Mixed patterns of transmission of human herpesvirus-8 (Kaposi’s sarcoma-associated herpesvirus) in Malawian families

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.Kaposi's sarcoma-associated herpesvirus (KSHV; also called human herpesvirus 8 ) is the etiologic agent of Kaposi's sarcoma (KS) (12) and peripheral effusion lymphoma (PEL) (10). The human herpesviruses are generally ubiquitous among human populations, are generally shed in the oral cavity, and are primarily transmitted via the salivary route. However, current serological tests for KSHV have determined that the seroprevalence within the general U.S. population is low, suggesting that, unlike the other human herpesviruses (with the exception of herpes simplex virus type 2 [HSV-2]), KSHV is not ubiquitous among healthy human populations.The primary mode of KSHV transmission remains unresolved, as extensive evidence exists for both sexual (8,25,30,31) and oral (5, 29, 34) transmission routes in immunosuppressed individuals. However, recent epidemiology studies suggested that oral transmission of KSHV does occur among healthy populations (14,15). Complicating the transmission issue, the identities of the cell types harboring KSHV in vivo in the oral cavity and producing the virus detected in saliva are not known. Endothelial cells are the primary infected cells in KS lesions (7). Recently, KSHV antigens were detected in oral epithelial cells of an early KS lesion (44). In addition, the ability of the virus to infect primary human keratinocytes has also been demonstrated (3, 9). Based on the above evidence, we hypothesized that the wild-type (WT) isol...

mentioning

confidence: 99%

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

Duus

Lentchitsky

Wagenaar

et al. 2004

“…Like HIV Env, the extracellular domain of the K1 protein is also extremely variable. Particularly, two 40-amino-acid blocks at the extracellular domain of K1, variable regions 1 and 2 (V1 and V2, respectively), show as much as 85% divergence at the nucleotide level and 60% divergence at the amino acid level (7,16,23,47). Different V1 and V2 regions seem to correspond to different geographic areas (7,16,23,47).…”

mentioning

confidence: 99%

“…Particularly, two 40-amino-acid blocks at the extracellular domain of K1, variable regions 1 and 2 (V1 and V2, respectively), show as much as 85% divergence at the nucleotide level and 60% divergence at the amino acid level (7,16,23,47). Different V1 and V2 regions seem to correspond to different geographic areas (7,16,23,47). Studies of K1 alleles from various KSHVinfected tissues have defined four major subtypes of K1 (A, B, C, and D) and 13 distinct variants and clades similar to those found for the HIV Env protein (16,19,47).…”

mentioning

confidence: 99%

Structural Analysis of the Kaposi's Sarcoma-Associated Herpesvirus K1 Protein

Lee¹,

Connole

Tang

et al. 2003

The K1 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) efficiently transduces extracellular signals to elicit cellular activation events through its cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM). In addition, the extracellular domain of K1 demonstrates regional homology with the immunoglobulin (Ig) family and contains conserved regions (C1 and C2) and variable regions (V1 and V2). To generate mouse monoclonal antibodies directed against the KSHV K1 protein, BALB/c mice were primed and given boosters with K1 protein purified from mammalian cells. Twenty-eight hybridomas were tested for reactivity with K1 protein by enzyme-linked immunosorbent assay, immunofluorescence, flow cytometry, immunohistochemistry, and immunoblotting. Deletion mutants of the K1 extracellular domain were used to map the epitope of each antibody. All antibodies were directed to the Ig, C1, and C2 regions of K1. Furthermore, antibody recognition of a short sequence (amino acids 92 to 125) of the C2 region overlapping with the Ig region of K1 efficiently induced intracellular free calcium mobilization; antibody recognition of the other regions of K1 did not. The efficient signal transduction of K1 induced by antibody stimulation required both the ITAM sequence of the cytoplasmic domain and the normal structure of the extracellular domain. Finally, immunological assays showed that K1 was expressed during the early lytic cycle of viral replication in primary effusion lymphoma cells. K1 was readily detected in multicentric Castleman's disease tissues, whereas it was not detected in Kaposi's sarcoma lesions, suggesting that K1 is preferentially expressed in lymphoid cells. Thus, these results indicate that the conserved regions, particularly the Ig and C2 regions, of the K1 extracellular domain are exposed on the outer surface and play an important role in K1 structure and signal transduction, whereas the variable regions of K1 appear to be away from the surface.

“…Besides its regional homology with the Ig family, the extracellular domain of K1 also contains conserved regions (C1 and C2) and variable regions (V1 and V2). Particularly, two 40-amino-acid blocks at the extracellular V1 and V2 domains of K1 show as much as 85% divergence at the nucleotide level and 60% divergence at the amino acid level (7,20,27,53). In addition, the amino-terminal region of K1 specifically interacts with the chains of B-cell antigen receptor (BCR) complexes, and this interaction inhibits the intracellular transport of BCR, resulting in downregulation of BCR surface expression (31).…”

mentioning

confidence: 99%

Characterization of the Kaposi's Sarcoma-Associated Herpesvirus K1 Signalosome

Lee

Feng

et al. 2005

Kaposi's sarcoma (KS) is a multifocal angiogenic tumor and appears to be a hyperplastic disorder caused, in part, by local production of inflammatory cytokines. The K1 lymphocyte receptor-like protein of KSassociated herpesvirus (KSHV) efficiently transduces extracellular signals to elicit cellular activation events through its cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM). To further delineate K1-mediated signal transduction, we purified K1 signaling complexes and identified its cellular components. Upon stimulation, the K1 ITAM was efficiently tyrosine phosphorylated and subsequently interacted with cellular Src homology 2 (SH2)-containing signaling proteins Lyn, Syk, p85, PLC␥2, RasGAP, Vav, SH2 domain-containing protein tyrosine phosphatase 1/2, and Grab2 through its phosphorylated tyrosine residues. Mutational analysis demonstrated that each tyrosine residue of K1 ITAM contributed to the interactions with cellular signaling proteins in distinctive ways. Consequently, these interactions led to the marked augmentation of cellular signal transduction activity, evidenced by the increase of cellular tyrosine phosphorylation and intracellular calcium mobilization, the activation of NF-AT and AP-1 transcription factor activities, and the production of inflammatory cytokines. These results demonstrate that KSHV K1 effectively recruits a set of cellular SH2-containing signaling molecules to form the K1 signalosome, which elicits downstream signal transduction and induces inflammatory cytokine production.