Intracellular Kaposi's sarcoma-associated herpesvirus load determines early loss of immune synapse components.

Lifelong infection is a hallmark of all herpesviruses, and their survival depends on countering host immune defenses. The human gammaherpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) encodes an array of proteins that contribute to immune evasion, including modulator of immune recognition 2 (MIR2), an E3 ubiquitin ligase. Exogenously expressed MIR2 downregulates the surface expression of several immune synapse proteins, including major histocompatibility complex (MHC) class 1, ICAM-1 (CD54), and PECAM (CD31). Although immunofluorescence assays detect this lytic gene in only 1 to 5% of cells within infected cultures, we have found that de novo infection of naive cells leads to the downregulation of these immune synapse components in a major proportion of the population. Investigating the possibility that low levels of MIR2 are responsible for this downregulation in the context of viral infection, we found that MIR2 transduction recapitulated the patterns of surface downregulation following de novo infection and that both MIR2 promoter activation, MIR2 expression level, and immune synapse component downregulation were proportional to the concentration of KSHV added to the culture. Additionally, MIR2-specific small interfering RNA reversed the downregulation effects. Finally, using a sensitive, high-throughput assay to detect levels of the virus in individual cells, we also observed that downregulation of MHC class I and ICAM-1 correlated with intracellular viral load. Together, these results suggest that the effects of MIR2 are gene dosage dependent and that low levels of this viral protein contribute to the widespread downregulation of immune-modulating cell surface proteins during the initial stages of KSHV infection.

Surface downregulation of major histocompatibility complex class I, PE-CAM, and ICAM-1 following de novo infection of endothelial cells with Kaposi's sarcoma-associated herpesvirus.

Under selective pressure from host cytotoxic T lymphocytes, many viruses have evolved to downregulate major histocompatibility complex (MHC) class I and/or T-cell costimulatory molecules from the surface of infected cells. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes two proteins, MIR-1 and MIR-2, that serve this function during lytic replication. In vivo, however, KSHV exists in a predominantly latent state, with less than 5% of infected cells expressing discernible lytic gene products. Thus, mechanisms of immune evasion that depend on genes expressed only during lytic replication are unlikely to be active in most KSHV-infected cells. As a result, we searched for evidence of similar defensive strategies extant during latency, employing culture systems that strongly favor latent KSHV infection. We measured cell surface levels of immunomodulatory proteins on both primary dermal microvascular endothelial cells (pDMVEC) infected through coculture with induced primary effusion lymphoma cells and telomerase-immortalized DMVEC infected directly with cell-free virus. Employing a panel of antibodies against several endothelial cell surface proteins, we show that de novo infection with KSHV leads to the downregulation of MHC class I, CD31 (PE-CAM), and CD54 (ICAM-I) but not CD58 (LFA-3) or CD95 (Fas). Furthermore, flow cytometry with a fluorescently labeled monoclonal antibody to the latency-associated nuclear antigen (LANA) revealed that downregulation occurred predominantly on KSHV-infected (LANA-positive) cells. Although the vast majority of infected cells displayed this downregulation, less than 1% expressed either immediate-early or late lytic proteins detectable by immunofluorescence. Together, these results suggest that downregulation of immunomodulatory proteins on the surface of target cells may represent a constitutive mode of immune evasion employed by KSHV following de novo infection.

Regulation of human osteocalcin promoter in hormone-independent human prostate cancer cells.

Osteocalcin (OC) is a small (6 kDa) polypeptide whose expression was thought to be limited to mature osteoblasts. The discovery of OC expression in prostate cancer specimens led us to study the regulation of OC gene in androgen-independent metastatic human prostate PC3 cells. An 800-bp human OC (hOC) promoter-luciferase construct exhibited strong basal and vitamin D-induced activity in OC-positive human prostate and osteosarcoma cell lines. Through deletion analysis of the hOC promoter, the functional hierarchy of the cis-acting elements, OSE1, OSE2, and AP-1/VDRE, was established in PC3 cells (OSE1 > AP-1/VDRE > OSE2). By juxtaposing dimers of these 3 cis-elements, we produced a minimal hOC promoter capable of displaying high tissue specific activity in prostate cancer cells. Our study demonstrated three groups of transcription factors, Runx2, JunD/Fra-2, and Sp1, responsible for the high hOC promoter activity in PC3 cells by binding to the OSE2, AP-1/VDRE, and OSE1 elements, respectively. Among the three groups of transcription factors, the expression levels of Runx2 and Fra-2 are higher in the OC-positive PC3 cells and osteoblasts, compared with the OC-negative LNCaP cells. Interestingly, unlike the mouse OC promoter, the OSE1 site in hOC promoter is regulated by members of Sp1 family instead of the osteoblast-specific factor Osf1. The molecular basis for androgen-independent prostate cancer cells behaving like mature osteoblasts may be explained by the interplay and coordination of these transcription factors under the tight regulation of autocrine and paracrine mediators.