Our laboratory is interested in understanding how retroviruses assemble, mature, and selectively package their RNA genomes. Nuclear magnetic resonance (NMR) and biophysical methods are the primary tools employed in our laboratory that allow us to study the structural and dynamical properties of viral constituents and their interactions under native-like solution conditions. Our efforts focus primarily on the viral Gag proteins and RNA genome of the human immunodeficiency virus (HIV), which causes AIDS, and nonhuman pathogenic retroviruses, some of which are used as vectors in human gene therapy trials and for the treatment of severe combined immunodeficiency (SCID).
Retroviral Genome Packaging
As retroviruses assemble in infected cells, two copies of the full-length genome are selected from a cellular milieu that contains a substantial excess of spliced viral and nonviral RNAs. Genome selection is mediated by interactions between the NC domain of Gag and a portion of the 5´-untranslated region (5´-UTR) of the full-length genome called the Ψ site. The Ψ sites generally overlap with regions that promote RNA dimerization, suggesting that RNA dimerization and packaging may be intimately coupled. The packaging elements also generally overlap with the major splice donor site, a region of the RNA that undergoes splicing to generate the messenger RNAs required for synthesis of the envelope proteins and, for complex retroviruses such as HIV, the accessory proteins. Understanding how these RNA elements participate in, and possibly regulate, such diverse functions is a major goal of our laboratory.
NMR is a powerful tool for probing RNA structure, but its application to larger RNAs is complicated by several factors. We have developed a suite of 2H-edited NMR approaches and applied it to the intact HIV-1 5´-leader—a 356-nucleotide RNA that is packaged into virions as a 712-nucleotide dimer. In some cases, structural elements could be identified from two-dimensional 1H NMR NOESY spectra obtained for RNAs prepared with different combinations of nucleotide- and atom-specific 2H substitutions. Assignments are facilitated by analysis of 2D NOESY spectra obtained for differentially labeled, annealed RNA fragments. In addition, elements with signals in crowded regions of the 2H-edited spectra are identified through an approach that involves replacement of a short stretch of adjacent base pairs by A-U base pairs (long-range probing by adenosine interaction detection, or lr-AID). NMR signal assignments and validation are facilitated by a computational database approach that we are helping to develop.
Our laboratory is using these NMR methods to study the three-dimensional structure of the HIV-1 5Ž-UTR. This region of the genome contains a conserved element spanning the 5'-UTR–gag junction (G328-A356, AUG), which is critical for genome dimerization and packaging and has been proposed to function as a regulatory element. We showed by NMR that AUG adopts a hairpin structure in the monomeric 5Ž-UTR (356 nt; 115 kDa) and forms intramolecular base pairs with an upstream element (U5) in the dimer. U5:AUG formation promotes dimerization and enhances binding by the NC, the cognate domain of the viral Gag protein required for packaging. Mutations that stabilize the hairpin inhibit dimerization and NC binding in vitro and strongly attenuate RNA packaging in vivo, whereas mutations that enhance U5:AUG formation promote 5Ž-UTR dimerization, NC binding, and RNA packaging. We recently determined the structure of a 155-nucleotide region of the leader that is independently capable of directing packaging (Core Encapsidation Signal; ΨCES). The RNA adopts an unexpected tandem three-way junction structure, in which residues of the major splice donor and translation initiation sites are sequestered by long-range base pairing, and guanosines essential for both packaging and high-affinity binding to the cognate Gag protein are exposed in helical junctions. The structure reveals how translation is attenuated, Gag binding promoted, and unspliced dimeric genomes selected, by the RNA conformer that directs packaging. Three-dimensional structural studies of the intact, monomeric and dimeric forms of 5Ž-UTR (under way) should provide insights into the mechanisms that differentially and temporally regulate splicing, transcription, nuclear export, packaging, and other RNA-dependent activities that are critical for viral replication.
Intracellular Trafficking and Membrane Targeting by HIV-1 Gag
Assembly of HIV-1 particles is initiated by the trafficking of viral Gag polyproteins from the cytoplasm to the plasma membrane (PM), where they co-localize and bud to form immature particles. Membrane targeting is mediated by the N-terminally myristoylated matrix (MA) domain of Gag and is dependent on the PM marker phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. Recent studies revealed that PI(4,5)P2 molecules containing truncated acyl chains [tr-PI(4,5)P2] are capable of binding MA in an “extended lipid” conformation and promoting myristoyl exposure. Here we report that tr-PI(4,5)P2 molecules also readily bind to non-membrane proteins, including HIV-1 capsid (CA), which prompted us to re-examine MA-PI(4,5)P2 interactions using native lipids and membrane mimetic liposomes and bicelles. Liposome binding trends observed using a recently developed NMR approach paralleled results of flotation assays, although the affinities measured under the equilibrium conditions of NMR experiments were significantly higher. Native PI(4,5)P2 enhanced MA binding to liposomes designed to mimic non-raft-like regions of the membrane, supporting proposals that Gag binding may nucleate raft formation. Studies with bicelles revealed a subset of surface and myr-associated MA residues that are sensitive to native PI(4,5)P2, but cleft residues that interact with the 2´-acyl chains of tr-PI(4,5)P2 molecules in aqueous solution were insensitive to native PI(4,5)P2 in bicelles. Our findings call to question extended-lipid MA:membrane binding models, and instead support a model put forward from coarse-grained simulations indicating that binding is mediated predominantly by dynamic, electrostatic interactions between conserved basic residues of MA and multiple PI(4,5)P2 and phosphatidylserine molecules. Efforts are currently being made to determine the 3D structure of HIV-1 MA bound to PI(4,5)P2- and phosphatidylseriine-containing bicelles. We are also now studying MA interactions with the intraviral domain of the HIV-1 envelope glycoprotein, and tRNAs, to understand how Gag is chaperoned to the plasma membrane and recruits viral factors during particle assembly.
Grants from the National Institutes of Health provided support for these projects.
As of April 8, 2016