Fu-Zhang Wang

Integrin α3β1 (CD 49c/29) Is a Cellular Receptor for Kaposi's Sarcoma-Associated Herpesvirus (KSHV/HHV-8) Entry into the Target Cells

Abstract Human herpesvirus-8 (HHV-8) is implicated in the pathogenesis of Kaposis sarcoma. HHV-8 envelope glycoprotein B possesses the RGD motif known to interact with integrin molecules, and HHV-8 infectivity was inhibited by RGD peptides, antibodies against RGD-dependent α3 and β1 integrins, and by soluble α3β1 integrin. Expression of human α3 integrin increased the infectivity of virus for Chinese hamster ovary cells. Anti-gB antibodies immunoprecipitated the virus-α3 and -β1 complexes, and virus binding studies suggest a role for α3β1 in HHV-8 entry. Further, HHV-8 infection induced the integrin-mediated activation of focal adhesion kinase (FAK). These findings implicate a role for α3β1 integrin and the associated signaling pathways in HHV-8 entry into the target cells.

Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Infection of Human Fibroblast Cells Occurs through Endocytosis

ABSTRACT Kaposis sarcoma (KS)-associated herpesvirus or human herpesvirus 8 (HHV-8) DNA and transcripts have been detected in the B cells, macrophages, keratinocytes, and endothelial and epithelial cells of KS patients. In vitro, HHV-8 infects human B, endothelial, epithelial, and fibroblast cells, as well as animal cells, and the infection is characterized by (i) absence of lytic replication by the input virus and (ii) latent infection. For its initial binding to target cells, HHV-8 uses ubiquitous heparan sulfate molecules via its envelope-associated glycoproteins gB and gpK8.1A. HHV-8 also interacts with the α3β1 integrin via its glycoprotein gB, and virus binding studies suggest that α3β1 is one of the HHV-8 entry receptors (S. M. Akula, N. P. Pramod, F. Z. Wang, and B. Chandran, Cell 108:407-419, 2002). In this study, morphological and biochemical techniques were used to examine the entry of HHV-8 into human foreskin fibroblasts (HFF). HHV-8 was detected in coated vesicles and in large, smooth-surfaced endocytic vesicles. Fusion of viral envelope with the vesicle wall was also observed. In immune electron microscopy, anti-HHV-8 gB antibodies colocalized with virus-containing endocytic vesicles. In fluorescence microscopic analyses, transferrin was colocalized with HHV-8. HHV-8 infection was significantly inhibited by preincubation of cells with chlorpromazine HCl, which blocks endocytosis via clathrin-coated pits, but not by nystatin and cholera toxin B, which blocks endocytosis via caveolae and induces the dissociation of lipid rafts, respectively. Infection was also inhibited by blocking the acidification of endosomes by NH4Cl and bafilomycin A. Inhibition of HHV-8 open reading frame 73 gene expression by chlorpromazine HCl, bafilomycin A, and NH4Cl demonstrated that the virions in the vesicles could proceed to cause an infection. Taken together, these findings suggest that for its infectious entry into HFF, HHV-8 uses clathrin-mediated endocytosis and a low-pH intracellular environment.

Human cytomegalovirus infection alters the expression of cellular MicroRNA species that affect its replication

ABSTRACT The human genome encodes over 500 microRNAs (miRNAs), small RNAs (19 to 26 nucleotides [nt]) that regulate the expressions of diverse cellular genes. Many cellular processes are altered through a variety of mechanisms by human cytomegalovirus (HCMV) infection. We asked whether HCMV infection leads to changes in the expression of cellular miRNAs and whether HCMV-regulated miRNAs are important for HCMV replication. Levels of most miRNAs did not change markedly during infection, but some were positively or negatively regulated. Patterns of miRNA expression were linked to the time course of infection. Some similarly reregulated miRNAs share identical or similar seed sequences, suggesting coordinated regulation of miRNA species that have shared targets. miRNAs miR-100 and miR-101 were chosen for further analyses based on their reproducible changes in expression after infection and on the basis of having predicted targets in the 3′ untranslated regions (3′-UTR) of genes encoding components of the mammalian target of rapamycin (mTOR) pathway, which is important during HCMV infection. Reporter genes that contain the 3′-UTR of mTOR (predicted targets for miR-100 and miR-101) or raptor (a component of the mTOR pathway; predicted site for miR-100) were constructed. Mimics of miR-100 and miR-101 inhibited expression from the mTOR construct, while only miR-100 inhibited the raptor construct. Together, miR-100 and miR-101 reduced mTOR protein levels. While the miR-100 and miR-101 mimics individually modestly inhibited production of infectious progeny, much greater inhibition was achieved with a combination of both (33-fold). Our key finding is that HCMV selectively manipulates the expression of some cellular miRNAs to help its own replication.

Human Herpesvirus 8 Envelope Glycoprotein K8.1A Interaction with the Target Cells Involves Heparan Sulfate

ABSTRACT Human herpesvirus-8 (HHV-8) or Kaposis sarcoma-associated herpesvirus K8.1 gene encodes for two immunogenic glycoproteins, gpK8.1A and gpK8.1B, originating from spliced messages. The 228-amino-acid (aa) gpK8.1A is the predominant form associated with the virion envelope, consisting of a 167-aa region identical to gpK8.1B and a 61-aa unique region (L. Zhu, V. Puri, and B. Chandran, Virology 262:237–249, 1999). HHV-8 has a broad in vivo and in vitro cellular tropism, and our studies showed that this may be in part due to HHV-8s interaction with the ubiquitous host cell surface molecule, heparan sulfate (HS). Since HHV-8 K8.1 gene is positionally colinear to the Epstein-Barr virus (EBV) gene encoding the gp350/gp220 protein involved in EBV binding to the target cells, gpK8.1As ability to interact with the target cells was examined. The gpK8.1A without the transmembrane and carboxyl domains (ΔTMgpK8.1A) was expressed in a baculovirus system and purified. Radiolabeled purified ΔTMgpK8.1A protein bound to the target cells, which was blocked by unlabeled ΔTMgpK8.1A. Unlabeled ΔTMgpK8.1A blocked the binding of [3H]thymidine-labeled purified HHV-8 to the target cells. Binding of radiolabeled ΔTMgpK8.1A to the target cells was inhibited in a dose-dependent manner by soluble heparin, a glycosaminoglycan (GAG) closely related to HS, but not by other GAGs such as chondroitin sulfate A and C, N-acetyl heparin and de-N-sulfated heparin. Cell surface absorbed ΔTMgpK8.1A was displaced by soluble heparin. Radiolabeled ΔTMgpK8.1A also bound to HS expressing Chinese hamster ovary (CHO-K1) cells, and binding to mutant CHO cell lines deficient in HS was significantly reduced. The ΔTMgpK8.1A specifically bound to heparin-agarose beads, which was inhibited by HS and heparin, but not by other GAGs. Virion envelope-associated gpK8.1A was specifically precipitated by heparin-agarose beads. These findings suggest that gpK8.1A interaction with target cells involves cell surface HS-like moieties, and HHV-8 interaction with HS could be in part mediated by virion envelope-associated gpK8.1A.

Human Herpesvirus 8 Envelope Glycoprotein B Mediates Cell Adhesion via Its RGD Sequence

ABSTRACT Human herpesvirus 8 (HHV-8) or Kaposis sarcoma-associated herpesvirus, implicated in the pathogenesis of Kaposis sarcoma, utilizes heparan sulfate-like molecules to bind the target cells via its envelope-associated glycoproteins gB and gpK8.1A. HHV-8-gB possesses the Arg-Gly-Asp (RGD) motif, the minimal peptide region of many proteins known to interact with subsets of host cell surface integrins. HHV-8 utilizes α3β1 integrin as one of the receptors for its entry into the target cells via its gB interaction and induces the activation of focal adhesion kinase (FAK) (S. M. Akula, N. P. Pramod, F.-Z. Wang, and B. Chandran, Cell 108:407-419, 2002). Since FAK activation is the first step in the outside-in signaling necessary for integrin-mediated cytoskeletal rearrangements, cell adhesions, motility, and proliferation, the ability of HHV-8-gB to mediate the target cell adhesion was examined. A truncated form of gB without the transmembrane and carboxyl domains (gBΔTM) and a gBΔTM mutant (gBΔTM-RGA) with a single amino acid mutation (RGD to RGA) were expressed in a baculovirus system and purified. Radiolabeled HHV-8-gBΔTM, gBΔTM-RGA, and ΔTMgpK8.1A proteins bound to the human foreskin fibroblasts (HFFs), human dermal microvascular endothelial (HMVEC-d) cells, human B (BJAB) cells, and Chinese hamster ovary (CHO-K1) cells with equal efficiency, which was blocked by preincubation of proteins with soluble heparin. Maxisorp plate-bound gBΔTM protein induced the adhesion of HFFs and HMVEC-d and monkey kidney epithelial (CV-1) cells in a dose-dependent manner. In contrast, the gBΔTM-RGA and ΔTMgpK8.1A proteins did not mediate adhesion. Adhesion mediated by gBΔTM was blocked by the preincubation of target cells with RGD-containing peptides or by the preincubation of plate-bound gBΔTM protein with rabbit antibodies against gB peptide containing the RGD sequence. In contrast, adhesion was not blocked by the preincubation of plate-bound gBΔTM protein with heparin, suggesting that the adhesion is mediated by the RGD amino acids of gB, which is independent of the heparin-binding domain of gB. Integrin-ligand interaction is dependent on divalent cations. Adhesion induced by the gBΔTM was blocked by EDTA, thus suggesting the role of integrins in the observed adhesions. Focal adhesion components such as FAK and paxillin were activated by the binding of gBΔTM protein to the target cells but not by gBΔTM-RGA protein binding. Inhibition of FAK phosphorylation by genistein blocked gBΔTM-induced FAK activation and cell adhesion. These findings suggest that HHV-8-gB could mediate cell adhesion via its RGD motif interaction with the cell surface integrin molecules and indicate the induction of cellular signaling pathways, which may play roles in the infection of target cells and in Kaposis sarcoma pathogenesis.

Kaposi's Sarcoma-Associated Herpesvirus Forms a Multimolecular Complex of Integrins (αVβ5, αVβ3, and α3β1) and CD98-xCT during Infection of Human Dermal Microvascular Endothelial Cells, and CD98-xCT Is Essential for the Postentry Stage of Infection

ABSTRACT Kaposis sarcoma-associated herpesvirus (KSHV) interacts with cell surface heparan sulfate (HS) and α3β1 integrin during the early stages of infection of human dermal microvascular endothelial cells (HMVEC-d) and human foreskin fibroblasts (HFF), and these interactions are followed by virus entry overlapping with the induction of preexisting host cell signal pathways. KSHV also utilizes the amino acid transporter protein xCT for infection of adherent cells, and the xCT molecule is part of the cell surface heterodimeric membrane glycoprotein CD98 (4F2 antigen) complex known to interact with α3β1 and αVβ3 integrins. KSHV gB mediates adhesion of HMVEC-d, CV-1, and HT-1080 cells and HFF via its RGD sequence. Anti-αV and -β1 integrin antibodies inhibited the cell adhesion mediated by KSHV-gB. Variable levels of neutralization of HMVEC-d and HFF infection were observed with antibodies against αVβ3 and αVβ5 integrins. Similarly, variable levels of inhibition of virus entry into adherent HMVEC-d, 293 and Vero cells, and HFF was observed by preincubating virus with soluble α3β1, αVβ3, and αVβ5 integrins, and cumulative inhibition was observed with a combination of integrins. We were unable to infect HT1080 cells. Virus binding and DNA internalization studies suggest that αVβ3 and αVβ5 integrins also play roles in KSHV entry. We observed time-dependent temporal KSHV interactions with HMVEC-d integrins and CD98/xCT with three different patterns of association and dissociation. Integrin αVβ5 interaction with CD98/xCT predominantly occurred by 1 min postinfection (p.i.) and dissociated at 10 min p.i., whereas α3β1-CD98/xCT interaction was maximal at 10 min p.i. and dissociated at 30 min p.i., and αVβ3-CD98/xCT interaction was maximal at 10 min p.i. and remained at the observed 30 min p.i. Fluorescence microscopy also showed a similar time-dependent interaction of αVβ5-CD98. Confocal-microscopy studies confirmed the association of CD98/xCT with α3β1 and KSHV. Preincubation of KSHV with soluble heparin and α3β1 significantly inhibited this association, suggesting that the first contact with HS and integrin is an essential element in subsequent CD98-xCT interactions. Anti-CD98 and xCT antibodies did not block virus binding and entry and nuclear delivery of viral DNA; however, viral-gene expression was significantly inhibited, suggesting that CD98-xCT play roles in the post-entry stage of infection, possibly in mediating signal cascades essential for viral-gene expression. Together, these studies suggest that KSHV interacts with functionally related integrins (αVβ3, α3β1, and αVβ5) and CD98/xCT molecules in a temporal fashion to form a multimolecular complex during the early stages of endothelial cell infection, probably mediating multiple roles in entry, signal transduction, and viral-gene expression.

Reactivation of human herpesvirus 6 during pregnancy.

Reactivation of human herpesvirus 6 (HHV-6) and cytomegalovirus (CMV) during pregnancy and transmission of the viruses to the fetus were investigated by polymerase chain reaction (PCR) and serology. In all, 104 blood samples were obtained 3 times during pregnancy and once at delivery. In another 107 women, samples were obtained only at delivery. Cord blood samples were obtained from both groups of women. HHV-6 DNA was detected in 41%-44% of the samples during months 3-8 of pregnancy, in 25% at delivery, and in 24% of age-matched controls. HHV-6 DNA was found in 1.0% of the cord blood samples. CMV DNA was detected in 1.7% of leukocytes from 104 pregnant women but in no cord blood sample. IgG antibodies to HHV-6 were found in 96% and CMV IgG in 62.5% of the women. HHV-6 IgG titers were significantly higher in HHV-6 PCR-positive women. Thus, HHV-6 reactivation seems common during pregnancy, and transfer of HHV-6 to the fetus may occur in approximately 1% of pregnancies.

Human herpesvirus 6 infection inhibits specific lymphocyte proliferation responses and is related to lymphocytopenia after allogeneic stem cell transplantation

Human herpesvirus 6 (HHV-6) infection and the HHV-6-specific lymphocyte proliferation response were studied longitudinally in 24 patients in the first 3 months after allogeneic stem cell transplantation (allo-SCT). HHV-6 DNAemia was analyzed by a nested PCR method, and the HHV-6-specific lymphocyte proliferation responses were evaluated with a standard lymphocyte proliferation assay. All patients who responded to HHV-6 GS (variant A) antigen also responded to HHV-6 Z29 (variant B) antigen, and a response to HHV-6 Z29 antigen was detected more often than to HHV-6 GS antigen after allo-SCT (P = 0.048). HHV-6 DNA was detected in more patients after than before transplantation (P = 0.01) and in more patients with acute GVHD grades II–IV than those without (P = 0.009). An HHV-6-specific proliferative response was more often detected in patients without, than in those with persistent HHV-6 infection (three consecutively positive PBL samples; P < 0.001). patients with persistent hhv-6 infection had lower lymphocyte counts from the 8th week after transplantation than those without (P = 0.03). No HHV-6-specific proliferation responses were detected in the three patients who developed HHV-6 disease. HHV-6 infection was associated with persistent lymphocyto- penia and might thereby inhibit immune function.

Human herpesvirus 6 infection and cytomegalovirus-specific lymphoproliferative responses in allogeneic stem cell transplant recipients.

The aim of this study was to investigate the effects of HHV-6 DNAemia on the CMV specific lymphoproliferative response after allogeneic stem cell transplantation. Twenty-one allogeneic stem cell transplantation (allo-SCT) patients were included in the study. The patients were either CMV seropositive and/or had CMV seropositive donors. We studied the effects of HHV-6 infection, documented by PCR, on CMV-specific lymphocyte proliferation response and on CMV infection documented by PCR. HHV-6 DNAemia correlated with the absence of CMV-specific lymphocyte proliferation responses after allo-SCT. Three of nine patients with persistent HHV-6 DNA had a CMV-specific lymphocyte proliferative response compared to 11 of 12 patients without persistent HHV-6 DNAemia (P = 0.02). Patients with higher HHV-6 DNA levels (>100 copies) were more likely than those with lower copy numbers not to develop a CMV-specific immune response (six of nine vs one of eight; P < 0.05). Patients who were repeatedly HHV-6 positive in three or more consecutive blood samples were also more likely to need repeated courses of preemptive antiviral therapy against CMV during the first 6 months after transplantation (P < 0.001). Our data indicate the possibility that HHV-6 can suppress the development of CMV-specific immune responses and thereby could predispose to development of late CMV disease.

Maribavir Inhibits Epstein-Barr Virus Transcription in Addition to Viral DNA Replication

ABSTRACT Although many drugs inhibit the replication of Epstein-Barr virus (EBV) in cell culture systems, there is still no drug that is effective and approved for use in primary EBV infection. More recently, maribavir (MBV), an l-ribofuranoside benzimidazole, has been shown to be a potent and nontoxic inhibitor of EBV replication and to have a mode of action quite distinct from that of acyclic nucleoside analogs such as acyclovir (ACV) that is based primarily on MBVs ability to block the phosphorylation of target proteins by EBV and human cytomegalovirus protein kinases. However, since the antiviral mechanisms of the drug are complex, we have carried out a comprehensive analysis of the effects of MBV on the RNA expression levels of all EBV genes with a quantitative real-time reverse transcription-PCR-based array. We show that in comparisons with ACV, the RNA expression profiles produced by the two drugs are entirely different, with MBV causing a pronounced inhibition of multiple viral mRNAs and with ACV causing virtually none. The results emphasize the different modes of action of the two drugs and suggest that the action of MBV may be linked to indirect effects on the transcription of EBV genes through the interaction of BGLF4 with multiple viral proteins.