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

Abstract

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) utilizes the NDP52/CALCOCO2 selective autophagy receptor to disassemble processing bodies

“…In our previous work, we showed that KSHV infection induced PB disassembly (52) and more recently we confirmed that KapB was not only sufficient but also necessary for this effect with the delB BAC16 virus (93). To determine if autophagic machinery is also required for PB disassembly in the context of KSHV infection, we silenced Atg5 or NDP52 in HUVECs prior to infecting with rKSHV.219 for 96 hours to establish latency, then stained for PBs.…”

“…In parallel, we immunoblotted infected cell lysates and observed that KSHV increased phosphorylation of Ser90 of Beclin 1 (Fig 7B), consistent with earlier observations of Ser90 phosphorylation after ectopic expression of KapB (Fig 3D). To confirm if KapB was necessary for upregulated autophagic flux after KSHV infection, we used a recombinant KSHV that does not express KapB (delB BAC16) and matched wild-type control (BAC16 KSHV), described in our recent publication (93). We infected HUVECs with WT BAC16 KSHV or delB BAC16 virus for 96 hours to permit latency establishment.…”

“…Cloning and creation of the iSLK-BAC16ΔKapB cell line is described in (93). iSLK.219 or iSLK-BAC16 cells were sub-cultured into a 10 cm cell culture dish without puromycin and reactivated from latency with either 1 μg/mL doxycycline (Sigma-Aldrich) alone (iSLK.219) and 1 μg/mL doxycycline and 1 mM sodium butyrate (Sigma-Aldrich) (iSLK-BAC16).…”

“…Plates were centrifuged at 800 x g for 2 hours at room temperature (123), the inoculum was removed and replaced with either EGM-2. For KSHV BAC16 infections, WT and ΔKapB inoculums were normalized empirically prior to infection to circumvent our previously observed impairment in latency establishment during ΔKapB infection (93). To establish robust and stable latency in Figure 7, de novo infected iSLKs were expanded from a 6-well to a 10cm dish, selected with 500 μg/mL hygromycin B (ThermoFisher), split once, then selected with 1200 μg/mL hygromycin B for a combined period of one week prior to seeding.…”

Kaposi’s sarcoma-associated herpesvirus (KSHV) utilizes the NDP52/CALCOCO2 selective autophagy receptor to disassemble processing bodies

“…In parallel, we immunoblotted infected cell lysates and observed that KSHV increased phosphorylation of Ser90 of Beclin ( Fig 7B ), consistent with earlier observations of Ser90 phosphorylation after ectopic expression of KapB ( Fig 3D ). To confirm if KapB was necessary for upregulated autophagic flux after KSHV infection, we used a recombinant KSHV that does not express KapB (delB BAC16) and matched wild-type control (BAC16 KSHV), described in [ 102 ]. We infected HUVECs with WT BAC16 KSHV or delB BAC16 virus for 96 hours to permit latency establishment.…”