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.

Figure 1.

Architecture of the KSHV PAN RNA. (A) Location of three main domains: i (blue), ii (orange), iii (green) and selected motifs in PAN RNA structure is indicated (top). Ex vivo 1M7 reactivities obtained for nuclear PAN RNA shown as the median reactivity over 55-nt sliding windows (middle). Shannon entropy values for the ex vivo secondary structure model smoothed over 55-nt sliding windows (bottom). High values indicate regions that probe many possible conformations, and low values indicate a well-defined structure. Yellow line indicates the global median. Gray shading marks well-defined structures with low SHAPE and low Shannon entropy. (B) Secondary structure model of PAN RNA color-coded according to SHAPE-MaP reactivity. Red notations are predicted to fall into single-stranded regions, whereas bases indicated in white correspond to constrained residues. Gray nucleotides correspond to residues for which no reactivity values were obtained, due to either pausing during reverse transcription or the position of primer hybridization. Bases are labeled every 20 nts. Helix numbering (H) is from the 5΄ to 3΄ end, and helices are differentiated if they are separated by either (i) a junction, (ii) an internal loop >12 nt in total or (iii) an asymmetric internal loop with zero residues present on one side and >6 nt on the other side. The three major domains (i–iii), together with cis-acting motifs (MRE core, ENE and poly(A) tail) are indicated.

Figure 2.

The modulating effect of (B) nuclear, (C) cytoplasmic and (D) viral environments on PAN RNA structure (A), location of three main domains: i (blue), ii (orange), iii (green) and selected motifs in PAN RNA structure. Charts represent the ratio between positive (blue) and negative (orange) reactivity differences within regions of substantial reactivity change. Blue and orange peaks indicate regions where protections or enhancements, respectively, are most pronounced. Red bars represent the position of RT stops. Bases are labeled every 100 nts.

Figure 3.

Protein binding sites detected by ΔSHAPE analysis indicated on secondary structure of (A) ex vivo nuclear, (B) ex vivo cytoplasmic and (C) ex virio PAN RNA. Nucleotides protected in cellulo are colored dark blue for strong and stable interactions (Z-factor >0, |S| ≥1) and light blue for weaker, transient interactions (Z-factor >0, 0.75 ≥|S| ≥1). The blue gradient scale under each structure indicates the standard score values.

Figure 4.

Secondary structure of polyadenylated in vitro-transcribed PAN RNA with mapped KSHV ORFs interaction sites. (A) RNP sites for each ORFs are color-coded as follows ORF26, orange), ORF57, blue, ORF59, green and ORF73/LANA, turquois. (B) Non-denaturing agarose gel indicating efficiency of PAN RNA poly(A) tailing.

Figure 5.

Three-dimensional projection model of the PAN RNA Domain III. (A) The ENE motif (green), 3΄ poly(A) tail (blue) and A9 residues involved in triple helix formation are indicated (light blue). Surrounding helices (H) are labeled accordingly (green). (B) The insert indicates the crystal structure of PAN ENE:A9 complex obtained previously (20).