Gene dosage of KSHV determines potential for immune evasion

Kaposi's sarcoma-associated herpesvirus (KSHV, human herpesvirus 8 or HHV8) is the etiologic agent of three distinct tumors in immunocompromised populations. Once infection is established, KSHV exhibits a biphasic lifecycle composed of latent and productive states. We were interested in the critical period following de novo infection but before the establishment of latency when the virus might be vulnerable to the host immune response. We refer to this period as pre-latency and sought to identify any potential mechanisms by which the virus could avoid elimination by the immune system. To this end, we characterized the role of modulator of immune recognition 2 (MIR2), a virally encoded E3 ubiquitin ligase, in downregulation of the immune synapse during pre-latency through promoter studies and targeted siRNA. Using multispectral imaging flow cytometry (MIFC), we investigated the effects of viral load on MIR2 gene expression. We found that viral load positively correlated with the loss of the immune synapse molecules MHC-I and ICAM-1. Importantly, the levels of infection (3-5 viral copies per cell) found in KS lesions were sufficient for loss of immune synapse components in vitro. We also characterized the viral lifecycle in a KSHV + primary effusion lymphoma (PEL) cell line, BCBL-1, using MIFC. By simultaneously measuring the lytic switch protein (replication and transcription activator, RTA), the late lytic viral 3 envelope protein K8.1, and a cellular surface marker (CD138 or syndecan-1), we characterized 5 distinct sub-phases of the viral lifecycle. Finally, we sought to explore the control of KSHV tumors by rapamycin, explaining the apparent paradox of how an immunosuppressant drug so effectively suppresses the pathogenesis of an opportunistic viral infection. To this end, we treated KSHV + cell PEL lines with rapamycin and found a decrease in the percentage of cells in lytic replication and a loss of virion production. These results indicate that mTOR may play a universal role in the regulation of RTA expression and, therefore, KSHV production, and provide a molecular explanation for the marked clinical success of rapamycin in the treatment of posttransplant Kaposi's sarcoma.

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