Kaposi's Sarcoma-associated Herpesvirus Virions Research Articles

Structures of Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus virions reveal species-specific tegument and envelope features.

Discovered in 1964, Epstein-Barr virus (EBV) is the first identified human oncogenic virus and the founding member of the gammaherpesvirus subfamily. In 1994, another cancer-causing virus was discovered in lesions of AIDS patients and later named Kaposi's sarcoma-associated herpesvirus (KSHV), the second human gammaherpesvirus. Despite the historical importance of EBV and KSHV, technical difficulties with isolating large quantities of these viruses and the pleiomorphic nature of their envelope and tegument layers have limited structural characterization of their virions. In this study, we employed the latest technologies in cryogenic electron microscopy (cryoEM) and tomography (cryoET) supplemented with an artificial intelligence-powered data processing software package to reconstruct 3D structures of the EBV and KSHV virions. We uncovered unique properties of the envelope glycoproteins and tegument layers of both EBV and KSHV. Comparison of these features with their non-tumorigenic counterparts provides insights into their relevance during infection.

Narciclasine Display Preferential Pre-Clinical Activity Against KSHV-Associated Primary Effusion Lymphoma By Targeting MYC Via NF-κB

Primary effusion lymphoma (PEL) is an aggressive type of non-Hodgkin lymphoma with extremely poor prognosis when treated with conventional chemotherapy. Kaposi sarcoma-associated herpesvirus (KSHV) is the etiologic agent of PEL. KSHV-encoded viral FLICE-inhibitory protein (vFLIP)-K13 constitutively activates the NF-κB pathway, which has been shown to be essential for the survival and proliferation of PEL cells. In a K13-isogenic cell line based screening of small molecule libraries, we have identified the natural products narciclasine and its structural analog, 7-deoxynarciclasine (found in daffodils), as having preferential cytotoxicity towards K13-expressing isogenic cell line. Further, the structurally closely related lycorine (another alkaloid found in daffodils) also exhibited similar activity. All the three compounds (Narciclasine, 7-deoxynarciclasine, and lycorine) display preferential cytotoxicity against PEL in a panel of cell lines comprising 6 hematological malignancies. Narciclasine is approximately 10 and 100-fold much effective than its structural analogs, 7-deoxynarciclasine, and lycorine, respectively. Importantly, IC50 of narciclasine in PEL cell lines (⁓10 nM) are well below the physiological concentration that was achieved in murine pharmacokinetic studies (250 nM). Narciclasine arrested cell cycle progression of PEL cells at the G0/G1 phase, with a concomitant decrease in the percentage of cells in S-phase. Further, there is a significant increase in the number of cells in sub-G0 peak, representative of apoptotic cells, which was confirmed by activation of caspase-3/7, AnnexinV staining, and PARP cleavage. Narciclasine blocked the NF-κB promoter activity in PEL cells as observed by a drastic decrease in firefly luciferase activity of PEL cells stably transduced with a lentiviral NF-κB-Luc reporter. IL-6 and IL-10 are known NF-κB targets and growth factors that are constitutively secreted by PEL, striking decrease in their secretory levels were observed upon treatment with narciclasine. Narciclasine preferentially blocked K13-induced NF-κB over the constitutive NF-κB activity in cells, which were engineered to express K13 conditionally (tet-on inducible lentiviral system). IKKγ/NEMO is essential for K13-induced NF-κB. The preference of narciclasine for K13-induced NF-κB is completely abolished upon NEMO knockout by CRISPR-Cas9 system. While probing the effect of narciclasine on the levels of putative NF-κB target genes by qRT-PCR in PEL, we found that there is a significant decrease in mRNA level of MYC, with a concomitant decrease in its protein level. Subsequently, all the direct target genes of MYC (MYB, TYRO3, TERT, PMM2, SLC19A1, and HK2) were significantly down regulated upon treatment with narciclasine. Furthermore, ectopic expression of MYC from a retroviral promoter significantly diminished the anti-proliferative effect of narciclasine and its analogs in PEL. It has been shown that inhibition of MYC is toxic to PEL and compounds that target MYC display selective activity against PEL, which explains the preferential activity of narciclasine and its analogs in PEL. MYC is required for maintenance of KSHV latency, and its down regulation results in the induction of KSHV Replication and Transcription Activator (RTA/ORF50) in PEL. However, narciclasine neither induced the expression of RTA nor the production of infectious KSHV virions in PEL. Most importantly, narciclasine doubled the survival of NSG mice bearing orthotopic - Cell Line Xenograft (CLX) and Patient Derived Xenograft (PDX) models of PEL. Finally, the IC50 of narciclasine in PBMCs from normal healthy donors is approximately 4-fold higher than the PEL cells directly isolated from the mice bearing the PDX model of the disease and PEL cell lines, indicating that there is a significant therapeutic window for narciclasine, which can be exploited for the treatment of KSHV-associated malignancies. DisclosuresChaudhary:University of Southern California: Patents & Royalties: P.M.C. have filed patent application related to the technology described in this manuscript.

An Update of the Virion Proteome of Kaposi Sarcoma-Associated Herpesvirus.

The virion proteins of Kaposi sarcoma-associated herpesvirus (KSHV) were initially characterized in 2005 in two separate studies that combined the detection of 24 viral proteins and a few cellular components via LC-MS/MS or MALDI-TOF. Despite considerable advances in the sensitivity and specificity of mass spectrometry instrumentation in recent years, leading to significantly higher yields in detections, the KSHV virion proteome has not been revisited. In this study, we have re-examined the protein composition of purified KSHV virions via ultra-high resolution Qq time-of-flight mass spectrometry (UHR-QqTOF). Our results confirm the detection of all previously reported virion proteins, in addition to 17 other viral proteins, some of which have been characterized as virion-associated using other methods, and 10 novel proteins identified as virion-associated for the first time in this study. These results add KSHV ORF9, ORF23, ORF35, ORF48, ORF58, ORF72/vCyclin, K3, K9/vIRF1, K10/vIRF4, and K10.5/vIRF3 to the list of KSHV proteins that can be incorporated into virions. The addition of these proteins to the KSHV virion proteome provides novel and important insight into early events in KSHV infection mediated by virion-associated proteins. Data are available via ProteomeXchange with identifier PXD022626.

Narciclasine, an isocarbostyril alkaloid, has preferential activity against primary effusion lymphoma

Primary effusion lymphoma (PEL) is a subtype of non-Hodgkin lymphoma associated with infection by Kaposi sarcoma-associated herpes virus (KSHV). PEL is an aggressive disease with extremely poor prognosis when treated with conventional chemotherapy. Narciclasine, a natural product present in Amaryllidaceae family of flowering plants including daffodils, belongs to a class of molecules termed ‘isocarbostyril alkaloid’. We have found that narciclasine displays preferential cytotoxicity towards PEL at low nanomolar concentrations and is approximately 10 and 100-fold more potent than its structural analogs lycoricidine and lycorine, respectively. Narciclasine arrested cell-cycle progression at the G1 phase and induced apoptosis in PEL, which is accompanied by activation of caspase-3/7, cleavage of PARP and increase in the surface expression of Annexin-V. Although narciclasine treatment resulted in a marked decrease in the expression of MYC and its direct target genes,time-course experiments revealed that MYC is not a direct target of narciclasine. Narciclasine treatment neither induces the expression of KSHV-RTA/ORF50 nor the production of infectious KSHV virions in PEL. Finally, narciclasine provides dramatic survival advantages to mice in two distinct mouse xenograft models of PEL. In conclusion, our results suggest that narciclasine could be a promising agent for the treatment of PEL.

Kaposi's Sarcoma-Associated Herpesvirus-Encoded circRNAs Are Expressed in Infected Tumor Tissues and Are Incorporated into Virions.

Kaposi's sarcoma-associated herpesvirus (KSHV) has recently been found to generate circular RNAs (circRNAs) from several KSHV genes, most abundantly from K10 (viral interferon regulatory factor 4 [vIRF4]), K7.3, and polyadenylated nuclear (PAN) RNA. To define expression of these circRNAs, KSHV-infected cell lines, patient tissues, and purified virions were examined. KSHV circRNA expression was universally detected in tests of six primary effusion lymphoma (PEL) cell lines but ranged from low-level expression in BC-1 cells dually infected with tightly latent KSHV and Epstein-Barr virus to abundant expression in KSHV-only BCBL-1 cells with spontaneous virus production. Generally, the PAN/K7.3 locus broadly and bidirectionally generated circRNA levels that paralleled the corresponding linear RNA levels. However, RNA corresponding to a particular KSHV circularization site (circ-vIRF4) was minimally induced, despite linear vIRF4 RNA being activated by virus induction. In situ hybridization showed abundant circ-vIRF4 in noninduced PEL cells. All three KSHV circRNAs were isolated as nuclease-protected forms from gradient-purified virions collected from BrK.219 cells infected with a KSHV molecular clone. For circ-vIRF4, the fully processed form that is exported to the cytoplasm was incorporated into virus particles but the nuclear, intron-retaining form was not. The half-life of circ-vIRF4 was twice as long as that of its linear counterpart. The KSHV circRNAs could be detected at a higher rate than their corresponding linear counterparts by in situ hybridization in archival tissues and by reverse transcription-PCR (RT-PCR) in sera stored for over 25 years. In summary, KSHV circRNAs are expressed in infection-associated diseases, can be regulated depending on virus life cycle, and are incorporated into viral particles for preformed delivery, suggesting a potential function in early infection.IMPORTANCE KSHV has recently been found to encode circRNAs. circRNAs result from back-splicing of an upstream pre-mRNA splice donor exon-intron junction to an acceptor site, generating a covalently closed circle. This study revealed that for one KSHV region, the PAN/K7.3 locus, broadly and bidirectionally generated circRNA levels parallel corresponding linear RNA levels. Another KSHV circularization site (circ-vIRF4), however, showed expression that differed from that of the corresponding linear RNA. All KSHV circRNAs are incorporated into KSHV virions and are potentially expressed as immediate early products in newly infected cells.

KSHV activates unfolded protein response sensors but suppresses downstream transcriptional responses to support lytic replication.

Herpesviruses usurp host cell protein synthesis machinery to convert viral mRNAs into proteins, and the endoplasmic reticulum (ER) to ensure proper folding, post-translational modification and trafficking of secreted and transmembrane viral proteins. Overloading ER folding capacity activates the unfolded protein response (UPR), whereby sensor proteins ATF6, PERK and IRE1 initiate a stress-mitigating transcription program that accelerates catabolism of misfolded proteins while increasing ER folding capacity. Kaposi's sarcoma-associated herpesvirus (KSHV) can be reactivated from latency by chemical induction of ER stress, which causes accumulation of the XBP1s transcription factor that transactivates the viral RTA lytic switch gene. The presence of XBP1s-responsive elements in the RTA promoter suggests that KSHV evolved a mechanism to respond to ER stress. Here, we report that ATF6, PERK and IRE1 were activated upon reactivation from latency and required for efficient KSHV lytic replication; genetic or pharmacologic inhibition of each UPR sensor diminished virion production. Despite UPR sensor activation during KSHV lytic replication, downstream UPR transcriptional responses were restricted; 1) ATF6 was cleaved to activate the ATF6(N) transcription factor but ATF6(N)-responsive genes were not transcribed; 2) PERK phosphorylated eIF2α but ATF4 did not accumulate; 3) IRE1 caused XBP1 mRNA splicing, but XBP1s protein did not accumulate and XBP1s-responsive genes were not transcribed. Ectopic expression of the KSHV host shutoff protein SOX did not affect UPR gene expression, suggesting that alternative viral mechanisms likely mediate UPR suppression during lytic replication. Complementation of XBP1s deficiency during KSHV lytic replication inhibited virion production in a dose-dependent manner in iSLK.219 cells but not in TREx-BCBL1-RTA cells. However, genetically distinct KSHV virions harvested from these two cell lines were equally susceptible to XBP1s restriction following infection of naïve iSLK cells. This suggests that cell-intrinsic properties of BCBL1 cells may circumvent the antiviral effect of ectopic XBP1s expression. Taken together, these findings indicate that while XBP1s plays an important role in reactivation from latency, it can inhibit virus replication at a later step, which the virus overcomes by preventing its synthesis. These findings suggest that KSHV hijacks UPR sensors to promote efficient viral replication while sustaining ER stress.

KSHV RNA-binding protein ORF57 inhibits P-body formation to promote viral multiplication by interaction with Ago2 and GW182.

Cellular non-membranous RNA-granules, P-bodies (RNA processing bodies, PB) and stress granules (SG), are important components of the innate immune response to virus invasion. Mechanisms governing how a virus modulates PB formation remain elusive. Here, we report the important roles of GW182 and DDX6, but not Dicer, Ago2 and DCP1A, in PB formation, and that Kaposi's sarcoma-associated herpesvirus (KSHV) lytic infection reduces PB formation through several specific interactions with viral RNA-binding protein ORF57. The wild-type ORF57, but not its N-terminal dysfunctional mutant, inhibits PB formation by interacting with the N-terminal GW-domain of GW182 and the N-terminal domain of Ago2, two major components of PB. KSHV ORF57 also induces nuclear Ago2 speckles. Homologous HSV-1 ICP27, but not EBV EB2, shares this conserved inhibitory function with KSHV ORF57. By using time-lapse confocal microscopy of HeLa cells co-expressing GFP-tagged GW182, we demonstrated that viral ORF57 inhibits primarily the scaffolding of GW182 at the initial stage of PB formation. Consistently, KSHV-infected iSLK/Bac16 cells with reduced GW182 expression produced far fewer PB and SG, but 100-fold higher titer of infectious KSHV virions when compared to cells with normal GW182 expression. Altogether, our data provide the first evidence that a DNA virus evades host innate immunity by encoding an RNA-binding protein that promotes its replication by blocking PB formation.

A multivalent Kaposi sarcoma-associated herpesvirus-like particle vaccine capable of eliciting high titers of neutralizing antibodies in immunized rabbits

Kaposi sarcoma-associated herpesvirus (KSHV) is an emerging pathogen and the causative agent of multiple cancers in immunocompromised patients. To date, there is no licensed prophylactic KSHV vaccine. In this study, we generated a novel subunit vaccine that incorporates four key KSHV envelope glycoproteins required for viral entry in diverse cell types (gpK8.1, gB, and gH/gL) into a single multivalent KSHV-like particle (KSHV-LP). Purified KSHV-LPs were similar in size, shape, and morphology to KSHV virions. Vaccination of rabbits with adjuvanted KSHV-LPs generated strong glycoprotein-specific antibody responses, and purified immunoglobulins from KSHV-LP-immunized rabbits neutralized KSHV infection in epithelial, endothelial, fibroblast, and B cell lines (60–90% at the highest concentration tested). These findings suggest that KSHV-LPs may be an ideal platform for developing a safe and effective prophylactic KSHV vaccine. We envision performing future studies in animal models that are susceptible to KSHV infection, to determine correlates of immune protection in vivo.

Guanylate-Binding Protein 1 Inhibits Nuclear Delivery of Kaposi's Sarcoma-Associated Herpesvirus Virions by Disrupting Formation of Actin Filament.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a typical gammaherpesvirus that establishes persistent lifelong infection in host cells. In order to establish successful infection, KSHV has evolved numerous immune evasion strategies to bypass or hijack the host immune system. However, host cells still produce immune cytokines abundantly during primary KSHV infection. Whether the immune effectors produced are able to inhibit viral infection and how KSHV successfully conquers these immune effectors remain largely unknown. The guanylate-binding protein 1 (GBP1) gene is an interferon-stimulated gene and exerts antiviral functions on several RNA viruses; however, its function in DNA virus infection is less well understood. In this study, we found that KSHV infection increases both the transcriptional and protein levels of GBP1 at the early stage of primary infection by activating the NF-κB pathway. The overexpression of GBP1 significantly inhibited KSHV infection, while the knockdown of GBP1 promoted KSHV infection. The GTPase activity and dimerization of GBP1 were demonstrated to be responsible for its anti-KSHV activity. Furthermore, we found that GBP1 inhibited the nuclear delivery of KSHV virions by disrupting the formation of actin filaments. Finally, we demonstrated that replication and transcription activator (RTA) promotes the degradation of GBP1 through a proteasome pathway. Taken together, these results provide a new understanding of the antiviral mechanism of GBP1, which possesses potent anti-KSHV activity, and suggest the critical role of RTA in the evasion of the innate immune response during primary infection by KSHV.IMPORTANCE GBP1 can be induced by various cytokines and exerts antiviral activities against several RNA viruses. Our study demonstrated that GBP1 can exert anti-KSHV function by inhibiting the nuclear delivery of KSHV virions via the disruption of actin filaments. Moreover, we found that KSHV RTA can promote the degradation of GBP1 through a proteasome-mediated pathway. Taken together, our results elucidate a novel mechanism of GBP1 anti-KSHV activity and emphasize the critical role of RTA in KSHV evasion of the host immune system during primary infection.

A Role for Host Activation-Induced Cytidine Deaminase in Innate Immune Defense against KSHV

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.

MicroRNAs and Unusual Small RNAs Discovered in Kaposi's Sarcoma-Associated Herpesvirus Virions

It is widely held that any given virus uses only one type of nucleic acid for genetic information storage. However, this consensus has been challenged slightly by several recent studies showing that many RNA species are present within a range of DNA viruses that include Kaposi's sarcoma-associated herpesvirus (KSHV). RNAs extracted from purified DNA virus particles exhibit great diversity in terms of length, abundance, temporal expression, cellular localization, and coding capacity during viral infection. In addition to known RNA species, the current study showed that small regulatory RNAs were present in KSHV virions. A large number of viral and cellular microRNAs (miRNAs), as well as unusual small RNAs (usRNAs), were detected in KSHV virions by using deep sequencing. Both viral and host miRNAs detected in small RNAs extracted from KSHV virions were further shown to colocalize with KSHV virions directly by in situ hybridization (ISH)-electron microscopy (EM) (ISH-EM). Some of these miRNAs were differentially present in the host cells and KSHV virions, suggesting that they are not randomly present in KSHV virions. The virional miRNAs could be transported into host cells, and they are biologically functional during de novo viral infection. Our study revealed miRNAs and usRNAs as a novel group of components in KSHV virions.

The ephrin receptor tyrosine kinase A2 is a cellular receptor for Kaposi's sarcoma–associated herpesvirus

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma(1), a highly vascularized tumor originating from lymphatic endothelial cells, and of at least two different B cell malignancies(2,3). A dimeric complex formed by the envelope glycoproteins H and L (gH-gL) is required for entry of herpesviruses into host cells(4). We show that the ephrin receptor tyrosine kinase A2 (EphA2) is a cellular receptor for KSHV gH-gL. EphA2 co-precipitated with both gH-gL and KSHV virions. Infection of human epithelial cells with a GFP-expressing recombinant KSHV strain, as measured by FACS analysis, was increased upon overexpression of EphA2. Antibodies against EphA(2) and siRNAs directed against EphA2 inhibited infection of endothelial cells. Pretreatment of KSHV with soluble EphA2 resulted in inhibition of KSHV infection by up to 90%. This marked reduction of KSHV infection was seen with all the different epithelial and endothelial cells used in this study. Similarly, pretreating epithelial or endothelial cells with the soluble EphA2 ligand ephrinA4 impaired KSHV infection. Deletion of the gene encoding EphA2 essentially abolished KSHV infection of mouse endothelial cells. Binding of gH-gL to EphA2 triggered EphA2 phosphorylation and endocytosis, a major pathway of KSHV entry(5,6). Quantitative RT-PCR and in situ histochemistry revealed a close correlation between KSHV infection and EphA2 expression both in cultured cells derived from human Kaposi's sarcoma lesions or unaffected human lymphatic endothelium, and in situ in Kaposi's sarcoma specimens, respectively. Taken together, our results identify EphA2, a tyrosine kinase with known functions in neovascularization and oncogenesis, as an entry receptor for KSHV.

KSHV infection of B-cell lymphoma using a modified KSHV BAC36 and coculturing system

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of two B cell lymphoproliferative diseases, namely primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). KSHV infection of B cell lymphoma in vitro has been a long-standing battle in advancing human KSHV biology. In this study, a modified form of KSHV BAC36 named BAC36A significantly increased the fidelity of gene-targeted site-directed mutagenesis in the KSHV genome. This modification eliminates tedious screening steps required to obtain mutant clones when a KSHV BAC36 reverse genetic system is used. Coculturing B-cell lymphoma BJAB cells with KSHV BAC36A stably transfected 293T cells enabled us to infect BJAB cells with a KSHV virion derived from the KSHV BAC36A. The coculture system produced substantial amounts of KSHV infection to BJAB, meaning that KSHV virions were released from 293T cells and then infected neighboring BJAB cells. Owing to our success with the KSHV BAC36A and coculture system, we propose a new genetic system for the study of KSHV gene expression and regulation in B-cell lymphoma.

Antagonism of host antiviral responses by Kaposi's sarcoma-associated herpesvirus tegument protein ORF45.

Virus infection of a cell generally evokes an immune response by the host to defeat the intruder in its effort. Many viruses have developed an array of strategies to evade or antagonize host antiviral responses. Kaposi's sarcoma-associated herpesvirus (KSHV) is demonstrated in this report to be able to prevent activation of host antiviral defense mechanisms upon infection. Cells infected with wild-type KSHV were permissive for superinfection with vesicular stomatitis virus (VSV), suggesting that KSHV virions fail to induce host antiviral responses. We previously showed that ORF45, a KSHV immediate-early protein as well as a tegument protein of virions, interacts with IRF-7 and inhibits virus-mediated type I interferon induction by blocking IRF-7 phosphorylation and nuclear translocation (Zhu et al., Proc. Natl. Acad. Sci. USA. 99:5573-5578, 2002). Here, using an ORF45-null recombinant virus, we demonstrate a profound role of ORF45 in inhibiting host antiviral responses. Infection of cells with an ORF45-null mutant recombinant KSHV (BAC-stop45) triggered an immune response that resisted VSV super-infection, concomitantly associated with appreciable increases in transcription of type I IFN and downstream anti-viral effector genes. Gain-of-function analysis showed that ectopic expression of ORF45 in human fibroblast cells by a lentivirus vector decreased the antiviral responses of the cells. shRNA-mediated silencing of IRF-7, that predominantly regulates both the early and late phase induction of type I IFNs, clearly indicated its critical contribution to the innate antiviral responses generated against incoming KSHV particles. Thus ORF45 through its targeting of the crucial IRF-7 regulated type I IFN antiviral responses significantly contributes to the KSHV survival immediately following a primary infection allowing for progression onto subsequent stages in its life-cycle.

Kaposi's sarcoma-associated herpes virus disrupts adherens junctions and increases endothelial permeability by inducing degradation of VE-cadherin

Kaposi's sarcoma (KS) is a vascular tumor of proliferative endothelial cells caused by infection of Kaposi's sarcoma-associated herpesvirus (KSHV). Aberrant vascular permeability is a hallmark of KS manifested as multifocal edematous skin and visceral lesions with dysregulated angiogenesis and vast inflammatory infiltrations. In this study, we showed that KSHV infection increased the permeability of confluent endothelial monolayers to serum albumin, blood-derived cells, KSHV-infected cells and KSHV virions. KSHV-induced permeability was associated with the disruption of adherens junctions and degradation of vascular endothelial (VE)-cadherin protein. Ultraviolet irradiation that inactivated KSHV virions and cycloheximide that blocked de novo protein synthesis failed to reverse KSHV-induced disruption of adherens junctions. However, soluble heparin that blocked KSHV entry into cells completely inhibited KSHV-induced permeability. Furthermore, KSHV-induced degradation of VE-cadherin was dose-dependent on the internalized virus particles. Together, these results indicate that KSHV infection induces vascular permeability by inducing VE-cadherin degradation during virus entry into cells. KSHV-induced aberrant vascular permeability could facilitate virus spread, promote inflammation and angiogenesis, and contribute to the pathogenesis of KSHV-induced malignancies.

Transient induction of lytic Kaposi's sarcoma-associated herpesvirus (KSHV) genes in KSHV-infected endothelial cells exposed to hypoxia

Kaposi's sarcoma-associated herpesvirus (KSHV), also called human herpesvirus-8 (HHV-8) is the causative agent of Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and a form of multicentric Castleman's disease (MCD). Two of these tumors, KS and PEL, tend to arise in areas of the body that are relatively hypoxic. Exploring this phenomenon, our group has previously found that KSHV in PEL tumor cell lines is activated to lytic replication by hypoxia and that certain KSHV genes are activated by hypoxia inducible factors (HIF-1 and HIF-2) through functional hypoxia response elements (HRE) (Davis et al., Blood, 2001, 15, 3244–50; Haque et al., J. Virol., 2006, 80, 7037–51). KS tumor cells are thought to be derived from endothelial cells or their precursors, and in the current study, we have addressed the question of whether KSHV was similarly activated to lytic replication in KSHV-infected endothelial cells. To investigate this, we infected primary dermal microvascular endothelial cells (DMEC) derived from human foreskin with a recombinant KSHV (rKSHV.219) that was engineered to contain red fluorescent protein (RFP) controlled by the lytic PAN RNA promoter as a lytic marker and green fluorescent protein under the control of human elongation factor 1-α promoter as a latent marker (gift of Dr. Jeff Vieira). Short-term ( 24 hours, there was a subsequent downregulation of KSHV lytic genes and of the production of virus despite continued upregulation of HIF-2 in the cells. We hypothesized that this late downregulation might be from production of one or more cellular factors with KSHV-suppressive activity in these cells upon exposure to hypoxia. We focused on IL-1β, IL-6, IL-8, tumor necrosis factor alpha (TNFα) and vascular endothelial growth factor (VEGF), as these are known to be produced by hypoxic endothelial cells and to be expressed by KSHV-infected cells in vitro and in KS lesions. IL-6, IL-1β and VEGF had no effect on lytic replication and IL8 increased replication in the KSHV-infected DMEC. However, TNFα blocked the expression of RTA, vIL6, ORF45 and K5 proteins and suppressed production of KSHV virions in the supernatant. Furthermore, when the cells were treated with blocking antibodies directed against human TNFα, KSHV lytic activation in the hypoxic DMEC was partially restored. Interestingly, established monolayers of KSHV-infected DMEC cells cultured in hypoxia out-survived normoxic cells cultured in parallel; this provided evidence that suppression of KSHV by TNFα was not simply from cell killing. These results suggest that production of TNFα by KSHV-infected endothelial cells may limit activation of lytic KSHV replication by hypoxia and that this effect may promote survival of KSHV-infected cells under hypoxic conditions and contribute to the pathogenesis of KS. This research was supported in part by the Intramural Research Program of the NIH, NCI.

Kaposi's Sarcoma-Associated Herpesvirus Virions Inhibit Interferon Responses Induced by Envelope Glycoprotein gpK8.1

The Kaposi's sarcoma-associated herpesvirus (KSHV) envelope glycoprotein gpK8.1 contributes to cellular attachment through binding cell surface heparan sulfate proteoglycans. By using a soluble recombinant form of gpK8.1, we discovered that a consequence of gpK8.1 interaction with human fibroblasts is the induction of an antiviral response, as characterized by the activation of interferon regulatory factor 3 (IRF-3), production of interferon beta (IFN-beta), and expression of interferon-stimulated antiviral genes. In contrast, neither IFN-beta expression nor a functional antiviral response is observed in cells treated with KSHV virions. The interferon response induced by soluble gpK8.1 can be inhibited by simultaneous treatment with UV-inactivated virions, while the induction of an indicator inflammatory cytokine, interleukin-6, was readily evident in the response to both gpK8.1 and KSHV. In addition, KSHV virions abrogate gpK8.1-mediated activation of IRF-3, an early transcriptional regulator for cellular antiviral responses. Although innate immune responses are initiated during contact between gpK8.1 and cellular receptor(s), these results suggest that the virion contains one or more structural elements that selectively repress an effective antiviral response while allowing cellular responses favorable to the KSHV life cycle.

Dissecting the Regions of Virion-Associated Kaposi's Sarcoma-Associated Herpesvirus Complement Control Protein Required for Complement Regulation and Cell Binding

Complement, which bridges innate and adaptive immune responses as well as humoral and cell-mediated immunity, is antiviral. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a lytic cycle protein called KSHV complement control protein (KCP) that inhibits activation of the complement cascade. It does so by regulating C3 convertases, accelerating their decay, and acting as a cofactor for factor I degradation of C4b and C3b, two components of the C3 and C5 convertases. These complement regulatory activities require the short consensus repeat (SCR) motifs, of which KCP has four (SCRs 1 to 4). We found that in addition to KCP being expressed on the surfaces of experimentally infected endothelial cells, it is associated with the envelope of purified KSHV virions, potentially protecting them from complement-mediated immunity. Furthermore, recombinant KCP binds heparin, an analogue of the known KSHV cell attachment receptor heparan sulfate, facilitating infection. Treating virus with an anti-KCP monoclonal antibody (MAb), BSF8, inhibited KSHV infection of cells by 35%. Epitope mapping of MAb BSF8 revealed that it binds within SCR domains 1 and 2, also the region of the protein involved in heparin binding. This MAb strongly inhibited classical C3 convertase decay acceleration by KCP and cofactor activity for C4b cleavage but not C3b cleavage. Our data suggest similar topological requirements for cell binding by KSHV, heparin binding, and regulation of C4b-containing C3 convertases but not for factor I-mediated cleavage of C3b. Importantly, they suggest KCP confers at least two functions on the virion: cell binding with concomitant infection and immune evasion.

Kaposi's Sarcoma-Associated Herpesvirus Virion-Induced Transcription Activation of the ORF50 Immediate-Early Promoter

Lytic cycle reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) can be initiated by transcription activation of the ORF50 immediate-early (IE) gene promoter (ORF50p). We provide evidence that KSHV virions stimulate transcription of ORF50p. Virion activation was resistant to UV inactivation and cycloheximide treatment. The virion-responsive element was mapped to core promoter region -150 to + 1 relative to the ORF50 initiation codon. Electrophoretic mobility shift assays and chromatin immunoprecipitation suggest that KSHV virions indirectly alter the protein composition and chromatin modifications at ORF50p. These data suggest that KSHV virions possess an IE trans-inducing function similar to that observed in alpha- and betaherpesviruses.

RNAs in the Virion of Kaposi's Sarcoma-Associated Herpesvirus

De novo infection of cultured cells with Kaposi's sarcoma-associated herpesvirus (KSHV) typically results in a latent infection. Recently, however, it has been reported that a subset of lytic mRNAs can be detected in cells shortly after KSHV infection; this expression is transient and eventually subsides, leading to latent infection (H. H. Krishnan et al., J. Virol 78:3601-3620, 2004). Since it has been shown that viral RNAs can be packaged into other herpesvirus virions, we sought to determine if KSHV virions contained RNAs and, if so, whether these RNAs contributed to the pool of lytic transcripts detected immediately after infection. Using DNA microarray, reverse transcription (RT)-PCR, and Northern blotting analyses, we identified 11 virally encoded RNAs in KSHV virions. These corresponded in size to the full-length mRNAs found in cytoplasmic RNA, and at least one was directly demonstrated to be translated upon infection in the presence of actinomycin D. Ten of these RNAs correspond to transcripts reported by Krishnan et al. at early times of infection, representing ca. 30% of such RNAs. Thus, import of RNAs in virions can account for some but not all of the early-appearing lytic transcripts. Quantitative RT-PCR analysis of infected-cell RNA demonstrated that most of the virion RNAs were very abundant at late times of infection, consistent with nonspecific incorporation during budding. However, the intracellular levels of one virion mRNA, encoding the viral protease, were much lower than those of transcripts not packaged in the virus particle, strongly suggesting that it may be incorporated by a specific mechanism.