Suppression of Kaposi's Sarcoma-Associated Herpesvirus Infection and Replication by 5′-AMP-Activated Protein Kinase

ABSTRACTThe host intracellular antiviral restriction factors inhibit viral infection and replication. The 5′-AMP-activated protein kinase (AMPK) is a cellular energy sensor regulating metabolic homeostasis. Activated AMPK inhibits the replication of numerous RNA viruses but enhances the entry of vaccinia virus. However, the role of AMPK in herpesvirus infection is unclear. In this study, we showed that the constitutive AMPK activity restricted Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication in primary human umbilical vein endothelial cells while KSHV infection did not markedly affect the endogenous AMPK activity. Knockdown of the AMPKα1 considerably enhanced the expression of viral lytic genes and the production of infectious virions, while overexpression of a constitutively active AMPK had the opposite effects. Accordingly, an AMPK inhibitor, compound C, augmented viral lytic gene expressions and virion productions but an AMPK agonist, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), suppressed both. Furthermore, a common diabetes drug, metformin, which carries an AMPK-agonistic activity, drastically inhibited the expression of viral lytic genes and the production of infectious virions, suggesting the use of metformin as a therapeutic agent for KSHV infection and replication. Together, these results identify the host AMPK as a KSHV restriction factor that can serve as a potential therapeutic target.IMPORTANCEHost cells encode specific proteins to restrict viral infection and replication. Kaposi's sarcoma-associated herpesvirus (KSHV) is a human tumor virus associated with several cancers. In this study, we have identified 5′-AMP-activated protein kinase (AMPK), a cellular energy sensor, as a restriction factor of KSHV lytic replication during primary infection. Activation of AMPK suppresses, while inhibition of AMPK enhances, KSHV lytic replication by regulating the expression of viral genes. AICAR and metformin, both of which are AMPK agonists currently used in clinics for the treatment of conditions associated with metabolic disorders, inhibit KSHV lytic replication. Thus, our work has identified AMPK as a potential therapeutic target and AICAR and metformin as potential therapeutic agents for KSHV-associated cancers.

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High Glucose Induces Reactivation of Latent Kaposi's Sarcoma-Associated Herpesvirus

Journal of Virology ◽

10.1128/jvi.01049-16 ◽

2016 ◽

Vol 90 (21) ◽

pp. 9654-9663 ◽

Cited By ~ 12

Author(s):

Fengchun Ye ◽

Yan Zeng ◽

Jingfeng Sha ◽

Tiffany Jones ◽

Kurt Kuhne ◽

...

Keyword(s):

High Glucose ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Lytic Replication ◽

Diabetic Patients ◽

Glucose Levels ◽

Production Of Hydrogen ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus ◽

Lytic Genes

ABSTRACT A high prevalence of Kaposi's sarcoma (KS) is seen in diabetic patients. It is unknown if the physiological conditions of diabetes contribute to KS development. We found elevated levels of viral lytic gene expression when Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells were cultured in high-glucose medium. To demonstrate the association between high glucose levels and KSHV replication, we xenografted telomerase-immortalized human umbilical vein endothelial cells that are infected with KSHV (TIVE-KSHV cells) into hyperglycemic and normal nude mice. The injected cells expressed significantly higher levels of KSHV lytic genes in hyperglycemic mice than in normal mice. We further demonstrated that high glucose levels induced the production of hydrogen peroxide (H 2 O 2 ), which downregulated silent information regulator 1 (SIRT1), a class III histone deacetylase (HDAC), resulting in the epigenetic transactivation of KSHV lytic genes. These results suggest that high blood glucose levels in diabetic patients contribute to the development of KS by promoting KSHV lytic replication and infection. IMPORTANCE Multiple epidemiological studies have reported a higher prevalence of classic KS in diabetic patients. By using both in vitro and in vivo models, we demonstrated an association between high glucose levels and KSHV lytic replication. High glucose levels induce oxidative stress and the production of H 2 O 2 , which mediates the reactivation of latent KSHV through multiple mechanisms. Our results provide the first experimental evidence and mechanistic support for the association of classic KS with diabetes.

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Signaling Cascades Triggered by Bacterial Metabolic End Products during Reactivation of Kaposi's Sarcoma-Associated Herpesvirus

Journal of Virology ◽

10.1128/jvi.02504-06 ◽

2007 ◽

Vol 81 (11) ◽

pp. 6032-6042 ◽

Cited By ~ 22

Author(s):

T. L. Morris ◽

R. R. Arnold ◽

J. Webster-Cyriaque

Keyword(s):

Protein Kinase ◽

Kaposi's Sarcoma ◽

Mapk Pathway ◽

Kaposi’S Sarcoma ◽

Lytic Replication ◽

Viral Reactivation ◽

Mitogen Activated Protein Kinase ◽

End Products ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT The present studies explore the role of polymicrobial infection in the reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) and analyze signaling pathways activated upon this induction. We hypothesized that activation of the cellular stress-activated mitogen-activated protein kinase (MAPK) p38 pathway would play a key role in the bacterium-mediated disruption of viral latency similar to that of previously reported results obtained with other inducers of gammaherpesvirus lytic replication. KSHV within infected BCBL-1 cells was induced to replicate following exposure to metabolic end products from gram-negative or -positive bacteria that were then simultaneously exposed to specific inhibitors of signal transduction pathways. We have determined that bacterium-mediated induction of lytic KSHV infection is significantly reduced by the inhibition of the p38 MAPK pathway. In contrast, inhibition of the phosphatidylinositol 3-kinase pathway did not impair induction of lytic replication or p38 phosphorylation. Protein kinase C, though activated, was not the major pathway used for bacterium-induced viral reactivation. Furthermore, hyperacetylation of histones 3 and 4 was detected. Collectively, our results show that metabolic end products from these pathogens induce lytic replication of KSHV in BCBL-1 cells primarily via the activation of a stress-activated MAPK pathway. Importantly, we demonstrate for the first time a mechanism by which polymicrobial bacterial infections result in KSHV reactivation and pathogenesis.

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Screening of the Human Kinome Identifies MSK1/2-CREB1 as an Essential Pathway Mediating Kaposi's Sarcoma-Associated Herpesvirus Lytic Replication during Primary Infection

Journal of Virology ◽

10.1128/jvi.01098-15 ◽

2015 ◽

Vol 89 (18) ◽

pp. 9262-9280 ◽

Cited By ~ 19

Author(s):

Fan Cheng ◽

Tanvee Vinod Sawant ◽

Ke Lan ◽

Chun Lu ◽

Jae U. Jung ◽

...

Keyword(s):

Endothelial Cells ◽

Protein Kinase ◽

Primary Infection ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Lytic Replication ◽

Kshv Infection ◽

Cellular Pathways ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACTViruses often hijack cellular pathways to facilitate infection and replication. Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic gammaherpesvirus etiologically associated with Kaposi's sarcoma, a vascular tumor of endothelial cells. Despite intensive studies, cellular pathways mediating KSHV infection and replication are still not well defined. Using an antibody array approach, we examined cellular proteins phosphorylated during primary KSHV infection of primary human umbilical vein endothelial cells. Enrichment analysis identified integrin/mitogen-activated protein kinase (integrin/MAPK), insulin/epidermal growth factor receptor (insulin/EGFR), and JAK/STAT as the activated networks during primary KSHV infection. The transcriptional factor CREB1 (cyclic AMP [cAMP]-responsive element-binding protein 1) had the strongest increase in phosphorylation. While knockdown of CREB1 had no effect on KSHV entry and trafficking, it drastically reduced the expression of lytic transcripts and proteins and the production of infectious virions. Chemical activation of CREB1 significantly enhanced viral lytic replication. In contrast, CREB1 neither influenced the expression of the latent gene LANA nor affected KSHV infectivity. Mechanistically, CREB1 was not activated through the classic cAMP/protein kinase A (cAMP/PKA) pathway or via the AKT, MK2, and RSK pathways. Rather, CREB1 was activated by the mitogen- and stress-activated protein kinases 1 and 2 (MSK1/2). Consequently, chemical inhibition or knockdown of MSKs significantly inhibited the KSHV lytic replication program; however, it had a minimal effect on LANA expression and KSHV infectivity. Together, these results identify the MSK1/2-CREB1 proteins as novel essential effectors of KSHV lytic replication during primary infection. The differential effect of the MSK1/2-CREB1 pathway on the expression of viral latent and lytic genes might control the robustness of viral lytic replication, and therefore the KSHV replication program, during primary infection.IMPORTANCEKaposi's sarcoma-associated herpesvirus (KSHV) is a human tumor virus associated with several cancers. Through genome-wide kinase screening, we found that KSHV activates the MSK1/2-CREB1 pathway during primary infection and that it depends on this pathway for viral lytic replication. Inhibition of this pathway blocks KSHV lytic replication. These results illustrate a mechanism by which KSHV hijacks a cellular pathway for its replication, and they identify a potential therapeutic target.

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Discovery of a Coregulatory Interaction between Kaposi's Sarcoma-Associated Herpesvirus ORF45 and the Viral Protein Kinase ORF36

Journal of Virology ◽

10.1128/jvi.00516-16 ◽

2016 ◽

Vol 90 (13) ◽

pp. 5953-5964 ◽

Cited By ~ 11

Author(s):

Denis Avey ◽

Sarah Tepper ◽

Benjamin Pifer ◽

Amritpal Bahga ◽

Hunter Williams ◽

...

Keyword(s):

Protein Kinase ◽

Viral Protein ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Lytic Replication ◽

Viral Protein Kinase ◽

Important Interaction ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACTKaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of three human malignancies. KSHV ORF36 encodes a serine/threonine viral protein kinase, which is conserved throughout all herpesviruses. Although several studies have identified the viral and cellular substrates of conserved herpesvirus protein kinases (CHPKs), the precise functions of KSHV ORF36 during lytic replication remain elusive. Here, we report that ORF36 interacts with another lytic protein, ORF45, in a manner dependent on ORF36 kinase activity. We mapped the regions of ORF36 and ORF45 involved in the binding. Their association appears to be mediated by electrostatic interactions, since deletion of either the highly basic N terminus of ORF36 or an acidic patch of ORF45 abolished the binding. In addition, the dephosphorylation of ORF45 protein dramatically reduced its association with ORF36. Importantly, ORF45 enhances both the stability and kinase activity of ORF36. Consistent with previous studies of CHPK homologs, we detected ORF36 protein in extracellular virions. To investigate the roles of ORF36 in the context of KSHV lytic replication, we used bacterial artificial chromosome mutagenesis to engineer both ORF36-null and kinase-dead mutants. We found that ORF36-null/mutant virions are moderately defective in viral particle production and are further deficient in primary infection. In summary, our results uncover a functionally important interaction between ORF36 and ORF45 and indicate a significant role of ORF36 in the production of infectious progeny virions.IMPORTANCEKaposi's sarcoma-associated herpesvirus (KSHV) is a human tumor virus with a significant public health burden. KSHV ORF36 encodes a serine/threonine viral protein kinase, whose functions throughout the viral life cycle have not been elucidated. Here, we report that ORF36 interacts with another KSHV protein, ORF45. We mapped the regions of ORF36 and ORF45 involved in their association and further characterized the consequences of this interaction. We engineered ORF36 mutant viruses in order to investigate the functional roles of ORF36 in the context of KSHV lytic replication, and we confirmed that ORF36 is a component of KSHV virions. Moreover, we found that ORF36 mutants are defective in virion production and primary infection. In summary, we discovered and characterized a functionally important interaction between KSHV ORF36 and ORF45, and our results suggest a significant role of ORF36 in the production of infectious progeny virions, a process critical for KSHV pathogenesis.

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A Kaposi's Sarcoma-Associated Herpesvirus Protein That Forms Inhibitory Complexes with Type I Interferon Receptor Subunits, Jak and STAT Proteins, and Blocks Interferon-Mediated Signal Transduction

Journal of Virology ◽

10.1128/jvi.02516-08 ◽

2009 ◽

Vol 83 (10) ◽

pp. 5056-5066 ◽

Cited By ~ 42

Author(s):

Sabine A. Bisson ◽

Anne-Laure Page ◽

Don Ganem

Keyword(s):

Kaposi's Sarcoma ◽

Human Tumor ◽

Kaposi’S Sarcoma ◽

Lytic Replication ◽

Type I Interferons ◽

Type I ◽

Type I Ifn ◽

Reading Frame ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Type I interferons (IFNs) are important mediators of innate antiviral defense and function by activating a signaling pathway through their cognate type I receptor (IFNAR). Here we report that lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV) efficiently blocks type I IFN signaling and that an important effector of this blockade is the viral protein RIF, the product of open reading frame 10. RIF blocks IFN signaling by formation of inhibitory complexes that contain IFNAR subunits, the Janus kinases Jak1 and Tyk2, and the STAT2 transcription factor. Activation of both Tyk2 and Jak1 is inhibited, and abnormal recruitment of STAT2 to IFNAR1 occurs despite the decrement in Tyk2 activity. As a result of these actions, phosphorylation of both STAT2 and STAT1 is impaired, with subsequent failure of ISGF3 accumulation in the nucleus. The presence in the viral genome of potent inhibitors of type I IFN signaling, along with several viral genes that block IFN induction, highlights the importance of the IFN pathway in the control of this human tumor virus infection.

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Kaposi's Sarcoma-Associated Herpesvirus ori-Lyt-Dependent DNA Replication: DualRole of Replication and Transcription Activator

Journal of Virology ◽

10.1128/jvi.00990-06 ◽

2006 ◽

Vol 80 (24) ◽

pp. 12171-12186 ◽

Cited By ~ 51

Author(s):

Yan Wang ◽

Qiyi Tang ◽

Gerd G. Maul ◽

Yan Yuan

Keyword(s):

Dna Replication ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Lytic Replication ◽

Transcription Activator ◽

Binding Motifs ◽

Viral Propagation ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus ◽

Lytic Dna Replication

ABSTRACT Lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV) is essential for viral propagation and pathogenicity. In Kaposi's sarcoma lesions, constant lytic replication plays a role in sustaining the population of latently infected cells that otherwise are quickly lost by segregation of latent viral episomes as spindle cells divide. Lytic DNA replication initiates from an origin (ori-Lyt) and requires trans-acting elements. Two functional ori-Lyts have been identified in the KSHV genome. Some cis-acting and trans-acting elements for ori-Lyt-dependent DNA replication have been found. Among these, K8 binding sites, a cluster of C/EBP binding motifs, and a replication and transcription activator (RTA) responsive element (RRE) are crucial cis-acting elements. Binding of K8 and RTA proteins to these motifs in ori-Lyt DNA was demonstrated to be absolutely essential for DNA replication. In the present study, functional roles of RTA in ori-Lyt-dependent DNA replication have been investigated. Two distinct functions of RTA were revealed. First, RTA activates an ori-Lyt promoter and initiates transcription across GC-rich tandem repeats. This RTA-mediated transcription is indispensable for DNA replication. Second, RTA is a component of the replication compartment, where RTA interacts with prereplication complexes composed of at least six core machinery proteins and K8. The prereplication complexes are recruited to ori-Lyt DNA through RTA, which interacts with the RRE, as well as K8, which binds to a cluster of C/EBP binding motifs with the aid of C/EBP α. The revelation of these two functions of RTA, together with its role in initiation of a transcriptional cascade that leads to transcription of all viral lytic genes, shows that RTA is a critical initiator and regulator of KSHV lytic DNA replication and viral propagation.

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Lytic Replication-Defective Kaposi's Sarcoma-Associated Herpesvirus: Potential Role in Infection and Malignant Transformation

Journal of Virology ◽

10.1128/jvi.78.20.11108-11120.2004 ◽

2004 ◽

Vol 78 (20) ◽

pp. 11108-11120 ◽

Cited By ~ 14

Author(s):

Jian-Hong Deng ◽

Yan-Jin Zhang ◽

Xin-Ping Wang ◽

Shou-Jiang Gao

Keyword(s):

Malignant Transformation ◽

Cell Lines ◽

Kaposi's Sarcoma ◽

Potential Role ◽

Primary Effusion Lymphoma ◽

Kaposi’S Sarcoma ◽

Lytic Replication ◽

Screening Methods ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Defective viruses often have pivotal roles in virus-induced diseases. Although Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL), defective KSHV has not been reported. Using differential genetic screening methods, we show that defective KSHV is present in KS tumors and PEL cell lines. To investigate the role of defective viruses in KSHV-induced pathogenesis, we isolated and characterized a lytic replication-defective KSHV, KV-1, containing an 82-kb genomic deletion of solely lytic genes. Cells harboring KV-1 escaped G0/G1 apoptosis induced by spontaneous lytic replication occurred in cells infected with regular KSHV but maintained efficient latent replication. Consequently, KV-1-infected cells had phenotypes of enhanced cell proliferation and transformation potentials. Importantly, KV-1 was packaged as infectious virions by using regular KSHV as helpers, and KV-1-like variants were detected in cultures of two of five KSHV cell lines and 1 of 18 KS tumors. These results point to a potential role for defective viruses in the regulation of KSHV infection and malignant transformation.

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Activation of the Unfolded Protein Response by 2-Deoxy-d-Glucose Inhibits Kaposi's Sarcoma-Associated Herpesvirus Replication and Gene Expression

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01126-12 ◽

2012 ◽

Vol 56 (11) ◽

pp. 5794-5803 ◽

Cited By ~ 27

Author(s):

Howard J. Leung ◽

Elda M. Duran ◽

Metin Kurtoglu ◽

Samita Andreansky ◽

Theodore J. Lampidis ◽

...

Keyword(s):

Protein Synthesis ◽

Er Stress ◽

Unfolded Protein Response ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Lytic Replication ◽

Unfolded Protein ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Protein Response ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACTLytic replication of the Kaposi's sarcoma-associated herpesvirus (KSHV) is essential for the maintenance of both the infected state and characteristic angiogenic phenotype of Kaposi's sarcoma and thus represents a desirable therapeutic target. During the peak of herpesvirus lytic replication, viral glycoproteins are mass produced in the endoplasmic reticulum (ER). Normally, this leads to ER stress which, through an unfolded protein response (UPR), triggers phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α), resulting in inhibition of protein synthesis to maintain ER and cellular homeostasis. However, in order to replicate, herpesviruses have acquired the ability to prevent eIF2α phosphorylation. Here we show that clinically achievable nontoxic doses of the glucose analog 2-deoxy-d-glucose (2-DG) stimulate ER stress, thereby shutting down eIF2α and inhibiting KSHV and murine herpesvirus 68 replication and KSHV reactivation from latency. Viral cascade genes that are involved in reactivation, including the master transactivator (RTA) gene, glycoprotein B, K8.1, and angiogenesis-regulating genes are markedly decreased with 2-DG treatment. Overall, our data suggest that activation of UPR by 2-DG elicits an early antiviral response via eIF2α inactivation, which impairs protein synthesis required to drive viral replication and oncogenesis. Thus, induction of ER stress by 2-DG provides a new antiherpesviral strategy that may be applicable to other viruses.

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Identification of the Nuclear Export and Adjacent Nuclear Localization Signals for ORF45 of Kaposi's Sarcoma-Associated Herpesvirus

Journal of Virology ◽

10.1128/jvi.02209-08 ◽

2008 ◽

Vol 83 (6) ◽

pp. 2531-2539 ◽

Cited By ~ 22

Author(s):

Xiaojuan Li ◽

Fanxiu Zhu

Keyword(s):

Subcellular Localization ◽

Nuclear Localization ◽

Nuclear Export ◽

Kaposi's Sarcoma ◽

Nuclear Export Signal ◽

Kaposi’S Sarcoma ◽

Lytic Replication ◽

Wild Type ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Open reading frame 45 (ORF45) of Kaposi's sarcoma-associated herpesvirus 8 (KSHV) is an immediate-early phosphorylated tegument protein and has been shown to play important roles at both early and late stages of viral infection. Homologues of ORF45 exist only in gammaherpesviruses, and their homology is limited. These homologues differ in their protein lengths and subcellular localizations. We and others have reported that KSHV ORF45 is localized predominantly in the cytoplasm, whereas its homologue in murine herpesvirus 68 is localized exclusively in the nucleus. We observed that ORF45s of rhesus rhadinovirus and herpesvirus saimiri are found exclusively in the nucleus. As a first step toward understanding the mechanism underlying the distinct intracellular distribution of KSHV ORF45, we identified the signals that control its subcellular localization. We found that KSHV ORF45 accumulated rapidly in the nucleus in the presence of leptomycin B, an inhibitor of CRM1 (exportin 1)-dependent nuclear export, suggesting that it could shuttle between the nucleus and cytoplasm. Mutational analysis revealed that KSHV ORF45 contains a CRM1-dependent, leucine-rich-like nuclear export signal and an adjacent nuclear localization signal. Replacement of the key residues with alanines in these motifs of ORF45 disrupts its shuttling between the cytoplasm and nucleus. The resulting ORF45 mutants have restricted subcellular localizations, being found exclusively either in the cytoplasm or in the nucleus. Recombinant viruses were reconstituted by introduction of these mutations into KSHV bacterial artificial chromosome BAC36. The resultant viruses have distinct phenotypes. A mutant virus in which ORF45 is restricted to the cytoplasm behaves as an ORF45-null mutant and produces 5- to 10-fold fewer progeny viruses than the wild type. In contrast, mutants in which the ORF45 protein is mostly restricted to the nucleus produce numbers of progeny viruses similar to those produced by the wild type. These data suggest that the subcellular localization signals of ORF45 have important functional roles in KSHV lytic replication.

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Activation of Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Lytic Replication by Human Cytomegalovirus

Journal of Virology ◽

10.1128/jvi.75.3.1378-1386.2001 ◽

2001 ◽

Vol 75 (3) ◽

pp. 1378-1386 ◽

Cited By ~ 134

Author(s):

Jeffrey Vieira ◽

Patricia O'Hearn ◽

Louise Kimball ◽

Bala Chandran ◽

Lawrence Corey

Keyword(s):

Human Cytomegalovirus ◽

Kaposi's Sarcoma ◽

Human Fibroblasts ◽

Human Herpesvirus ◽

Kaposi’S Sarcoma ◽

Lytic Replication ◽

Lytic Cycle ◽

Nuclear Antigen ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT The majority of Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells identified in vivo contain latent KSHV, with lytic replication in only a few percent of cells, as is the case for the cells of Kaposi's sarcoma (KS) lesions. Factors that influence KSHV latent or lytic replication are not well defined. Because persons with KS are often immunosuppressed and susceptible to many infectious agents, including human cytomegalovirus (HCMV), we have investigated the potential for HCMV to influence the replication of KSHV. Important to this work was the construction of a recombinant KSHV, rKSHV.152, expressing the green fluorescent protein (GFP) andneo (conferring resistance to G418). The expression of GFP was a marker of KSHV infection in cells of both epithelial and endothelial origin. The rKSHV.152 virus was used to establish cells, including human fibroblasts (HF), containing only latent KSHV, as demonstrated by latency-associated nuclear antigen expression and Gardella gel analysis. HCMV infection of KSHV latently infected HF activated KSHV lytic replication with the production of infectious KSHV. Dual-color immunofluorescence detected both the KSHV lytic open reading frame 59 protein and the HCMV glycoprotein B in coinfected cells, and UV-inactivated HCMV did not activate the production of infectious KSHV-GFP. In addition, HCMV coinfection increased the production of KSHV from endothelial cells and activated lytic cycle gene expression in keratinocytes. These data demonstrate that HCMV can activate KSHV lytic replication and suggest that HCMV could influence KSHV pathogenesis.

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