HnRNP A1 Alters the Structure of a Conserved Enterovirus IRES Domain to Stimulate Viral Translation

Abstract

Enteroviruses use a type I IRES structure to facilitate protein synthesis and promote genome replication. Type I IRES elements require auxiliary host proteins to organize RNA structure for 40S ribosomal subunit assembly. Heterogeneous nuclear ribonucleoprotein A1 stimulates Enterovirus 71 (EV71) translation in part through specific interactions with its stem loop II (SLII) IRES domain. Here, we determined a conjoined NMR-SAXS structure of the EV71 SLII domain and a mutant that significantly attenuates viral replication by abrogating hnRNP A1 interactions. Native SLII adopts a locally compact structure wherein stacking interactions in a conserved 5′-AUAGC-3′ bulge preorganize the adjacent helices at nearly orthogonal orientations. Mutating the bulge sequence to 5′-ACCCC-3′ ablates base stacking in the loop and globally reorients the SLII structure. Biophysical titrations reveal that the 5′-AUAGC-3′ bulge undergoes a conformational change to assemble a functional hnRNP A1-RNA complex. Importantly, IRES mutations that delete the bulge impair viral translation and completely inhibit replication. Thus, this work provides key details into how an EV71 IRES structure adapts to hijack a cellular protein and it suggests the SLII domain is a potential target for antiviral therapy.

Role of MAPK/MNK1 signaling in virus replication

“…MNK1 substrate hnRNPA1 has been shown to colocalize with viral nucleoprotein in the cytoplasm, particularly in the perinuclear region of virus-infected cells (Shi et al, 2000;Wang and Zhang, 1999) and is believed to regulates not only IRES-dependent translation initiation in enteroviruses (Tolbert et al, 2017), but also non-IRES-dependent translation initiation in SINV (Lin et al, 2009) and HCV (Kim et al, 2007;Li et al, 1997).…”

Small Molecule Targeting IRES Domain Inhibits Enterovirus 71 Replication via an Allosteric Mechanism that Stabilizes a Ternary Complex

“…Extensive studies of the EV71 IRES have revealed functional contributions of different RNA structures along with the collection of cellular proteins that interact with those structures [5][6][7][8][9][10][11][12][13][14][15] . Of particular importance, the stem loop II (SLII) domain interacts specifically with at least four cellular RNA binding proteins (RBPs) and a viral derived small RNA to modulate translation levels 5,7,9,10,16 . The secondary structure of the EV71 SLII domain folds into a phylogenetically conserved hairpin with a 7-nt apical loop and 5-nt bulge (Fig 1B) 9 .…”

“…Of particular importance, the stem loop II (SLII) domain interacts specifically with at least four cellular RNA binding proteins (RBPs) and a viral derived small RNA to modulate translation levels 5,7,9,10,16 . The secondary structure of the EV71 SLII domain folds into a phylogenetically conserved hairpin with a 7-nt apical loop and 5-nt bulge (Fig 1B) 9 . The three-dimensional structure of SLII shows that the apical loop and bulge form well-defined structural motifs (Fig 1C) 9 .…”

“…The secondary structure of the EV71 SLII domain folds into a phylogenetically conserved hairpin with a 7-nt apical loop and 5-nt bulge (Fig 1B) 9 . The three-dimensional structure of SLII shows that the apical loop and bulge form well-defined structural motifs (Fig 1C) 9 . Functional binding sites for the cellular hnRNP A1 and AUF1 proteins reside within the structured bulge, and these sites undergo a conformational change to form stable protein-RNA complexes 5,9 .…”

“…The three-dimensional structure of SLII shows that the apical loop and bulge form well-defined structural motifs (Fig 1C) 9 . Functional binding sites for the cellular hnRNP A1 and AUF1 proteins reside within the structured bulge, and these sites undergo a conformational change to form stable protein-RNA complexes 5,9 . HnRNP A1 binds the SLII bulge to stimulate translation whereas AUF1 competes for the bulge to counteract this stimulatory activity so that the levels of IRES-dependent translation are tuned to meet the replication needs of EV71.…”