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 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).
Gag is a multidomain polyprotein that is responsible for capturing the viral genome and self-associating at appropriate cellular membranes. With the assistance of cellular machinery, several thousand copies of Gag bud from the membrane to form an immature virus particle. Subsequent to budding, the Gag proteins are cleaved by the viral protease into the matrix (MA), capsid (CA), and nucleocapsid (NC) proteins, which rearrange to form the mature and infectious virus.
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 database approach that is under development.
Our laboratory is using these NMR methods to determine the three-dimensional structure of the HIV-1 5´-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 5´-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 5´-UTR dimerization, NC binding, and RNA packaging. Our findings suggest that diploid genome selection is mediated by an RNA switch mechanism, in which conformational changes induced by U5:AUG formation expose residues that promote dimerization and NC binding. Three-dimensional structural studies of monomeric and dimeric forms of 5´-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 HIV-1 Assembly
During the late phase of HIV-1 replication, newly synthesized retroviral Gag proteins are targeted to the plasma membrane of most hematopoietic cell types, where they colocalize at lipid rafts and assemble into immature virions. Membrane binding is mediated by the MA domain of Gag, a 132-residue polypeptide containing an amino-terminal myristyl group that can adopt sequestered and exposed conformations. Recent studies indicate that cellular phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] plays a major role in regulating Gag localization and assembly.
We recently demonstrated that soluble forms of PI(4,5)P2 that contain truncated acyl chains can bind directly to HIV-1 MA, inducing a conformational change that triggers myristate exposure. Other phosphatidylinositides do not bind MA with significant affinity or trigger myristate exposure. Structural studies revealed that PI(4,5)P2 adopts an "extended lipid" conformation upon binding, in which the inositol head group and 2´–fatty acid chain bind to a hydrophobic cleft, and the 1´–fatty acid and exposed myristyl group bracket a conserved basic surface patch previously implicated in membrane binding. This PI(4,5)P2 conformation, and the predicted membrane-binding mode, are strikingly similar to those predicted in "extended lipid" phospholipid–cytochrome c models and could be used to anchor other proteins to membranes as well. We are testing this model in more robust and biologically relevant systems, with the ultimate goal of determining the 3D structure of MA bound to a PI(4,5)P2-containing membrane.
Studies of HIV-2 MA revealed similarities with HIV-1, as well as some surprising and significant differences. HIV-1 and -2 both infect CD4+ T cells and macrophages and are capable of causing AIDS. However, HIV-2 is less pathogenic than HIV-1, and most HIV-2–infected individuals live relatively normal life spans, even in the absence of antiretroviral therapy. As observed for HIV-1 MA, the myristyl group of HIV-2 MA is partially sequestered within a narrow hydrophobic tunnel. However, the myristate of HIV-2 MA is more tightly sequestered than that of the HIV-1 protein and does not exhibit concentration- or PI(4,5)P2-dependent exposure. Despite these differences, the site of HIV-2 assembly in vivo can be manipulated by enzymes that regulate PI(4,5)P2 localization. Since HIV-2 MA has a weaker myristyl switch, one might expect the HIV-2 proteins to bind membranes with reduced affinity compared to the HIV-1 proteins. Interestingly, several strains of HIV-2 were recently shown to be unable to assemble and bud from some cells that readily support HIV-1 assembly, and the defect was attributed to a reduced ability of HIV-2 Gag proteins to remain stably associated with the plasma membrane at virus assembly sites. The poor assembly properties of HIV-2 in these cells may be due to the weaker myristyl switch.
As we showed recently, single, conservative substitutions near the amino terminus of HIV-1 MA can dramatically influence the behavior of its myristyl switch in vitro, and these mutations also lead to a retargeting of HIV-1 Gag in vivo. The differences in the ability of HIV-1 and HIV-2 Gag to assemble and bud from certain cell types may be due to differences in the efficiency of the myristyl switch. A weak myristyl switch might also explain why HIV-2 replicates much more slowly than HIV-1 in vitro and may thus contribute to the lower pathogenicity of HIV-2 relative to HIV-1.
Grants from the National Institutes of Health provided support for these projects.
As of April 14, 2014