of the KSHV genome. Hence, simultaneous targeting of the receptor EphA2 and scaffolding action of p130Cas can potentially uncouple the signal cross talk of the KSHV entry-associated upstream signal complex from the immediate downstream trafficking-associated signalosome, consequently routing KSHV toward lysosomal degradation and eventually blocking KSHV infection and associated malignancies.

Identification of properties of the Kaposi's sarcoma-associated herpesvirus latent origin of replication that are essential for the efficient establishment and maintenance of intact plasmids.

The maintenance of latent Kaposi's sarcoma-associated herpesvirus (KSHV) genomes is mediated in cis by their terminal repeats (TR). A KSHV genome can have 16 to 50 copies of the 801-bp TR, each of which harbors a 71-bp-long minimal replicator element (MRE). A single MRE can support replication in transient assays, and the presence of as few as two TRs appears to support establishment of KSHV-derived plasmids. Why then does KSHV have such redundancy and heterogeneity in the number of TRs? By determining the abilities of KSHV-derived plasmids containing various numbers of the TRs and MREs to be established and maintained in the long term, we have found that plasmids with fewer than 16 TRs or those with tandem repeats of the MREs are maintained inefficiently, as shown by both their decreased abilities to support formation of colonies and their instability, resulting in frequent rearrangements yielding larger plasmids during and after establishment. These defects often can be overcome by adding the Epstein-Barr virus (EBV) partitioning element, FR (i.e., family of repeats), in cis to these plasmids. In addition we have found that the spacing between MREs is important for their functions, too. Thus, two properties of KSHV's origin of latent replication essential for the efficient establishment and maintenance of viral plasmids stably are (i) the presence of approximately 16 copies of the TR, which are needed for efficient partitioning, and (ii) the presence of at least 2 MRE units separated by 801 bp of center-to-center spacing, which are required for efficient synthesis. KSHV is a human tumor virus that maintains its genome as a plasmid in lymphoid tumor cells. Each plasmid DNA molecule encodes many origins of synthesis. Here we show that these many origins provide an essential advantage to KSHV, allowing the DNAs to be maintained without rearrangement. We find also that the correct spacing between KSHV's origins of DNA synthesis is required for them to support synthesis efficiently. The identification of these properties illuminates plasmid replication in mammalian cells and should lead to the development of rational means to inhibit these tumorigenic replicons.