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

Abstract

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.

Increased tumorigenicity of cells carrying recombinant human herpesvirus 8

“…1D). K1 staining was cytoplasmic, in agreement with previous work reporting K1 as a membrane-bound lytic protein [15]. Once again, no staining was observed in control 293T cells.…”

“…Cells were first reacted with a rat anti-LANA, mouse anti-K1 (2H5) (generously provided by Dr Jung) [15], and rabbit anti-vIL-6 for 1 h at room temperature. Slides were washed three times for 5 min in PBS and then incubated with Alexa 568-labeled anti-rat IgG, Alexa 568-labeled anti-mouse IgG or Alexa 568-labeled anti-rabbit IgG (Invitrogen) for 1 h at room temperature.…”

K1 protein of human herpesvirus 8 suppresses lymphoma cell Fas-mediated apoptosis

“…BJAB transfectants were incubated with the primary anti-K1 antibody 2H5 (a gift from Dr Jae U. Jung) 1:5 in dilution buffer, at ambient temperature for 30 minutes as reported previously. 20 Slides were washed 3 times with PBS and incubated at ambient temperature for 15 minutes with a secondary antibody at a concentration of 20 g/mL in dilution buffer. Slides were washed again and coverslips were mounted onto them using Vectashield mounting medium (Vector Laboratories, Burlingame, CA), followed by 4Ј, 6-diamidino-2-phenylindole (DAPI) staining.…”

Activation of Src kinase Lyn by the Kaposi sarcoma–associated herpesvirus K1 protein: implications for lymphomagenesis

“…Moreover, K1 expression has been detected in MCD tissues, and in PEL cells during the lytic viral life cycle on induction with 12-O-tetradecanoylphorbol-13-acetate (TPA). 9,13,14 It is possible that K1 produced during lytic replication has a lasting role in the development of KSHVassociated lymphoproliferative disorders. This is further supported by the finding that ITAM-dependent signaling by K1, although not critical, moderately augments lytic replication in B lymphocytes.…”