Solution Structure of the HIV-1 Intron Splicing Silencer and Its Interactions with the UP1 Domain of Heterogeneous Nuclear Ribonucleoprotein (hnRNP) A1

Abstract

Splicing patterns in human immunodeficiency virus type 1 (HIV-1) are maintained through cis regulatory elements that recruit antagonistic host RNA-binding proteins. The activity of the 3 acceptor site A7 is tightly regulated through a complex network of an intronic splicing silencer (ISS), a bipartite exonic splicing silencer (ESS3a/b), and an exonic splicing enhancer (ESE3). Because HIV-1 splicing depends on protein-RNA interactions, it is important to know the tertiary structures surrounding the splice sites. Herein, we present the NMR solution structure of the phylogenetically conserved ISS stem loop. ISS adopts a stable structure consisting of conserved UG wobble pairs, a folded 2X2 (GU/UA) internal loop, a UU bulge, and a flexible AGUGA apical loop. Calorimetric and biochemical titrations indicate that the UP1 domain of heterogeneous nuclear ribonucleoprotein A1 binds the ISS apical loop site-specifically and with nanomolar affinity. Collectively, this work provides additional insights into how HIV-1 uses a conserved RNA structure to commandeer a host RNA-binding protein.Human immunodeficiency virus type 1 (HIV-1) 2 requires controlled synthesis of its protein complement for persistent infection and successful virion production. Genome expression is tightly regulated at the levels of transcription, splicing, mRNA nuclear export, and translation (1). RNA polymerase II-dependent transcription yields a 9-kilobase (kb) polycistronic transcript that undergoes multiple rounds of alternative splicing to produce upward of 100 different viral mRNAs that are classified by their intron composition: unspliced, incompletely spliced (4 kb), and completely spliced (2 kb) (2, 3). During early phase HIV-1 replication, cytoplasmic levels of the 2-kb transcripts predominate to encode Tat, Rev, and Nef. Accumulation of unspliced and 4-kb transcripts coincides with the transition to late phase replication and expression of Gag, Gag/Pol, Vpr, Vif, and Env/Vpu. Thus, HIV-1 splicing pathways are essential components of the viral replication cycle and represent new targets for therapeutic intervention (3, 4).The identities of HIV-1 transcripts are determined by the combinatorial use of several 5Ј donor and 3Ј acceptor splice sites. Efforts to understand mechanisms of HIV-1 splicing reveal that all of the acceptor sites and splice donors D2-D4 are suboptimal due to non-consensus core splicing signals (4, 5). Proper spliced ratios are therefore established via auxiliary cis RNA features, collectively known as splicing regulatory elements. Splicing regulatory elements function either as enhancers or silencers of splicing by recruiting host proteins belonging to the serine-arginine-rich (SR) and hnRNP families, respectively. Splicing regulatory element location (intronic or exonic) influences how frequently a given splice site is utilized, thereby making it difficult to determine general rules of the mechanisms that regulate alternative splicing (6).Splicing from site D4 to A7 removes the Rev-responsive element, which leads to the accu...

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

“…An initial pool of 1024 structures was generated using Xplor-NIH 2.34[62] as previously described[63]. During this stage, hydrogen-bond, NOE, dihedral and planarity restraints were used.…”

“…NMR restraints (RDC, NOE, hydrogen bond, sugar pucker and chirality) and simulation parameters were identical to those used for the production run of the previous step, with the exception that the temperature was held at 0K for 100 ps. Inclusion of the SAXS data was accomplished by utilizing the SAXS ab initio model (creation of which is described in SAXS materials and methods section) and the emap function[72], incorporated in a manner as previously described[63]. First, Supcomb[73] was used to align the SAXS molecular envelope to each structure while allowing the search for enantiomers.…”

NMR Studies of the Structure and Function of the HIV-1 5′-Leader

“…Although NMR spectroscopy has historically been used to determine structures of relatively small RNAs that typically comprise fewer than 60 nucleotides [67,68,69,70,71,72,73,74,75], the above studies have shown that structures can be probed by NMR spectroscopy in RNAs comprising up to 688 nucleotides. When used in combination with cryo-electron microscopy (cryoEM) or small-angle X-ray scattering (SAXS), 3D structural information can be obtained for relatively large protein:RNA complexes [68,69].…”

“…When used in combination with cryo-electron microscopy (cryoEM) or small-angle X-ray scattering (SAXS), 3D structural information can be obtained for relatively large protein:RNA complexes [68,69]. An advantage of the Adenosine-H2 detected, 2 H-edited method described above is that it enables direct detection of structure, even in RNAs that exist as equilibrium mixtures of species.…”

“…Ultimately, it will be important to determine if these new NMR approaches can contribute to our understanding of the Gag:Ψ CES structures that assemble in cells and nucleate virus assembly. Although NMR alone will not likely be capable of providing all of the information needed to determine 3D structures of large Gag:5′-leader complexes, it could provide important complementary information when used in combination with other techniques such as cryo-EM [80] or SAXS [69,82]. …”