Divide and conquer: a viral bifunctional RNA utilizes both RNA and protein to modulate pro-tumourigenic pathways

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic herpesvirus that causes the endothelial cell neoplasm Kaposi's sarcoma (KS) and other B-cell malignancies. The viral transcript kaposin is largely composed of GC-rich direct repeats (DRs) and is expressed during all phases of KSHV replication. Kaposin is polycistronic, encoding for at least 3 kaposin (Kap) proteins; of these, KapB is the most abundant and well-studied. Recent work from our lab discovered that kaposin is found in nuclear speckles (NSs) and that DRs are required for NS retention. During latency, kaposin is predominantly in NSs and is not exported or translated into KapB. During viral reactivation, abundant kaposin correlates with kaposin escape from NSs and KapB protein production. Thus, although kaposin is a latent transcript, KapB protein production occurs only during lytic reactivation. The spatiotemporal regulation of kaposin translation by NS retention during KSHV latency likewise suggests that these two biomolecules may have distinct functions. Because historically kaposin was viewed as a protein-coding mRNA, overexpression studies attributed any function to KapB even though both kaposin and KapB were produced in these systems. Armed with the knowledge that kaposin is a bifunctional RNA which derives function from both the transcript and the protein(s) it encodes, it remains unclear whether prior phenotypes should be attributed to kaposin or KapB. In this study, we developed a reductionist overexpression system to uncouple the effects of these biomolecules. To mimic kaposin during viral latency, we used a construct capable of forming kaposin NSs that does not translate reported Kap proteins ({Delta}Starts). To mimic late lytic reactivation, we used a recoded construct, AltB, that does not form kaposin NSs, is readily exported from the nucleus, and translates abundant KapB. We found that KapB protein induced actin stress fibre formation, cell enlargement, increased total levels of Yes-associated protein (YAP) and increased YAP-target gene transcription including pro-growth and pro-inflammatory transcripts. Conversely, kaposin RNA induced cell autonomous p-body disassembly with an overall reduction in pro-growth and pro-inflammatory transcripts, suggesting kaposin might sustain cell survival while dampening immune signalling. Taken together, we demonstrate that kaposin RNA and KapB protein have distinct and sometimes opposing effects on cellular growth and inflammatory signalling pathways, highlighting the ability of a viral bifunctional RNA to use both its RNA and protein products to mediate viral tumourigenesis.