Our laboratory is interested in understanding how retroviruses assemble, mature, and selectively package their RNA genomes, and in developing therapeutic approaches for inhibiting these processes. 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 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 (SD), 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.
Phylogenetic studies identified base pair complementarity between residues of the unique 5' element and those near the gag start codon (gagAUG) that is conserved among evolutionarily distant retroviruses, suggesting a potential long-range RNA-RNA interaction. Nucleotide accessibility studies led, however, to conflicting conclusions about the presence of such interactions in virions and in infected cells. Our recent studies showed that an 11-nucleotide oligo-RNA spanning residues 105–115 of the 5'-UTR (U5) readily binds to oligoribonucleotides containing the gag start codon,disrupting a preexisting stem loop and forming a heteroduplex stabilized by 11 Watson-Crick base pairs. Addition of the HIV-1 NC, the trans-acting viral factor required for genome packaging, disrupts the heteroduplex by binding tightly to U5. The structure of the NC:U5 complex, determined by NMR, exhibits features similar to those observed in NC complexes with HIV-1 stem loop RNAs, including the insertion of guanosine nucleobases to hydrophobic clefts on the surface of the zinc fingers and a 3'-to-5' orientation of the RNA relative to protein.
Although studies with small RNA fragments provide insights into interactions that are feasible, they do not inform us about the structures that actually occur in the context of the intact 5'-UTR. We are therefore focusing on developing new NMR methods that will allow us to directly observe nucleotides in the context of the intact HIV-1 5'-UTR under a variety of experimental conditions.
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 PI(4,5)P2 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.
Although extrusion of the 2'-chain from lamellar membranes might intuitively be considered energetically expensive, a number of studies suggest that this can relieve conformational stress caused by lipids with propensities for negative membrane curvature. Since the inner leaflet of retroviral membranes exhibits a high degree of negative curvature, extrusion of the 2' chain may actually be favorable. Sequestration of the unsaturated 2'-acyl chain of PI(4,5)P2 and exposure of the myristic acid results in a structure that exposes two fully saturated acyl chains at the putative membrane-binding surface. Lipid rafts arecomprised mainly of constituents with saturated acyl chains, and cellular proteins that bind specifically to rafts are often targeted using two saturated acyl chains (for example, glycosylphosphatidylinositol [GPI]-anchored or dually acylated proteins). Thus, the PI(4,5)P2:MA structure may also explain how HIV-1 Gag proteins are targeted specifically to lipid rafts for virus assembly.
Similar 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 ofHIV-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.
