A panel of KSHV mutants in the polycistronic kaposin locus for precise analysis of individual protein products

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Abstract
Kaposi’s sarcoma-associated herpesvirus (KSHV) is the cause of several human cancers including the endothelial cell (EC) malignancy, Kaposi’s sarcoma. Unique KSHV genes absent from other human herpesvirus genomes, the “K-genes”, are important for KSHV replication and pathogenesis. Among these, the kaposin transcript is highly expressed in all phases of infection, but its complex polycistronic nature has hindered functional analysis to date. At least three proteins are produced from the kaposin transcript: Kaposin A (KapA), B (KapB), and C (KapC). To determine the relative contributions of kaposin proteins during KSHV infection, we created a collection of mutant viruses unable to produce kaposin proteins individually or in combination. Kaposin-deficient latent iSLK cell lines displayed reduced viral genome copy number and often exhibited small LANA nuclear bodies; despite this, all were capable of progeny virion production. Primary infection with ΔKapB virus revealed decreased LANA expression and viral genome copy number, yet providing KapB protein in trans failed to complement these defects, suggesting a requirement for the kaposin locus in cis . Our previous work showed that KapB was sufficient to recapitulate the elevated proinflammatory cytokine transcripts associated with KS via the disassembly of RNA granules called processing bodies (PBs). We now show that KapB is necessary for PB disassembly during latent KSHV infection. These findings demonstrate that our panel of kaposin-deficient viruses enables precise analysis of the respective contributions of individual kaposin proteins to KSHV replication. Moreover, our mutagenesis approach serves as a guide for the functional analysis of other complex multicistronic viral loci. Importance: Kaposi’s sarcoma-associated herpesvirus (KSHV) expresses high levels of the kaposin transcript during both latent and lytic phases of replication. Due to its repetitive, GC-rich nature and polycistronic coding capacity, until now no reagents existed to permit a methodical analysis of the role of individual kaposin proteins in KSHV replication. We report the creation of a panel of recombinant viruses and matched producer cell lines that delete kaposin proteins individually or in combination. We demonstrate the utility of this panel by confirming the requirement of one kaposin translation product to a key KSHV latency phenotype. This study describes a new panel of molecular tools for the KSHV field to enable precise analysis of the roles of individual kaposin proteins during KSHV infection.
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