Date Published: November 1, 2018
Publisher: Public Library of Science
Author(s): Johanna B. Withers, Eric S. Li, Tenaya K. Vallery, Therese A. Yario, Joan A. Steitz, Britt A. Glaunsinger.
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.
Kaposi’s sarcoma-associated herpesvirus (KSHV) is an opportunistic pathogen of human immunodeficiency virus (HIV) patients and the etiological agent of several human cancers, including Kaposi sarcoma and primary effusion lymphoma . The KSHV life cycle includes a latent phase, when viral gene expression is largely absent and no progeny virions are produced, and a lytic phase, characterized by robust viral gene expression and virus replication. The most abundant lytic phase viral RNA is a 1 kb, noncoding, polyadenylated nuclear RNA called PAN [2–4]; PAN RNA accounts for up to 80% of the polyadenylated RNA present in a lytically infected cell. A 3′-end element called the element for nuclear expression (ENE) is required to maintain PAN RNA at these elevated levels. Crystallographic analysis of the PAN ENE complexed with an A9 oligonucleotide revealed that the U-rich internal loop of the ENE forms a triple-stranded interaction with the poly(A) tail of its own transcript . This triple-helical RNA structure that shields the 3′ end [6–8] robustly inhibits nuclear RNA decay of PAN RNA.
We have shown that two herpesviral PAN RNA homologs are functionally interchangeable, despite lacking appreciable nucleotide sequence conservation. Knockdown or knockout of KSHV PAN RNA was previously shown to result in loss of late lytic protein expression, and consequently, a reduction in release of new virions into the surrounding media [2, 10]. In this study, we demonstrate that knockdown or knockout of RRV PAN RNA likewise causes a reduction in release of encapsulated viral DNA (Figs 1 and 2). Furthermore, by expressing either RRV or KSHV PAN RNA from the appropriate herpesvirus species-matched PAN RNA promoter, both PAN RNAs are capable of restoring progeny virion release from HEK293T cells when co-expressed with either a KSHV or RRV PAN RNA knockout bacmid in HEK293T cells (Fig 2). RRV PAN RNA is also capable of rescuing the deficiency in late lytic protein expression observed with the KSHVΔPAN bacmid. In contrast to BCBL-1 and BJAB cells, the level of intracellular viral DNA produced from a PAN RNA knockout bacmid expressed in HEK293T cells is reduced. This suggests that in addition to lacking late lytic viral proteins required for packaging viral DNA, the viral DNA itself is not available for incorporation into progeny virions. This is likely due to a deficiency in robust lytic induction, as reported ; we also observed reduced expression of early mRNA, as well as late mRNA (Fig 2). Such subtle differences in phenotype associated with the loss of PAN RNA could be attributable either to cell type differences–a phenomenon that has been observed for other herpesviral gene knockouts and viral gene expression analyses [39–41]–or to the residual level of PAN RNA present after knockdown in B-cells, which may be sufficient to promote robust viral DNA replication.