Kaposi's sarcoma-associated herpesvirus (KSHV, human herpesvirus 8) is the causative agent of Kaposi's sarcoma and other lymphoproliferative syndromes often associated with HIV/AIDS. Functional complementary DNA selection for a receptor mediating KSHV cell fusion identified xCT, the 12-transmembrane light chain of the human cystine/glutamate exchange transporter system x-c. Expression of recombinant xCT rendered otherwise not susceptible target cells permissive for both KSHV cell fusion and virion entry. Antibodies against xCT blocked KSHV fusion and entry with naturally permissive target cells. KSHV target cell permissiveness correlated closely with endogenous expression of xCT messenger RNA and protein in diverse human and nonhuman cell types.

Upregulation of xCT, the inducible subunit of a membrane-bound amino acid transporter, replenishes intracellular glutathione stores to maintain cell viability in an environment of oxidative stress. xCT also serves as a fusion-entry receptor for the Kaposi's sarcoma-associated herpesvirus (KSHV), the causative agent of Kaposi's sarcoma (KS). Ongoing KSHV replication and infection of new cell targets is important for KS progression, but whether xCT regulation within the tumor microenvironment plays a role in KS pathogenesis has not been determined. Using gene transfer and whole virus infection experiments, we found that KSHV-encoded microRNAs (KSHV miRNAs) upregulate xCT expression by macrophages and endothelial cells, largely through miR-K12-11 suppression of BACH-1—a negative regulator of transcription recognizing antioxidant response elements within gene promoters. Correlative functional studies reveal that upregulation of xCT by KSHV miRNAs increases cell permissiveness for KSHV infection and protects infected cells from death induced by reactive nitrogen species (RNS). Interestingly, KSHV miRNAs simultaneously upregulate macrophage secretion of RNS, and biochemical inhibition of RNS secretion by macrophages significantly reduces their permissiveness for KSHV infection. The clinical relevance of these findings is supported by our demonstration of increased xCT expression within more advanced human KS tumors containing a larger number of KSHV-infected cells. Collectively, these data support a role for KSHV itself in promoting de novo KSHV infection and the survival of KSHV-infected, RNS-secreting cells in the tumor microenvironment through the induction of xCT.

The OX-40 receptor, a member of the nerve growth factor/tumor necrosis factor receptor gene family, is expressed preferentially on autoreactive CD4+ T cells isolated from the site of inflammation in rats with clinical signs of experimental autoimmune encephalomyelitis (EAE). To examine whether the OX-40 receptor has biologic relevance to T cell function, we evaluated the ability of a rat OX-40 receptor-specific antibody to co-stimulate a myelin basic protein (MBP)-reactive CD4+ T cell line. The anti-OX-40 antibody provided a potent co-stimulatory signal to CD4+ T cells when added in conjunction with a submitogenic dose of anti-CD3, but the anti-OX-40 antibody alone did not produce a mitogenic response. The magnitude and dose-response of anti-OX-40 co-stimulation was virtually identical to the signal delivered to T cells when cultured with anti-CD28 in conjunction with anti-CD3. MBP-specific T cells stimulated with both anti-CD3 and anti-OX-40 antibodies expressed increased mRNA and protein for IL-2 when compared to anti-CD3 alone. MBP-specific T cells stimulated with both anti-CD3 and anti-OX-40 antibodies were also able to induce EAE when transferred into naive Lewis rats. In contrast, cells stimulated with anti-CD3 alone were not encephalitogenic. These data suggest that the function of the OX-40 receptor on activated T cells is to provide an alternative pathway for T cell co-stimulation that may be similar in potency to the CD28-mediated signal.

The molecular mechanism of Kaposi's sarcoma-associated herpesvirus (KSHV, human herpesvirus 8) entry is poorly understood. We tested a broad variety of cell types of diverse species and tissue origin for their ability to function as targets in a quantitative reporter gene assay for KSHV-glycoprotein-mediated cell fusion. Several human, non-human primate, and rabbit cell lines were efficient targets, whereas rodent and all human lymphoblastoid cell lines were weak targets. Parallel findings were obtained with a virion entry assay using a recombinant KSHV encoding a reporter gene. No correlation was observed between target cell activity and surface expression of alpha3beta1 integrin, a proposed KSHV receptor. We hypothesize that target cell permissiveness in both the cell fusion and virion entry assays reflects the presence of a putative KSHV fusion-entry receptor.