Kaposi's sarcoma-associated herpesvirus polyadenylated nuclear RNA: a structural scaffold for nuclear, cytoplasmic and viral proteins

Abstract

Kaposi's sarcoma-associated herpes virus (KSHV) polyadenylated nuclear (PAN) RNA facilitates lytic infection, modulating the cellular immune response by interacting with viral and cellular proteins and DNA. Although a number nucleoprotein interactions involving PAN have been implicated, our understanding of binding partners and PAN RNA binding motifs remains incomplete. Herein, we used SHAPE-mutational profiling (SHAPE-MaP) to probe PAN in its nuclear, cytoplasmic or viral environments or following cell/virion lysis and removal of proteins. We thus characterized and put into context discrete RNA structural elements, including the cis-acting Mta responsive element and expression and nuclear retention element (1,2). By comparing mutational profiles in different biological contexts, we identified sites on PAN either protected from chemical modification by protein binding or characterized by a loss of structure. While some protein binding sites were selectively localized, others were occupied in all three biological contexts. Individual binding sites of select KSHV gene products on PAN RNA were also identified in in vitro experiments. This work constitutes the most extensive structural characterization of a viral lncRNA and interactions with its protein partners in discrete biological contexts, providing a broad framework for understanding the roles of PAN RNA in KSHV infection.

Long non‐coding subgenomic flavivirus RNAs have extended 3D structures and are flexible in solution

“…The size of the minimal ensemble was varied from 1 to 20 (1,2,3,4,6,8,20) to test the effect of ensemble size on the quality of the fitting. Starting from the atomic models of the respective full-length sfRNAs, which were assembled with the available atomic models of individual subdomains as template in Rosetta, conformational pools were generated for the respective sfRNAs using the Xplor-NIH package, which follows a simulated annealing protocol driven by molecular dynamic simulation in torsion angle space that is subject to a target function comprising of bond length, bond angles, improper dihedral angles which specify chirality and planarity of functional groups, a quartic van der Waals repulsion, and R g terms to prevent atomic overlap, a multidimensional torsion angle database potential to improve backbone and sidechain conformation.…”

“…A variety of experimental techniques such as SHAPE chemical probing in combination with bioinformatics prediction methods have been developed to characterize the secondary structures of several lncRNAs in vitro (such as HOTAIR [28] and SRA [29]) or in vivo (such as PAN [4]) [30]; unfortunately, three-dimensional structural characterization of lncRNAs like sfRNAs is still challenging to traditional techniques like X-ray crystallography (XRC), nuclear magnetic resonance (NMR), and cryo-electron microscopy (cryo-EM) [31][32][33]. Recent progress in small angle X-ray scattering (SAXS) has made it a powerful tool in bridging the gap between the secondary and tertiary structures and characterizing the accessible 3D conformational space of large RNAs in solution [34,35], and SAXS might be the only reasonable method for directly acquiring structural data for large RNAs [36].…”

“…Like their host cells, many if not all viruses can make their own lncRNAs with multiple biological functions, including the regulation of viral replication, viral persistence, host immune evasion, and pathogenesis [2,3]. These diverse roles of lncRNAs are dictated by their propensities to form stable and complex secondary and higher-order structures, as evidenced by the recent research on Kaposi's sarcoma-associated herpesvirus (KSHV) polyadenylated nuclear (PAN) RNA which a viral lncRNA consists of complex structures [4].…”

Unveiling the druggable RNA targets and small molecule therapeutics

“…SHAPE and mutational profiling (SHAPE-MaP) is an example of RTmutate methods that have recently been applied in living cells to resolve the secondary structure of lncRNAs with emerging roles in human diseases. The metastasis associated lung adenocarcinoma transcript 1 (MALAT1), a highly abundant nuclear transcript associated with metastasis in non-small cell lung cancer, 48 polyadenylated nuclear (PAN) RNA, the key regulator of Kaposi's sarcoma associate herpesvirus lytic reactivation, 49,50 and X-inactive specific transcript (XIST) 51 a transcript that orchestrates mammalian X-chromosome inactivation (XCI), are just to name a few that led to increased appreciation of the functional capabilities of RNA structure. From the partition function of base pairing, a SHAPE-MaP data-informed Shannon entropy 52 can be calculated, which yields information on the diversity of RNA conformation within a given region ( Fig.…”

“…Viral genomes are highly economical when it comes to the genetic information they encode and, as such, provide only a narrow number of protein targets for therapeutic intervention. Recent studies aiming at elucidation of the structural conformation of viral RNA genomes 52 and virus-encoded coding and nc transcripts 49 greatly expanded the repertoire of drug targets.…”