scholarly journals Wild-Type Kaposi's Sarcoma-Associated Herpesvirus Isolated from the Oropharynx of Immune-Competent Individuals Has Tropism for Cultured Oral Epithelial Cells

2004 ◽  
Vol 78 (8) ◽  
pp. 4074-4084 ◽  
Author(s):  
Karen M. Duus ◽  
Vivian Lentchitsky ◽  
Timothy Wagenaar ◽  
Charles Grose ◽  
Jennifer Webster-Cyriaque
Keyword(s):  
Epithelial Cells ◽  
Neutralizing Antibodies ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Wild Type ◽  
Oral Epithelial Cells ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
Oral Epithelial

ABSTRACT Based on the observation that wild-type Kaposi's sarcoma-associated herpesvirus (KSHV) DNA can be detected in the oral cavity of healthy, immunocompetent individuals, we hypothesized that epithelial cells could be infected in vitro by wild-type (WT) KSHV isolated from immunocompetent individuals. Primary oral epithelial (P-EPI) cells and telomerase-immortalized oral epithelial cells were generated from human gingival tissue and were then infected in vitro with WT KSHV isolated from throat wash samples. Markers of lytic and latent KSHV infection were detected in cultures by 24 h postinfection by immunofluorescence confocal microscopic assays. The infectivity of the WT and BCBL virus was blocked by neutralizing antibodies against KSHV gB. The presence of KSHV DNA in these cells was confirmed by real-time PCR amplification of different regions of the viral genome. The significant in vitro viral replication that had occurred was inhibited by ganciclovir and by neutralizing antibodies against gB. When infected cultures were examined by scanning electron microscopy, thousands of KSHV particles were clearly visible across the surfaces of P-EPI cells. The detection of enveloped particles indicated that the infectious cycle had proceeded through assembly and egress. We thus demonstrated that oral WT KSHV isolated from immunocompetent individuals was able to infect and replicate in vitro in a relevant primary cell type. Furthermore, our results provide compelling evidence for KSHV transmission within infected oral epithelial cells derived from healthy, immunocompetent populations.

scholarly journals Human Immunodeficiency Virus-Associated Exosomes Promote Kaposi’s Sarcoma-Associated Herpesvirus Infection via the Epidermal Growth Factor Receptor

2020 ◽  
Vol 94 (9) ◽  
Author(s):  
Lechuang Chen ◽  
Zhimin Feng ◽  
Guoxiang Yuan ◽  
Corey C. Emerson ◽  
Phoebe L. Stewart ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Kshv Infection ◽  
Oral Epithelial Cells ◽  
Tar Rna ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
Oral Epithelial ◽  
Hiv Aids

ABSTRACT Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causal agent for Kaposi’s sarcoma (KS), the most common malignancy in people living with human immunodeficiency virus (HIV)/AIDS. The oral cavity is a major route for KSHV infection and transmission. However, how KSHV breaches the oral epithelial barrier for spreading to the body is not clear. Here, we show that exosomes purified from either the saliva of HIV-positive individuals or the culture supernatants of HIV-1-infected T-cell lines promote KSHV infectivity in immortalized and primary human oral epithelial cells. HIV-associated saliva exosomes contain the HIV trans-activation response element (TAR), Tat, and Nef RNAs but do not express Tat and Nef proteins. The TAR RNA in HIV-associated exosomes contributes to enhancing KSHV infectivity through the epidermal growth factor receptor (EGFR). An inhibitory aptamer against TAR RNA reduces KSHV infection facilitated by the synthetic TAR RNA in oral epithelial cells. Cetuximab, a monoclonal neutralizing antibody against EGFR, blocks HIV-associated exosome-enhanced KSHV infection. Our findings reveal that saliva containing HIV-associated exosomes is a risk factor for the enhancement of KSHV infection and that the inhibition of EGFR serves as a novel strategy for preventing KSHV infection and transmission in the oral cavity. IMPORTANCE Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causal agent for Kaposi’s sarcoma (KS), the most common malignancy in HIV/AIDS patients. Oral transmission through saliva is considered the most common route for spreading the virus among HIV/AIDS patients. However, the role of HIV-specific components in the cotransfection of KSHV is unclear. We demonstrate that exosomes purified from the saliva of HIV-positive patients and secreted by HIV-infected T-cell lines promote KSHV infectivity in immortalized and primary oral epithelial cells. HIV-associated exosomes promote KSHV infection, which depends on HIV trans-activation response element (TAR) RNA and EGFR of oral epithelial cells, which can be targeted for reducing KSHV infection. These results reveal that HIV-associated exosomes are a risk factor for KSHV infection in the HIV-infected population.

scholarly journals Construction of an Infectious Rhesus Rhadinovirus Bacterial Artificial Chromosome for the Analysis of Kaposi's Sarcoma-Associated Herpesvirus-Related Disease Development

2007 ◽  
Vol 81 (6) ◽  
pp. 2957-2969 ◽  
Author(s):  
Ryan D. Estep ◽  
Michael F. Powers ◽  
Bonnie K. Yen ◽  
He Li ◽  
Scott W. Wong
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Kaposi's Sarcoma ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Artificial Chromosome ◽  
Wild Type ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Rhesus rhadinovirus (RRV) is closely related to Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 (HHV-8) and causes KSHV-like diseases in immunocompromised rhesus macaques (RM) that resemble KSHV-associated diseases including multicentric Castleman's disease and non-Hodgkin's lymphoma. RRV retains a majority of open reading frames (ORFs) postulated to be involved in the pathogenesis of KSHV and is the closest available animal model to KSHV infection in humans. Here we describe the generation of a recombinant clone of RRV strain 17577 (RRV17577) utilizing bacterial artificial chromosome (BAC) technology. Characterization of the RRV BAC demonstrated that it is a pathogenic molecular clone of RRV17577, producing virus that behaves like wild-type RRV both in vitro and in vivo. Specifically, BAC-derived RRV displays wild-type growth properties in vitro and readily infects simian immunodeficiency virus-infected RM, inducing B cell hyperplasia, persistent lymphadenopathy, and persistent infection in these animals. This RRV BAC will allow for rapid genetic manipulation of the RRV genome, facilitating the creation of recombinant versions of RRV that harbor specific alterations and/or deletions of viral ORFs. This system will provide insights into the roles of specific RRV genes in various aspects of the viral life cycle and the RRV-associated pathogenesis in vivo in an RM model of infection. Furthermore, the generation of chimeric versions of RRV containing KSHV genes will allow analysis of the function and contributions of KSHV genes to viral pathogenesis by using a relevant primate model system.

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

2008 ◽  
Vol 83 (6) ◽  
pp. 2531-2539 ◽  
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.

scholarly journals The Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) gH/gL Complex Is the Predominant Neutralizing Antigenic Determinant in KSHV-Infected Individuals

Viruses ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 256 ◽  
Author(s):  
Yasaman Mortazavi ◽  
Salum J. Lidenge ◽  
Tara Tran ◽  
John T. West ◽  
Charles Wood ◽  
...  
Keyword(s):  
Antigenic Determinant ◽  
Neutralizing Antibodies ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Sub Saharan Africa ◽  
People Living With Hiv ◽  
Viral Glycoproteins ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Sub Saharan ◽  
Kaposi’S Sarcoma Associated Herpesvirus

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi’s sarcoma (KS), one of the most prevalent cancers of people living with HIV/AIDS in sub-Saharan Africa. The seroprevalence for KSHV is high in the region, and no prophylactic vaccine against the virus is available. In this study, we characterized the antigenic targets of KSHV-specific neutralizing antibodies (nAbs) in asymptomatic KSHV-infected individuals and KS patients with high nAbs titers. We quantified the extent to which various KSHV envelope glycoproteins (gB, ORF28, ORF68, gH, gL, gM, gN and gpK8.1) adsorbed/removed KSHV-specific nAbs from the plasma of infected individuals. Our study revealed that plasma from a majority of KSHV neutralizers recognizes multiple viral glycoproteins. Moreover, the breadth of nAbs responses against these viral glycoproteins varies among endemic KS, epidemic KS and asymptomatic KSHV-infected individuals. Importantly, among the KSHV glycoproteins, the gH/gL complex, but neither gH nor gL alone, showed the highest adsorption of KSHV-specific nAbs. This activity was detected in 80% of the KSHV-infected individuals regardless of their KS status. The findings suggest that the gH/gL complex is the predominant antigenic determinant of KSHV-specific nAbs. Therefore, gH/gL is a potential target for development of KSHV prophylactic vaccines.

scholarly journals Sensitivity of the C-Terminal Nuclease Domain of Kaposi's Sarcoma-Associated Herpesvirus ORF29 to Two Classes of Active-Site Ligands

2018 ◽  
Vol 62 (10) ◽  
Author(s):  
Jennifer T. Miller ◽  
Haiyan Zhao ◽  
Takashi Masaoka ◽  
Brittany Varnado ◽  
Elena M. Cornejo Castro ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Active Site ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Nuclease Activity ◽  
Divalent Metal ◽  
Wild Type ◽  
Nuclease Domain ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACTKaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent of Kaposi's sarcoma, belongs to theHerpesviridaefamily, whose members employ a multicomponent terminase to resolve nonparametric viral DNA into genome-length units prior to their packaging. Homology modeling of the ORF29 C-terminal nuclease domain (pORF29C) and bacteriophage Sf6 gp2 have suggested an active site clustered with four acidic residues, D476, E550, D661, and D662, that collectively sequester the catalytic divalent metal (Mn2+) and also provided important insight into a potential inhibitor binding mode. Using this model, we have expressed, purified, and characterized the wild-type pORF29C and variants with substitutions at the proposed active-site residues. Differential scanning calorimetry demonstrated divalent metal-induced stabilization of wild-type (WT) and D661A pORF29C, consistent with which these two enzymes exhibited Mn2+-dependent nuclease activity, although the latter mutant was significantly impaired. Thermal stability of WT and D661A pORF29C was also enhanced by binding of an α-hydroxytropolone (α-HT) inhibitor shown to replace divalent metal at the active site. For the remaining mutants, thermal stability was unaffected by divalent metal or α-HT binding, supporting their role in catalysis. pORF29C nuclease activity was also inhibited by two classes of small molecules reported to inhibit HIV RNase H and integrase, both of which belong to the superfamily of nucleotidyltransferases. Finally, α-HT inhibition of KSHV replication suggests ORF29 nuclease function as an antiviral target that could be combined with latency-activating compounds as a shock-and-kill antiviral strategy.

scholarly journals Early Events in Kaposi's Sarcoma-Associated Herpesvirus Infection of Target Cells

2009 ◽  
Vol 84 (5) ◽  
pp. 2188-2199 ◽  
Author(s):  
Bala Chandran
Keyword(s):  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Target Cells ◽  
Mode Of Entry ◽  
Successful Infection ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
Cell Signaling Pathways

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV), the most recently identified member of the herpesvirus family, infects a variety of target cells in vitro and in vivo. This minireview surveys current information on the early events of KSHV infection, including virus-receptor interactions, involved envelope glycoproteins, mode of entry, intracellular trafficking, and initial viral and host gene expression programs. We describe data supporting the hypothesis that KSHV manipulates preexisting host cell signaling pathways to allow successful infection. The various signaling events triggered by infection, and their potential roles in the different stages of infection and disease pathogenesis, are summarized.

scholarly journals Clinical-Grade Functional Dendritic Cells From Patients With Multiple Myeloma Are Not Infected With Kaposi's Sarcoma-Associated Herpesvirus

Blood ◽  
1998 ◽  
Vol 91 (6) ◽  
pp. 1852-1857 ◽  
Author(s):  
Karin Tarte ◽  
Sonja J. Olsen ◽  
Zhao Yang Lu ◽  
Eric Legouffe ◽  
Jean-François Rossi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Dendritic Cells ◽  
Kaposi's Sarcoma ◽  
Fluorescein Isothiocyanate ◽  
Kaposi’S Sarcoma ◽  
Interleukin 4 ◽  
Clinical Grade ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

Abstract Bone marrow dendritic cells (DC) from patients with multiple myeloma (MM) were recently reported to be infected with Kaposi's sarcoma-associated herpesvirus (KSHV). Because immunotherapy strategies using DC are very promising in this disease, we looked for KSHV DNA in clinical-grade DC generated in vitro from MM patients. Adherent apheresis cells from MM patients were maintained for 7 days in clinical-grade X-VIVO 15 culture medium supplemented with granulocyte-macrophage colony-stimulating factor, interleukin-4, or interleukin-13. Tumor necrosis factor α was added for the last 2 days. We obtained a cell population with a DC phenotype able to endocytose fluorescein isothiocyanate (FITC)-dextran and efficiently activate resting allogenic T lymphocytes. To detect KSHV DNA, we used polymerase chain reaction (PCR) followed by Southern blotting of PCR product with a sensitivity detecting a few copies of viral DNA. All the PCR were repeated in a blinded fashion three times, on 1 μg and 0.2 μg of genomic DNA, in two different laboratories. Clinical-grade DC from 10 (91%) of 11 patients were not infected with KSHV. The apheresis cells and the purified CD34+ cells from the same patients were also negative. A very weak PCR band was detected with DC from one patient, but the initial apheresis cells were negative. The detection of KSHV infection in 1 (9%) of 11 MM patients probably represents background seroprevalence. It seems likely that functional and clinical-grade DC from MM patients can safely be used in clinical trials.

scholarly journals Disintegrin-like domain of glycoprotein B regulates Kaposi’s sarcoma-associated herpesvirus infection of cells

2014 ◽  
Vol 95 (8) ◽  
pp. 1770-1782 ◽  
Author(s):  
Lia R. Walker ◽  
Hosni A. M. Hussein ◽  
Shaw M. Akula
Keyword(s):  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Glycoprotein B ◽  
Binding Motif ◽  
Wild Type ◽  
Infection Rates ◽  
Herpesvirus Infection ◽  
Kshv Infection ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

Kaposi's sarcoma-associated herpesvirus (KSHV) glycoprotein B (gB) is a lytic structural protein expressed on the envelope of mature virions and on the membrane of cells supporting lytic infection. In addition to this viral glycoprotein’s interaction with integrins via its RGD (Arg-Gly-Asp) motif, KSHV gB possesses a disintegrin-like domain (DLD), which binds integrins as well. Prior to this study, there has been minimal research involving the less common integrin-binding motif, DLD, of gB as it pertains to herpesvirus infection. By using phage display peptide library screening and molecular biology techniques, the DLD of KSHV gB was shown to interact specifically with non-RGD binding α9β1 integrins. Similarly, monitoring wild-type infection confirmed α9β1:DLD interactions to be critical to successful KSHV infection of human foreskin fibroblast (HFF) cells and human dermal microvascular endothelial cells (HMVEC-d) compared with 293 cells. To further demonstrate the importance of the DLD of gB in KSHV infection, two recombinant virus constructs were generated using a bacterial artificial chromosome (BAC) system harbouring the KSHV genome (BAC36): BAC36ΔD-KSHV (lacking a functionally intact DLD of gB and containing an introduced tetracycline cassette) and BAC36.T-KSHV (containing an intact DLD sequence and an introduced tetracycline cassette). Accordingly, BAC36ΔD-KSHV presented significantly lower infection rates in HFF and HMVEC-d cells compared with the comparable infection rates achieved by wild-type BAC36-KSHV and BAC36.T-KSHV. Thus, the present report has delineated a critical role for the DLD of gB in KSHV infection, which may lead to a broader knowledge regarding the sophisticated mechanisms utilized by virus-encoded structural proteins in KSHV entry and infection.

scholarly journals Functional Characterization of Kaposi's Sarcoma-Associated Herpesvirus ORF45 byBacterial Artificial Chromosome-Based Mutagenesis

2006 ◽  
Vol 80 (24) ◽  
pp. 12187-12196 ◽  
Author(s):  
Fan Xiu Zhu ◽  
Xiaojuan Li ◽  
Fuchun Zhou ◽  
Shou-Jiang Gao ◽  
Yan Yuan
Keyword(s):  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Artificial Chromosome ◽  
Viral Gene ◽  
Type I ◽  
Wild Type ◽  
Early Protein ◽  
Significant Difference ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Open reading frame 45 (ORF45) of Kaposi's sarcoma-associated herpesvirus (KSHV) encodes an immediate-early protein. This protein is also present in virions as a tegument protein. ORF45 protein interacts with interferon regulatory factor 7 (IRF-7) and inhibits virus-induced type I interferon production by blocking activation of IRF-7. To define further the function of ORF45 and the mechanism underlying its action, we constructed an ORF45-null recombinant virus genome (BAC-stop45) by using a bacterial artificial chromosome (BAC) system. Stable 293T cells carrying the BAC36 (wild type) and BAC-stop45 genomes were generated. When monolayers of 293T BAC36 and 293T BAC-stop45 cells were induced with 12-O-tetradecanoylphorbol-13-acetate and sodium butyrate, no significant difference was found between them in overall viral gene expression and lytic DNA replication, but induced 293T BAC-stop45 cells released 10-fold fewer virions to the medium than did 293T BAC36 cells. When ORF45-null virus was used to infect cells, lower infectivity was observed than for wild-type BAC36. These results suggest that KSHV ORF45 plays roles in both early and late stages of viral infection, probably in viral ingress and egress.

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