Serotype-dependent recombinant adeno-associated vector (AAV) infection of Epstein-Barr virus-positive B-cells, towards recombinant AAV-based therapy of focal EBV + lymphoproliferative disorders.

BACKGROUND: B-cell proliferative disorders, such as post-transplant lymphoproliferative disease (PTLD), are increased among persons afflicted by T-cell compromise. Most are Epstein-Barr virus (EBV) + and can first present with a focal lesion. Direct introduction of oncolytic viruses into localized tumors provides theoretical advantages over chemotherapy, immunotherapy and radiation therapy by reducing systemic toxicity. Despite extensive study as a vehicle for gene therapy, adeno-associated viruses (AAV) have rarely been applied to human cancer research due to technical and theoretical obstacles. Moreover, human B-cells have historically been described as resistant to AAV infection. Nonetheless, advances using different recombinant (r)AAV serotypes with unique tropisms to deliver cytotoxic therapy suggested a localized anti-tumor approach was feasible. METHODS: As a prelude to the development of a therapeutic vehicle, the ability of fifteen distinct EGFP-bearing rAAV serotypes to transduce human B-cells, including primary, immortalized, and B-cell tumor lines ± EBV was assessed by confocal microscopy, flow cytometry and subsequently cell viability assay. RESULTS: Rank order analysis revealed augmented transduction by rAAV6.2 and closely related virions. EBV infection of EBV-negative B-cell tumor lines and EBV immortalization of primary B-cells increased susceptibility to rAAV6.2 transduction. As a proof of concept, transduction by rAAV6.2 encoding herpes simplex virus type 1 (HSV1)-thymidine kinase (TK) eliminated TK-negative rhabdomyosarcoma cells and diminished viability of transduced B-cell lines upon incubation with ganciclovir. CONCLUSIONS: rAAV serotypes differentially transduce human B-cell lines reversing the dogma that human B-cells are refractory to AAV infection. EBV + B-cells display increased susceptibility to rAAV6.2 infection, uncovering a new method for improved nucleic acid transfer into transfection-resistant B-cell lines. The introduction of a functional suicide gene into the rAAV6.2 genome identifies a candidate vector for the development of rAAV-based oncolytic therapy targeting focal EBV-bearing B-lymphoproliferative disorders.

Analysis of host gene expression changes reveals distinct roles for the cytoplasmic domain of the Epstein-Barr virus receptor/CD21 in B-cell maturation, activation, and initiation of virus infection.

UNLABELLED: Epstein-Barr virus (EBV) attachment to human CD21 on the B-cell surface initiates infection. Whether CD21 is a simple tether or conveys vital information to the cell interior for production of host factors that promote infection of primary B cells is controversial, as the cytoplasmic fragment of CD21 is short, though highly conserved. The ubiquity of CD21 on normal B cells, the diversity of this population, and the well-known resistance of primary B cells to gene transfer technologies have all impeded resolution of this question. To uncover the role(s) of the CD21 cytoplasmic domain during infection initiation, the full-length receptor (CD21=CR), a mutant lacking the entire cytoplasmic tail (CT), and a control vector (NEO) were stably expressed in two pre-B-cell lines that lack endogenous receptor. Genome-wide transcriptional analysis demonstrated that stable CD21 surface expression alone (either CR or CT) produced multiple independent changes in gene expression, though both dramatically decreased class I melanoma-associated antigen (MAGE) family RNAs and upregulated genes associated with B-cell differentiation (e.g., C2TA, HLA-II, IL21R, MIC2, CD48, and PTPRCAP/CD45-associated protein). Temporal analysis spanning 72 h revealed that not only CR- but also CT-expressing lines initiated latency. In spite of this, the number and spectrum of transcripts altered in CR- compared with CT-bearing lines at 1 h after infection further diverged. Differential modulation of immediate early cellular transcripts (e.g., c-Jun and multiple histones), both novel and previously linked to CD21-initiated signaling, as well as distinct results from pathway analyses support a separate role for the cytoplasmic domain in initiation of intracellular signals. IMPORTANCE: Membrane proteins that mediate virus attachment tether virus particles to the cell surface, initiating infection. In addition, upon virus interaction such proteins may transmit signals to the interior of the cell that support subsequent steps in the infection process. Here we show that expression of the Epstein-Barr virus B-cell attachment receptor, CD21, in B cells that lack this receptor results in significant changes in gene expression, both before and rapidly following EBV-CD21 interaction. These changes translate into major signaling pathway alterations that are predicted to support stable infection.

A chimeric EBV gp350/220-based VLP replicates the virion B-cell attachment mechanism and elicits long-lasting neutralizing antibodies in mice.

Epstein-Barr virus (EBV), an oncogenic gammaherpesvirus, causes acute infectious mononucleosis (AIM) and is linked to the development of several human malignancies. There is an urgent need for a vaccine that is safe, prevents infection and/or limits disease. Unique among human herpesviruses, glycoprotein (gp)350/220, which initiates EBV attachment to susceptible host cells, is the major ligand on the EBV envelope and is highly conserved. Interaction between gp350/220 and complement receptor type 2 (CR2)/CD21 and/or (CR1)/CD35 on B-cells is required for infection. Potent antibody responses to gp350/220 occur in animal models and humans. Thus, gp350/220 provides an attractive candidate for prophylactic subunit vaccine development. However, in a recent Phase II clinical trial immunization with soluble recombinant gp350 reduced the incidence of AIM, but did not prevent infection. Despite various attempts to produce an EBV vaccine, no vaccine is licensed. Herein we describe a sub-unit vaccine against EBV based on a novel Newcastle disease virus (NDV)-virus-like particle (VLP) platform consisting of EBVgp350/220 ectodomain fused to NDV-fusion (F) protein. The chimeric protein EBVgp350/220-F is incorporated into the membrane of a VLP composed of the NDV matrix and nucleoprotein. The particles resemble native EBV in diameter and shape and bind CD21 and CD35. Immunization of BALB/c mice with EBVgp350/220-F VLPs elicited strong, long-lasting neutralizing antibody responses when assessed in vitro. This chimeric VLP is predicted to provide a superior safety profile as it is efficiently produced in Chinese hamster ovary (CHO) cells using a platform devoid of human nucleic acid and EBV-transforming genes.

Hsp90 inhibitor 17-DMAG decreases expression of conserved herpesvirus protein kinases and reduces virus production in Epstein-Barr virus-infected cells.

All eight human herpesviruses have a conserved herpesvirus protein kinase (CHPK) that is important for the lytic phase of the viral life cycle. In this study, we show that heat shock protein 90 (Hsp90) interacts directly with each of the eight CHPKs, and we demonstrate that an Hsp90 inhibitor drug, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), decreases expression of all eight CHPKs in transfected HeLa cells. 17-DMAG also decreases expression the of the endogenous Epstein-Barr virus protein kinase (EBV PK, encoded by the BGLF4 gene) in lytically infected EBV-positive cells and inhibits phosphorylation of several different known EBV PK target proteins. Furthermore, 17-DMAG treatment abrogates expression of the human cytomegalovirus (HCMV) kinase UL97 in HCMV-infected human fibroblasts. Importantly, 17-DMAG treatment decreased the EBV titer approximately 100-fold in lytically infected AGS-Akata cells without causing significant cellular toxicity during the same time frame. Increased EBV PK expression in 17-DMAG-treated AGS-Akata cells did not restore EBV titers, suggesting that 17-DMAG simultaneously targets multiple viral and/or cellular proteins required for efficient viral replication. These results suggest that Hsp90 inhibitors, including 17-DMAG, may be a promising group of drugs that could have profound antiviral effects on herpesviruses.

SIRPα/CD172a and FHOD1 are unique markers of littoral cells, a recently evolved major cell population of red pulp of human spleen.

Asplenic individuals are compromised not only in their ability to destroy infectious agents, but are at increased risk for death from autoimmune disease, certain tumors, and ischemic heart disease. Enhanced mortality is attributed to lack of phagocytes sequestered in spleen that efficiently engulf and destroy appropriate targets, although related cells are found elsewhere. To determine whether a unique population regulates RBC-pathogen clearance and filtration of altered self, we reviewed the anatomic literature and analyzed in situ by immunohistochemistry and immunofluorescence the expression patterns of a little-characterized cell that dominates the splenic red pulp of humans and closely related primates: the venous sinus-lining or littoral cell (LC). High expression of the formin homology domain protein 1 outlines the LC population. Although LCs are endothelial-like in distribution, they express several macrophage-directed proteins, the RBC Duffy Ag receptor for chemokines and T cell coreceptor CD8α/α, yet they lack lineage-associated markers CD34 and CD45. Strikingly, SIRPα (CD172a) expression in human spleen concentrates on LCs, consistent with recent demonstration of a key role in RBC turnover and elimination versus release of infected or altered self. Our results indicate human LCs (SIRPα(+), formin homology domain protein 1(+), CD8α/α(+), CD34(-), CD45(-)) comprise a highly plastic barrier cell population that emerged late in primate evolution coordinate with CD8 expression. Unique to Hominidae, LCs may be the ultimate determinant of which cells recirculate after passage through human spleen.

Lymphatic reprogramming of blood vascular endothelium by Kaposi sarcoma-associated herpesvirus.

Kaposi sarcoma is considered a neoplasm of lymphatic endothelium infected with Kaposi sarcoma-associated herpesvirus. It is characterized by the expression of lymphatic lineage-specific genes by Kaposi sarcoma tumor cells. Here we show that infection of differentiated blood vascular endothelial cells with Kaposi sarcoma-associated herpesvirus leads to their lymphatic reprogramming; induction of approximately 70% of the main lymphatic lineage-specific genes, including PROX1, a master regulator of lymphatic development; and downregulation of blood vascular genes.

The association of infectious mononucleosis and invasive breast cancer in The Health of Women (HOW) Study®.

BACKGROUND: The link between Epstein-Barr Virus (EBV) and breast cancer (BC) etiology remains unclear. We utilized the Health of Women (HOW) Study® to understand the association between infectious mononucleosis (IM), a surrogate for EBV infection, and invasive BC. METHODS: The HOW Study® was a web-based survey of BC risk factors with > 40, 000 participants; 183 had IM at < 10 years old, 3, 654 had IM between 10 and 22 years old, 764 had IM at > 22 years old, and 17, 026 never developed IM. Of these 21, 627 women, 2093 had Stages I-III BC and 14, 143 were cancer-free. Binary logistic regression ascertained the association between IM and invasive BC risk by controlling for confounders. RESULTS: A history of IM was associated with a lower likelihood of developing invasive BC compared to women who did not develop IM (adjusted OR = 0.83, 95% CI 0.72-0.94). That finding was driven by women who had IM between 10 and 22 years old (adjusted OR = 0.83, 95% CI 0.72-0.97) albeit no linear association between age at developing IM and breast cancer (p-trend > 0.05). Women who had IM between 10 and 22 years old were less likely to develop estrogen receptor positive (ER+ ; adjusted OR = 0.84, 95% CI 0.71-0.99) or hormone receptor positive (HR+ ; adjusted OR = 0.86, 95% CI 0.73-1.01) BC. There was no association between IM and ER- or HR- BC. CONCLUSION: In the HOW Study®, women diagnosed with IM between the ages of 10 and 22 had a lower risk of developing invasive BC compared to women who never developed IM.

The Epstein-Barr virus (EBV)-encoded protein kinase, EBV-PK, but not the thymidine kinase (EBV-TK), is required for ganciclovir and acyclovir inhibition of lytic viral production.

Ganciclovir (GCV) and acyclovir (ACV) are guanine nucleoside analogues that inhibit lytic herpesvirus replication. GCV and ACV must be monophosphorylated by virally encoded enzymes to be converted into nucleotides and incorporated into viral DNA. However, whether GCV and/or ACV phosphorylation in Epstein-Barr virus (EBV)-infected cells is mediated primarily by the EBV-encoded protein kinase (EBV-PK), the EBV-encoded thymidine kinase (EBV-TK), or both is controversial. To examine this question, we constructed EBV mutants containing stop codons in either the EBV-PK or EBV-TK open reading frame and selected for stable 293T clones latently infected with wild-type EBV or each of the mutant viruses. Cells were induced to the lytic form of viral replication with a BZLF1 expression vector in the presence and absence of various doses of GCV and ACV, and infectious viral titers were determined by a green Raji cell assay. As expected, virus production in wild-type EBV-infected 293T cells was inhibited by both GCV (50% inhibitory concentration [IC(50)] = 1.5 microM) and ACV (IC(50) = 4.1 microM). However, the EBV-PK mutant (which replicates as well as the wild-type (WT) virus in 293T cells) was resistant to both GCV (IC(50) = 19.6 microM) and ACV (IC(50) = 36.4 microM). Expression of the EBV-PK protein in trans restored GCV and ACV sensitivity in cells infected with the PK mutant virus. In contrast, in 293T cells infected with the TK mutant virus, viral replication remained sensitive to both GCV (IC(50) = 1.2 microM) and ACV (IC(50) = 2.8 microM), although susceptibility to the thymine nucleoside analogue, bromodeoxyuridine, was reduced. Thus, EBV-PK but not EBV-TK mediates ACV and GCV susceptibilities.

Comparative binding of soluble fragments (derCD23, sCD23, and exCD23) of recombinant human CD23 to CD21 (SCR 1-2) and native IgE, and their effect on IgE regulation.

IgE, responsible for type I hypersensitivities, is regulated by interactions between its receptor, CD23, and co-receptor CD21. To examine comparative binding of recombinant human CD21 SCR 1-2 and native human IgE to CD23 plus the effect of CD23 on IgE production, we engineered recombinant soluble human CD23 fragments; (1) derCD23, (2) sCD23 and (3) exCD23, formed in vivo by proteolysis. SPR analysis revealed a progressive increment in affinity of soluble fragments for IgE, upon increasing length of CD23 "stalk" domain, exCD23>sCD23>derCD23. Soluble CD23 fragments and their oligomeric state are shown to fine-tune the immune response. Oligomers appear more important in enhancing IgE synthesis and monomers lacking the tail residues fail to bind CD21 yet bind membrane IgE and down-regulate IgE synthesis. Co-ligation of membrane IgE and CD21 through soluble CD23 monomers is disturbed. This study supports anti-allergic therapies involving stabilizing membrane CD23, or preventing shedding of soluble CD23.

Complete and Durable Responses in Primary Central Nervous System Posttransplant Lymphoproliferative Disorder with Zidovudine, Ganciclovir, Rituximab, and Dexamethasone.

Purpose: Primary central nervous system posttransplant lymphoproliferative disorder (PCNS-PTLD) is a complication of solid organ transplantation with a poor prognosis and typically associated with Epstein-Barr virus (EBV). We hypothesized EBV lytic-phase protein expression would allow successful treatment with antiviral therapy.Patients and Methods: Thirteen patients were treated with zidovudine (AZT), ganciclovir (GCV), dexamethasone, and rituximab in EBV+ PCNS-PTLD. Twice-daily, intravenous AZT 1,500 mg, GCV 5 mg/kg, and dexamethasone 10 mg were given for 14 days. Weekly rituximab 375 mg/m2 was delivered for the first 4 weeks. Twice-daily valganciclovir 450 mg and AZT 300 mg started day 15. Lytic and latent protein expression was assessed using in situ hybridization and immunohistochemistry. Immunoblot assay assessed lytic gene activation. Cells transfected with lytic kinase vectors were assessed for sensitivity to our therapy using MTS tetrazolium and flow cytometry.Results: The median time to response was 2 months. Median therapy duration was 26.5 months. Median follow-up was 52 months. The estimated 2-year overall survival (OS) was 76.9% (95% CI, 44.2%-91.9%). Overall response rate (ORR) was 92% (95% CI, 64%-100%). BXLF1/vTK and BGLF4 expression was found in the seven tumor biopsies evaluated. Lytic gene expression was induced in vitro using the four-drug regimen. Transfection with viral kinase cDNA increased cellular sensitivity to antiviral therapy.Conclusions: EBV+ PCNS-PTLD expressed lytic kinases and therapy with AZT, GCV, rituximab and dexamethasone provided durable responses. Induction of the lytic protein expression and increased cellular sensitivity to antiviral therapy after transfection with viral kinase cDNA provides a mechanistic rationale for our approach. Clin Cancer Res; 24(14); 3273-81. ©2018 AACR.

A systematic review and meta-analysis on herpes zoster and the risk of cardiac and cerebrovascular events.

BACKGROUND: Patients who develop herpes zoster or herpes zoster ophthalmicus may be at risk for cerebrovascular and cardiac complications. We systematically reviewed the published literature to determine the association between herpes zoster and its subtypes with the occurrence of cerebrovascular and cardiac events. METHODS/RESULTS: Systematic searches of PubMed (MEDLINE), SCOPUS (Embase) and Google Scholar were performed in December 2016. Eligible studies were cohort, case-control, and self-controlled case-series examining the association between herpes zoster or subtypes of herpes zoster with the occurrence of cerebrovascular and cardiac events including stroke, transient ischemic attack, coronary heart disease, and myocardial infarction. Data on the occurrence of the examined events were abstracted. Odds ratios and their accompanying confidence intervals were estimated using random and fixed effects models with statistical heterogeneity estimated with the I2 statistic. Twelve studies examining 7.9 million patients up to 28 years after the onset of herpes zoster met our pre-defined eligibility criteria. Random and fixed effects meta-analyses showed that herpes zoster, type unspecified, and herpes zoster ophthalmicus were associated with a significantly increased risk of cerebrovascular events, without any evidence of statistical heterogeneity. Our meta-analysis also found a significantly increased risk of cardiac events associated with herpes zoster, type unspecified. CONCLUSIONS: Our results are consistent with the accumulating body of evidence that herpes zoster and herpes zoster ophthalmicus are significantly associated with cerebrovascular and cardiovascular events.

Epstein-Barr Virus Infection of Mammary Epithelial Cells Promotes Malignant Transformation.

Whether the human tumor virus, Epstein-Barr Virus (EBV), promotes breast cancer remains controversial and a potential mechanism has remained elusive. Here we show that EBV can infect primary mammary epithelial cells (MECs) that express the receptor CD21. EBV infection leads to the expansion of early MEC progenitor cells with a stem cell phenotype, activates MET signaling and enforces a differentiation block. When MECs were implanted as xenografts, EBV infection cooperated with activated Ras and accelerated the formation of breast cancer. Infection in EBV-related tumors was of a latency type II pattern, similar to nasopharyngeal carcinoma (NPC). A human gene expression signature for MECs infected with EBV, termed EBVness, was associated with high grade, estrogen-receptor-negative status, p53 mutation and poor survival. In 11/33 EBVness-positive tumors, EBV-DNA was detected by fluorescent in situ hybridization for the viral LMP1 and BXLF2 genes. In an analysis of the TCGA breast cancer data EBVness correlated with the presence of the APOBEC mutational signature. We conclude that a contribution of EBV to breast cancer etiology is plausible, through a mechanism in which EBV infection predisposes mammary epithelial cells to malignant transformation, but is no longer required once malignant transformation has occurred.

Inherited CHST11/MIR3922 deletion is associated with a novel recessive syndrome presenting with skeletal malformation and malignant lymphoproliferative disease.

Glycosaminoglycans (GAGs) such as chondroitin are ubiquitous disaccharide carbohydrate chains that contribute to the formation and function of proteoglycans at the cell membrane and in the extracellular matrix. Although GAG-modifying enzymes are required for diverse cellular functions, the role of these proteins in human development and disease is less well understood. Here, we describe two sisters out of seven siblings affected by congenital limb malformation and malignant lymphoproliferative disease. Using Whole-Genome Sequencing (WGS), we identified in the proband deletion of a 55 kb region within chromosome 12q23 that encompasses part of CHST11 (encoding chondroitin-4-sulfotransferase 1) and an embedded microRNA (MIR3922). The deletion was homozygous in the proband but not in each of three unaffected siblings. Genotyping data from the 1000 Genomes Project suggest that deletions inclusive of both CHST11 and MIR3922 are rare events. Given that CHST11 deficiency causes severe chondrodysplasia in mice that is similar to human limb malformation, these results underscore the importance of chondroitin modification in normal skeletal development. Our findings also potentially reveal an unexpected role for CHST11 and/or MIR3922 as tumor suppressors whose disruption may contribute to malignant lymphoproliferative disease.

Human complement receptor type 1/CD35 is an Epstein-Barr Virus receptor.

Epstein-Barr virus (EBV) attachment to primary B cells initiates virus entry. Although CD21 is the only known receptor for EBVgp350/220, a recent report documents EBV-infected B cells from a patient genetically deficient in CD21. On normal resting B cells, CD21 forms two membrane complexes: one with CD19 and another with CD35. Whereas the CD21/CD19 complex is widely retained on immortalized and B cell tumor lines, the related complement-regulatory protein CD35 is lost. To determine the role(s) of CD35 in initial infection, we transduced a CD21-negative pre-B cell and myeloid leukemia line with CD35, CD21, or both. Cells expressing CD35 alone bound gp350/220 and became latently infected when the fusion receptor HLA II was coexpressed. Temporal, biophysical, and structural characteristics of CD35-mediated infection were distinct from CD21. Identification of CD35 as an EBV receptor uncovers a salient role in primary infection, addresses unsettled questions of virus tropism, and underscores the importance of EBVgp350/220 for vaccine development.

Epstein-Barr virus thymidine kinase is a centrosomal resident precisely localized to the periphery of centrioles.

The thymidine kinase (TK) encoded by Epstein-Barr virus (EBV) differs not only from that of the alphaherpesviruses but also from that of the gamma-2 herpesvirus subfamily. Because cellular location is frequently a determinant of regulatory function, to gain insight into additional role(s) of EBV TK and to uncover how the lymphocryptovirus and rhadinovirus enzymes differ, the subcellular localizations of EBV TK and the related cercopithecine herpesvirus-15 TK were investigated. We show that in contrast to those of the other family members, the gamma-1 herpesvirus TKs localize to the centrosome and even more precisely to the periphery of the centriole, tightly encircling the tubulin-rich centrioles in a microtubule-independent fashion. Centrosomal localization is observed in diverse cell types and occurs whether the protein is expressed independently or in the context of lytic EBV infection. Surprisingly, analysis of mutants revealed that the unique N-terminal domain was not critical for targeting to the centrosome, but rather, peptide sequences located C terminal to this domain were key. This is the first herpesvirus protein documented to reside in the centrosome, or microtubule-organizing center, an amembranous organelle that regulates the structural biology of the cell cycle through control of chromosome separation and cytokinesis. More recently, proteasome-mediated degradation of cell cycle regulatory proteins, production and loading of antigenic peptides onto HLA molecules, and transient homing of diverse virion proteins required for entry and/or egress have been shown to be coordinated at the centrosome. Potential implications of centrosomal localization for EBV TK function are discussed.

A phase 1/2 trial of arginine butyrate and ganciclovir in patients with Epstein-Barr virus-associated lymphoid malignancies.

Malignancies associated with latent Epstein-Barr virus (EBV) are resistant to nucleoside-type antiviral agents because the viral enzyme target of these antiviral drugs, thymidine kinase (TK), is not expressed. Short-chain fatty acids, such as butyrate, induce EBV-TK expression in latently infected B cells. As butyrate has been shown to sensitize EBV(+) lymphoma cells in vitro to apoptosis induced by ganciclovir, arginine butyrate in combination with ganciclovir was administered in 15 patients with refractory EBV(+) lymphoid malignancies to evaluate the drug combination for toxicity, pharmacokinetics, and clinical responses. Ganciclovir was administered twice daily at standard doses, and arginine butyrate was administered by continuous infusion in an intrapatient dose escalation, from 500 mg/(kg/day) escalating to 2000 mg/(kg/day), as tolerated, for a 21-day cycle. The MTD for arginine butyrate in combination with ganciclovir was established as 1000 mg/(kg/day). Ten of 15 patients showed significant antitumor responses, with 4 CRs and 6 PRs within one treatment cycle. Complications from rapid tumor lysis occurred in 3 patients. Reversible somnolence or stupor occurred in 3 patients at arginine butyrate doses of greater than 1000 mg/(kg/day). The combination of arginine butyrate and ganciclovir was reasonably well-tolerated and appears to have significant biologic activity in vivo in EBV(+) lymphoid malignancies which are refractory to other regimens.

Functional divergence of Kaposi's sarcoma-associated herpesvirus and related gamma-2 herpesvirus thymidine kinases: novel cytoplasmic phosphoproteins that alter cellular morphology and disrupt adhesion.

The nucleoside kinase encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) is a relatively inefficient enzyme with substrate specificity for thymidine alone, unlike alphaherpesvirus thymidine kinases (TKs). Similar to all gammaherpesvirus TKs, KSHV TK is composed of two distinct domains, a conserved C-terminal kinase and a novel and uncharacterized N terminus. Ectopic expression of KSHV TK in adherent cells induced striking morphological changes and anchorage independence although cells survived, a property shared with the related rhadinovirus TKs of rhesus monkey rhadinovirus and herpesvirus saimiri. To determine whether KSHV TK served alternate functions relevant to the rhadinovirus life cycle and to reveal the contribution of the N terminus, an enhanced green fluorescent protein-tagged fusion protein and serial mutants were generated for investigation of intracellular localization and cell biology. Analysis of truncation mutants showed that a proline-rich region located within the N terminus cooperated with the conserved C-terminal kinase to tether KSHV TK to a reticular network in the cytoplasm and to induce morphological change. Fusion of the KSHV N terminus to herpes simplex virus type 1 TK, a nucleus-localized enzyme, similarly resulted in cytoplasmic redistribution of the chimeric protein but did not alter cell shape or adhesion. Unlike other human herpesvirus TKs, KSHV TKs and related rhadinovirus TKs are constitutively tyrosine phosphorylated; a KSHV TK mutant that was hypophosphorylated failed to detach and grow in suspension. Loss of adhesion may enhance terminal differentiation, viral replication, and egress at the cellular level and at the organism level may facilitate detachment and distant migration of KSHV-replicating cells within body fluids--promoting oropharyngeal transmission and perhaps contributing to the multifocal lesions that characterize KS.

Kaposi's sarcoma-associated herpesvirus activation of vascular endothelial growth factor receptor 3 alters endothelial function and enhances infection.

Kaposi's sarcoma-associated herpesvirus (KSHV; also known as human herpesvirus 8) is the etiologic agent of Kaposi's sarcoma, an endothelial neoplasm. This gamma-herpesvirus encodes for several unique proteins that alter target cell function, including the virion envelope-associated glycoprotein B (gB). Glycoprotein B has an RGD (Arg-Gly-Asp) motif at the extracellular amino terminus region and binds to the alpha3beta1 surface integrin, which enhances virus entry. We now report that gB can activate the vascular endothelial growth factor receptor 3 (VEGFR-3) on the surface of microvascular endothelial cells and trigger receptor signaling, which can modulate endothelial migration and proliferation. Furthermore, we observed that VEGFR-3 expression and activation enhance KSHV infection and participate in KSHV-mediated transformation. These functional changes in the endothelium may contribute to the pathogenesis of Kaposi's sarcoma and suggest that interventions that inhibit gB activation of VEGFR-3 could be useful in the treatment of this neoplasm.

Characterization of Kaposi sarcoma-associated herpesvirus/human herpesvirus-8 infection of human vascular endothelial cells: early events.

Kaposi sarcoma-associated herpesvirus (KSHV)/human herpesvirus-8 (HHV-8) is causally associated with Kaposi sarcoma (KS). The absence of a cell culture system that effectively reproduces the composite mechanisms governing initiation and maintenance of HHV-8 infection (lytic and latent) in KS endothelial cells, however, has left important questions unanswered. Here, we report a culture system in which the earliest events that accompany HHV-8 infection could be surveyed in primary endothelial cells. Binding of HHV-8 to microvascular dermal endothelial cells (MVDECs) was directly compared with other primary target cells implicated in HHV-8-associated diseases. Virus attachment, fusion, internalization and transport within MVDECs was monitored by electron microscopy. Studies of genome configuration revealed that rapid circularization of the viral DNA occurred on entry, though by 72 hours after infection linear DNAs accumulated and early as well as late lytic RNAs (T1.1, K8.1) could be detected. The latency transcripts (LT1/LT2) were first detected on day 8, demonstrating that both lytic and latent infection were initiated. Although most lytic transcripts accrued until passage, open-reading frame-74 RNAs fluctuated with a fixed periodicity, suggesting that early replication after infection of MVDECs was synchronous.

Methylation of the EBV genome and establishment of restricted latency in low-passage EBV-infected 293 epithelial cells.

Epstein-Barr virus (EBV) encodes multiple latency programs: a growth-transforming program (type III) latency program and restricted-latency (types I and II) programs. During type III latency, EBV expresses six nuclear antigens, all of which are encoded by a single complex transcriptional unit driven by two linked promoters, Cp and Wp, while restricted viral latency is characterized by the expression of a single nuclear antigen, EBNA1, whose expression is driven from a distinct transcription unit under the control of the Qp promoter. EBV infection of the 293 epithelial cell line frequently leads to the establishment of a type I/II latent infection. Here we report that during the initial stages of virus infection of the 293 cell line, both Cp and Wp are active. However, analysis of four established, low-passage EBV-infected 293 cell lines revealed that three of these exhibited Qp-driven transcription of the EBNA 1 gene and little or no detectable Cp and Wp activity, while the fourth cell line exhibited Cp activity. Notably, all four cell lines contained the necessary transcription factors to drive transcription initiation from Cp and Wp when transiently transfected with unmethylated reporter constructs. Furthermore, in the cell lines exhibiting restricted EBV latency the viral genomes were extensively methylated around Cp and Wp, but not Qp. In contrast, in the cell line exhibiting Cp activity the viral genomes were hypomethylated around Cp, Wp, and Qp. Taken together, these results provide evidence that the establishment of a restricted latent infection in the 293 epithelial cell line is not due to a failure to initiate the growth-transforming (type III) latency program, but rather may arise from a selection against the type III latency program. Furthermore, these results are consistent with the hypothesis that methylation of Cp and Wp is required for entry into the type I or II latency programs.