Two herpesviral noncoding PAN RNAs are functionally homologous but do not associate with common chromatin loci

Abstract

During lytic replication of Kaposi’s sarcoma-associated herpesvirus (KSHV), a nuclear viral long noncoding RNA known as PAN RNA becomes the most abundant polyadenylated transcript in the cell. Knockout or knockdown of KSHV PAN RNA results in loss of late lytic viral gene expression and, consequently, reduction of progeny virion release from the cell. Here, we demonstrate that knockdown of PAN RNA from the related Rhesus macaque rhadinovirus (RRV) phenocopies that of KSHV PAN RNA. These two PAN RNA homologs, although lacking significant nucleotide sequence conservation, can functionally substitute for each other to rescue phenotypes associated with the absence of PAN RNA expression. Because PAN RNA is exclusively nuclear, previous studies suggested that it directly interacts with host and viral chromatin to modulate gene expression. We studied KSHV and RRV PAN RNA homologs using capture hybridization analysis of RNA targets (CHART) and observed their association with host chromatin, but the loci differ between PAN RNA homologs. Accordingly, we find that KSHV PAN RNA is undetectable in chromatin following cell fractionation. Thus, modulation of gene expression at specific chromatin loci appears not to be the primary, nor the pertinent function of this viral long noncoding RNA. PAN RNA represents a cautionary tale for the investigation of RNA association with chromatin whereby cross-linking of DNA spatially adjacent to an abundant nuclear RNA gives the appearance of specific interactions. Similarly, PAN RNA expression does not affect viral transcription factor complex expression or activity, which is required for generation of the late lytic viral mRNAs. Rather, we provide evidence for an alternative model of PAN RNA function whereby knockdown of KSHV or RRV PAN RNA results in compromised nuclear mRNA export thereby reducing the cytoplasmic levels of viral mRNAs available for production of late lytic viral proteins.

Author summary

Herpesviruses produce noncoding RNAs, some of which are essential to the viral life cycle. One such noncoding RNA from Kaposi’s sarcoma-associated herpesvirus is the polyadenylated, nuclear (PAN) RNA, which is required for production and release of progeny virions from infected cells. In this study, we demonstrate that although lacking nucleotide sequence conservation, PAN RNAs from two related viruses–when knocked down–exhibit the same phenotype, loss of late lytic viral gene expression and progeny virion production. Moreover, they can functionally substitute for each other to rescue this phenotype. We demonstrate that, in contrast to published literature, the reduction in viral gene expression upon PAN RNA knockdown is not due to loss of PAN RNA association with conserved, specific chromatin loci, nor does PAN RNA expression affect the viral transcription factor complex required for generation of the late lytic viral mRNAs. We present data suggesting that PAN RNA instead serves as a binding platform to sequester cellular proteins that are mislocalized to the nucleoplasm. These herpesviral noncoding RNAs can serve as models for the mechanistic study of human noncoding RNAs.