A Panel of Kaposi’s Sarcoma-Associated Herpesvirus Mutants in the Polycistronic Kaposin Locus for Precise Analysis of Individual Protein Products

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. 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.

Human coronaviruses disassemble processing bodies

“…We individually transfected each plasmid and immunostained for each of the SARS-CoV-2 proteins using antibodies to the Strep-tag II and for PBs using anti-DDX6 (Figs 6A and S5). Relative to control cells, many SARS-CoV-2 ORF transfected cells still displayed DDX6-positive PBs; however, expression of some SARS-CoV-2 genes reduced DDX6-positive puncta, including N and ORF7b (Fig 6A ) to a similar or greater extent than our positive control, the KapB protein from Kaposi's sarcoma-associated herpesvirus, which we have previously shown causes PB disassembly (Fig 6B and 6C) [13,91,97,98]. We quantified the number of DDX6-positive PBs per cell for each transfection as in [91] and found that the average number of PBs per cell was reduced relative to our negative control after transfection of eight SARS-CoV-2 genes: nsp7, ORF7b, N, ORF9b, ORF3b, nsp6, nsp1, and nsp11 (Fig 6D and 6E).…”

Human coronaviruses disassemble processing bodies

“…We individually transfected each plasmid and immunostained for each of the SARS-CoV-2 proteins using antibodies to the Strep-tag II and for PBs using anti-DDX6 (Fig 6A, S5). Relative to control cells, many SARS-CoV-2 ORF transfected cells still displayed DDX6-positive PBs; however, expression of some SARS-CoV-2 genes reduced DDX6-positive puncta, including N and ORF7b (Fig 6A) to a similar or greater extent than our positive control, the KapB protein from Kaposi’s sarcoma-associated herpesvirus which we have previously shown causes PB disassembly (Fig 6B, C) [13,91,97,98]. We quantified the number of DDX6-positive PBs per cell for each transfection as in [91] and found that the average number of PBs per cell was reduced relative to our negative control after transfection of eight SARS-CoV-2 genes: nsp7, ORF7b, N, ORF9b, ORF3b, nsp6, nsp1, and nsp11 (Fig 6D-E).…”

Variation within major internal repeats of KSHV in vivo

“…2002 ), but the preceding DR6 sequence encodes an important protein-binding domain of Kaposin B ( McCormick and Ganem 2005 ). Recently, it has been found that KSHV expressing Kaposin A only, without the Kaposins from IR2, establishes fewer episome copies during latency ( Kleer et al 2022 ). This effect may be attributed to the alteration of the GC-rich repeats in IR2, to be discussed later.…”

“…This effect may be attributed to the alteration of the GC-rich repeats in IR2, to be discussed later. Nevertheless, viruses lacking Kaposins retain the ability to reactivate and produce infectious virions ( Kleer et al 2022 ). Additionally, the expression of Kaposin B was unnecessary for KSHV-infected endothelial cells to assume spindle morphology ( Kleer et al 2022 ).…”

“…While a noncoding RNA transcribed through the IR1 TRUs was found to be critical to K8-mediated viral DNA replication during de novo infection ( Liu, Wang, and Yuan 2018 ), whether degraded IR1 TRUs can affect its function and allow for successful infection of a new host is uncertain. It has also been shown that DNA-RNA hybrids, or R-loops, form at IR1 and IR2 of KSHV; recoding the IR2 kaposin locus repeats to reduce their GC-rich character results in significantly fewer viral episome copies during latency ( Kleer et al 2022 ). GC-rich repeats are prone to forming G-quadruplex DNA secondary structures, which are mutagenic at the single-nucleotide level ( Guiblet et al.…”