Kaposi's sarcoma-associated herpesvirus (KSHV) is the infectious cause of several human cancers including the endothelial cell (EC) malignancy, Kaposi's sarcoma. Unique KSHV genes absent from other human herpesvirus genomes, known as K-genes, are typically important for KSHV replication and pathogenesis. Among the K-genes, the kaposin mRNA is highly expressed in both latent and lytic phases of infection, but its polycistronic nature has hindered methodical analysis of the role of kaposin translation products in viral replication. At least three proteins are produced from the kaposin transcript, Kaposin A (KapA), B (KapB), and C (KapC). We have previously shown that KapB overexpression is sufficient to recapitulate two KS phenotypes, EC spindling and elevated proinflammatory cytokine transcripts, the latter which proceeds via the disassembly of RNA decay granules called processing bodies (PBs). To pinpoint the relative contributions of kaposin proteins at different stages of KSHV infection, we constructed four recombinant viruses by deleting or recoding the kaposin locus. Latent infection of iSLK cells with kaposin-deficient viruses resulted in reduced viral genome copy number and small LANA nuclear bodies; despite this, all iSLK cells were capable of progeny virion production. De novo infection of ECs revealed that KapB was dispensable for EC spindling but required for PB disassembly during KSHV latency, suggesting other viral proteins contribute to spindling. These findings demonstrate that our panel of viruses enables precise analysis of respective contributions of individual kaposin proteins to KSHV replication. This approach serves as a guide for the functional analysis of complex multicistronic viral loci.

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. In previous work, we showed KapB alone recapitulated the elevated proinflammatory cytokine transcripts associated with KS via the disassembly of RNA granules called processing bodies (PBs). Using the new ΔKapB virus, we showed that KapB was necessary for this effect during latent KSHV infection. Moreover, we observed that despite the ability of all kaposin-deficient latent iSLK cell lines to produce virions, all displayed low viral episome copy number, a defect that became more pronounced after primary infection of naïve ECs. For ΔKapB, provision of KapB in trans failed to complement the defect, suggesting a requirement for the kaposin locus in cis . 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.