Interplay between Polyadenylate-Binding Protein 1 and Kaposi's Sarcoma-Associated Herpesvirus ORF57 in Accumulation of Polyadenylated Nuclear RNA, a Viral Long Noncoding RNA

Abstract

Polyadenylate-binding protein cytoplasmic 1 (PABPC1) is a cytoplasmic-nuclear shuttling protein important for protein translation initiation and both RNA processing and stability. We report that PABPC1 forms a complex with the Kaposi's sarcomaassociated herpesvirus (KSHV) ORF57 protein, which allows ORF57 to interact with a 9-nucleotide (nt) core element of KSHV polyadenylated nuclear (PAN) RNA, a viral long noncoding RNA (lncRNA), and increase PAN stability. The N-terminal RNA recognition motifs (RRMs) of PABPC1 are necessary for the direct interaction with ORF57. During KSHV lytic infection, the expression of viral ORF57 leads to a substantial decrease in overall PABPC1 expression, along with a shift in the cellular distribution of the remaining PABPC1 to the nucleus. Interestingly, PABPC1 and ORF57 have opposing functions in modulating PAN steady-state accumulation. The suppressive effect of PABPC1 specific to PAN expression is alleviated by small interfering RNA knockdown of PABPC1 or by overexpression of ORF57. Conversely, ectopic PABPC1 reduces ORF57 steady-state protein levels and induces aberrant polyadenylation of PAN and thereby indirectly inhibits ORF57-mediated PAN accumulation. However, E1B-AP5 (heterogeneous nuclear ribonucleoprotein U-like 1), which interacts with a region outside the 9-nt core to stimulate PAN expression, does not interact or even colocalize with ORF57. Unlike PABPC1, the nuclear distribution of E1B-AP5 remains unchanged by viral lytic infection or overexpression of ORF57. Together, these data indicate that PABPC1 is an important cellular target of viral ORF57 to directly upregulate PAN accumulation during viral lytic infection, and the ability of host PABPC1 to disrupt ORF57 expression is a strategic host counterbalancing mechanism.

Stability of Structured Kaposi's Sarcoma-Associated Herpesvirus ORF57 Protein Is Regulated by Protein Phosphorylation and Homodimerization

“…With unique sequence composition enriched in charged and hydrophilic residues, IDPs and IDRs interact with numerous ligands to exert a high binding capacity and are common in multifunctional proteins, such as the ones in cell signaling and in the viral proteome (59). We confirmed in this study and based on other reports (3,5,7,14,16) that the identified N-terminal IDR within the first ϳ250 aa residues of ORF57 is a major binding interface for ORF57 to interact with numerous cellular partners and RNAs and is highly conserved among other homologues as determined with the ANCHOR software (60, 61) (data not shown). While the structured C-terminal domain of ORF57 might play a role for a cofactor to efficiently bind to the ORF57 IDR, the finding that RBM15 and SRSF3 bind to ORF57 in a mutually exclusive manner due to both proteins binding to the same site in the N-terminal IDR (7) excludes such a possibility.…”

“…Although all ORF57 functions involve ORF57 association with an RNA target, this association also requires cellular proteins to function as ORF57 cofactors (14,15), and each of the ORF57-specific functions depends on a specific cofactor(s). This has been demonstrated by the observation that ORF57 stabilizes PAN RNA via interaction with PABPC1 (16), that ORF57 mediates K8 splicing by interaction with SRSF3 (7), that ORF57 enhances ORF59 expression by the suppression of SPEN-induced nuclear hyperpolyadenylation (4), and that ORF57 promotes vIL-6 translation by preventing Ago2, a major component of RISC complexes, from interacting with a microRNA binding site in vIL-6 RNA (6).…”

Cell Type- and Tissue Context-dependent Nuclear Distribution of Human Ago2

“…Immunofluorescence Analysis (IFA) and Confocal Microscopy-Indirect immunofluorescence antibody staining was performed as described previously (16,17). Briefly HeLa and HaCaT cells grown on poly-D-lysine-treated glass coverslips were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature.…”

New insights into the expression and functions of the Kaposi's sarcoma-associated herpesvirus long noncoding PAN RNA

“…It remains unknown how ORF57 association with PAN RNA protects the transcript from cellular decay, but the cytoplasmic poly(A)-binding protein, PABPC1 (described below) and ALYREF have been implicated in its mechanism (Li et al, 2012; Massimelli et al, 2011; 2013; Stubbs et al, 2012). ALYREF is a component of the nuclear mRNA export machinery that binds to ORF57 (Boyne et al, 2008; Majerciak et al, 2006; Malik et al, 2004; Nekorchuk et al, 2007; Reed and Cheng, 2005).…”

“…Interestingly, PABPC1 was also pulled down from cellular extract by a biotinylated MRE element but not a size-matched control (Massimelli et al, 2011; 2013) and PABPC1 binds to ORF57 in vitro (Massimelli et al, 2013). The same study reported that PABPC1 knockdown increases the accumulation of PAN RNA, suggesting a negative role for PABPC1 in PAN RNA accumulation.…”