LL-37 disrupts the Kaposi's sarcoma-associated herpesvirus envelope and inhibits infection in oral epithelial cells

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.

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

Journal of Virology ◽

10.1128/jvi.78.8.4074-4084.2004 ◽

2004 ◽

Vol 78 (8) ◽

pp. 4074-4084 ◽

Cited By ~ 48

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.

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Macaque homologs of Kaposi's sarcoma-associated herpesvirus (KSHV) infect germinal center lymphoid cells, epithelial cells in skin and gastrointestinal tract and gonadal germ cells in naturally infected macaques

Virology ◽

10.1016/j.virol.2018.04.007 ◽

2018 ◽

Vol 519 ◽

pp. 106-120 ◽

Cited By ~ 1

Author(s):

Helle Bielefeldt-Ohmann ◽

A. Gregory Bruce ◽

Kellie Howard ◽

Minako Ikoma ◽

Margaret E. Thouless ◽

...

Keyword(s):

Gastrointestinal Tract ◽

Epithelial Cells ◽

Germ Cells ◽

Germinal Center ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Lymphoid Cells ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

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Ephrin Receptor A4 is a New Kaposi’s Sarcoma-Associated Herpesvirus Virus Entry Receptor

mBio ◽

10.1128/mbio.02892-18 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 10

Author(s):

Jia Chen ◽

Xianming Zhang ◽

Samantha Schaller ◽

Theodore S. Jardetzky ◽

Richard Longnecker

Keyword(s):

B Cells ◽

Epithelial Cells ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Target Cells ◽

Double Knockout ◽

Barr Virus ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus ◽

Entry Receptor

ABSTRACT Kaposi’s sarcoma-associated herpesvirus (KSHV) is a human gammaherpesvirus associated with the development of Kaposi’s sarcoma (KS). KSHV target cells include endothelial cells, B cells, monocytes, epithelial cells, dendritic cells, macrophages, and fibroblasts. KSHV entry into target cells is a complex multistep process and is initiated by the binding and interaction of viral envelope glycoproteins with the cellular receptors. In the current studies, we have found that EphA4 promotes KSHV glycoprotein H/glycoprotein L (gH/gL)-mediated fusion and infection better than does ephrin A2 (EphA2) in HEK293T cells, indicating that EphA4 is a new KSHV entry receptor. To confirm that epithelial cells express EphA2 and EphA4, we analyzed the expression of EphA2 and EphA4 in epithelial cells, endothelial cells, B cells, monocytes, fibroblasts using RNA sequencing (RNA-seq) data analysis of existing data sets. We found that these cell types broadly express both EphA2 and EphA4, with the exception of monocytes and B cells. To confirm EphA4 is important for KSHV fusion and infection, we generated EphA2 and EphA4 single- and double-knockout cells. We found that both EphA2 and EphA4 play a role in KSHV fusion and infection, since EphA2-EphA4 double-knockout cells had the greatest decrease in fusion activity and infection compared to single-knockout cells. Fusion and infection of KSHV were rescued in the EphA2-EphA4 double-knockout cells upon overexpression of EphA2 and/or EphA4. EphA2 binds to both Epstein-Barr virus (EBV) and KSHV gH/gL; however, EphA4 binds only to KSHV gH/gL. Taken together, our results identify EphA4 as a new entry receptor for KSHV. IMPORTANCE The overall entry mechanism for herpesviruses is not completely known, including those for the human gammaherpesviruses Kaposi’s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). To fully understand the herpesvirus entry process, functional receptors need to be identified. In the current study, we found that EphA4 can also function for a KSHV entry receptor along with EphA2. Interestingly, we found that EphA4 does not function as an entry receptor for EBV, whereas EphA2 does. The discovery of EphA4 as a KSHV entry receptor has important implications for KSHV pathogenesis in humans, may prove useful in understanding the unique pathogenesis of KSHV infection in humans, and may uncover new potential targets that can be used for the development of novel interventional strategies.

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Kaposi's Sarcoma-Associated Herpesvirus Can Productively Infect Primary Human Keratinocytes and Alter Their Growth Properties

Journal of Virology ◽

10.1128/jvi.75.5.2435-2443.2001 ◽

2001 ◽

Vol 75 (5) ◽

pp. 2435-2443 ◽

Cited By ~ 52

Author(s):

Francesca Cerimele ◽

Francesca Curreli ◽

Scott Ely ◽

Alvin E. Friedman-Kien ◽

Ethel Cesarman ◽

...

Keyword(s):

Endothelial Cells ◽

Epithelial Cells ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Human Keratinocytes ◽

Lytic Cycle ◽

Rt Pcr ◽

Eccrine Glands ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Previous studies have shown the presence of Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8) DNA in endothelial cells, in keratinocytes in the basal layer of the epidermis overlying plaque-stage nodular lesions of cutaneous Kaposi's sarcoma (KS), and in the epithelial cells of eccrine glands within KS lesions. We infected primary cell cultures of human keratinocytes with KSHV/HHV8. At 6 days post infection, transcription of viral genes was detected by reverse transcriptase PCR (RT-PCR), and protein expression was documented by an immunofluorescence assay with an anti-LANA monoclonal antibody. To determine whether the viral lytic cycle was inducible by chemical treatment, KSHV/HHV8-infected keratinocytes were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) and RT-PCR was performed to confirm the transcription of lytic genes such as open reading frame 26, (which encodes a capsid protein). Finally, to assess infectious viral production, other primary human cells (human umbilical vein endothelial cells), were infected with concentrated supernatant of KSHV-infected, TPA-induced keratinocytes and the presence of viral transcripts was confirmed by RT-PCR. The uninfected keratinocytes senesced 3 to 5 weeks after mock infection, while the KSHV/HHV8-infected keratinocytes continued to proliferate and to date are still in culture. However, 8 weeks after infection, viral genomes were no longer detectable by nested PCR. Although the previously KSHV/HHV8-infected keratinocytes still expressed epithelial markers, they acquired new characteristics such as contact inhibition loss, telomerase activity, anchorage-independent growth, and changes in cytokine production. These results show that KSHV/HHV8, like other herpesviruses, can infect and replicate in epithelial cells in vitro and suggest that in vivo these cells may play a significant role in the establishment of KSHV/HHV8 infection and viral transmission.

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Modulation of Host Gene Expression by the K15 Protein of Kaposi's Sarcoma-Associated Herpesvirus

Journal of Virology ◽

10.1128/jvi.00648-06 ◽

2006 ◽

Vol 81 (1) ◽

pp. 42-58 ◽

Cited By ~ 67

Author(s):

Melanie M. Brinkmann ◽

Marcel Pietrek ◽

Oliver Dittrich-Breiholz ◽

Michael Kracht ◽

Thomas F. Schulz

Keyword(s):

Epithelial Cells ◽

Kaposi's Sarcoma ◽

Target Genes ◽

Intracellular Localization ◽

Tumor Necrosis Factor Receptor ◽

Kaposi’S Sarcoma ◽

Kinetic Studies ◽

Cox 2 ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) contains several open reading frames (ORFs) encoding proteins capable of initiating signal transduction pathways. Among them is the K15 ORF, which consists of eight exons encoding a protein with 12 predicted transmembrane domains and a cytoplasmic C terminus. When transiently expressed, the 8-exon K15 transcript gives rise to a protein with an apparent molecular mass of 45 kDa. K15 interacts with cellular proteins, TRAF (tumor necrosis factor receptor-associated factor) and Src kinases, and activates AP-1, NF-κB, and the mitogen-activated protein kinases (MAPKs) c-jun-N-terminal kinase and extracellular signal-regulated kinase. This signaling activity of K15 is related to phosphorylation of Y481 of the K15 SH2-B motif Y481EEV. In this study we demonstrate the expression of an endogenous 45-kDa K15 protein in KSHV BAC36-infected epithelial cells. This endogenous K15 protein shows the same intracellular localization as transiently expressed K15, and expression kinetic studies suggest it to be a lytic gene. We have further determined the downstream target genes of K15 signaling using DNA oligonucleotide microarrays. We demonstrate that K15 is capable of inducing expression of multiple cytokines and chemokines, including interleukin-8 (IL-8), IL-6, CCL20, CCL2, CXCL3, and IL-1α/β, as well as expression of Dscr1 and Cox-2. In epithelial cells, K15-induced upregulation of most genes was dependent on phosphorylation of Y481, whereas in endothelial cells mutation of Y481 did not result in a complete loss of Dscr1 and Cox-2 expression and NFAT-activity. Our study establishes K15 as one of the KSHV lytic genes that are inducing expression of multiple cytokines, which have been shown to play an important role in KSHV-associated pathogenesis.

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Modulation of gene expression in Kaposi’s sarcoma-associated herpesvirus-infected lymphoid and epithelial cells

Future Virology ◽

10.2217/fvl-2016-0063 ◽

2016 ◽

Vol 11 (9) ◽

pp. 619-629

Author(s):

Barbara Matteoli ◽

Francesco Broccolo ◽

Massimo Oggioni ◽

Antonio Scaccino ◽

Francesco Formica ◽

...

Keyword(s):

Gene Expression ◽

Epithelial Cells ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Modulation Of Gene Expression ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

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Ephrin receptor A4 (EphA4) is a new Kaposi’s sarcoma-associated herpesvirus virus entry receptor

10.1101/510651 ◽

2019 ◽

Author(s):

Jia Chen ◽

Xianming Zhang ◽

Samantha Schaller ◽

Theodore S. Jardetzky ◽

Richard Longnecker

Keyword(s):

Endothelial Cells ◽

B Cells ◽

Epithelial Cells ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Target Cells ◽

Double Knockout ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus ◽

Entry Receptor

AbstractKaposi’s sarcoma-associated herpesvirus (KSHV) is a human γ-herpesvirus associated with the development of Kaposi’s sarcoma (KS). KSHV target cells include endothelial cells, B cells, monocytes, epithelial cells, dendritic cells, macrophages, and fibroblasts. KSHV entry into target cells is a complex multistep process and is initiated by the binding and interaction of viral envelope glycoproteins with the cellular receptors. In our current studies, we have found that EphA4 promotes KSHV gH/gL-mediated fusion and infection better than EphA2 in HEK293T cells indicating that EphA4 is a new KSHV entry receptor. To confirm that epithelial cells express EphA2 and EphA4, we analyzed the expression of EphA2 and EphA4 in epithelial cells, endothelial cells, B cells, monocytes, fibroblasts using RNA-seq data analysis of existing data sets. We found these cell types broadly express both EphA2 and EphA4 with the exception of monocytes and B cells. To confirm EphA4 is important for KSHV fusion and infection, we generated EphA2 and EphA4 single and double knockout cells. We found that both EphA2 and EphA4 play a role in KSHV fusion and infection, since EphA2/EphA4 double knockout cells had the greatest decrease in fusion activity and infection compared to single knockout cells. Fusion and infection of KSHV was rescued in the EphA2/EphA4 double knock cells upon overexpression of EphA2 and/or EphA4. EphA2 binds to both EBV and KSHV gH/gL; however, EphA4 binds only to KSHV gH/gL. Taken together, our results identify EphA4 as a new entry receptor for KSHV.ImportanceThe overall entry mechanism for herpesviruses is not completely known including that for the human γ-herpesviruses Kaposi’s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). To fully understand the herpesvirus entry process, functional receptors need to be identified. In our current study, we found that EphA4 can also function for a KSHV entry receptor along with EphA2. Interestingly, we found that EphA4 does not function as an entry receptor for EBV whereas EphA2 does. The discovery of EphA4 as KSHV entry receptor has important implications for KSHV pathogenesis in humans and may prove useful in understanding the unique pathogenesis of KSHV infection in humans and may uncover new potential targets that can be used for the development of novel interventional strategies.

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