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Nuclear Magnetic Resonance Studies of Retrovirus Assembly and Structure
Summary: Michael Summers is interested in the application of nuclear magnetic resonance to studies of retrovirus structure and function.
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.
Current understanding of the molecular mechanism of retroviral genome selection has been obtained mainly from studies of Moloney murine leukemia virus (MLV) and HIV-1. Mutagenesis studies have identified regions of the HIV and MLV 5'-UTRs that are essential for genome packaging. Deletion or mutation of residues near dimer-promoting elements can severely attenuate genome selection, although for both retroviruses, efficient packaging of heterologous RNAs requires most (MLV) or all (HIV) of the 5'-UTR.
The Rous sarcoma virus (RSV), an alpharetrovirus that induces connective tissue tumors in chickens via the integration of a viral src gene of cellular origin, is unusual in that efficient RNA packaging can be directed by relatively small fragments of the 5'-UTR. A 160-nucleotide fragment of the RSV 5'-UTR, termed MΨ, is capable of directing heterologous RNA packaging with efficiency that is only 2.6-fold less than that of the native, intact genome. Although the L3 stem loop is required for infectivity of the virus, the actual sequence of the L3 stem of MΨ appears not to be essential for genome packaging, and an even smaller 82-nucleotide segment (μΨ) was more recently shown to direct RNA packaging with efficiency equal to that of MΨ. The RSV 5'-UTR also contains three translational start codons (AUG-1, -2, and -3) that have been controvertibly implicated in translation initiation and genome packaging, one of which (AUG-3) resides within the μΨ sequence.
We recently used NMR data obtained for samples containing 13C,15N-labeled NC and 2H-enriched, nucleotide-specific, protonated RNAs to determine the solution structure of the NC:μΨ complex. Upon NC binding, μΨ adopts a stable secondary structure that consists of three stem loops (SL-A, SL-B, and SL-C) and an 8–base pair stem (O3). Binding is mediated by NC's two zinc knuckle domains. The amino-terminal knuckle interacts with a conserved U(217)GCG tetraloop (a member of the UNCG family; N = A, U, G, or C), and the carboxyl-terminal zinc knuckle binds to residues that flank SL-A, including residues of AUG-3. Mutations of critical nucleotides in these sequences compromise or abolish viral infectivity. Our studies reveal novel structural features important for NC:RNA binding and support the hypothesis that AUG-3 is conserved for genome packaging rather than translational control.
Membrane Recognition 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. Phosphatidylinositides comprise a class of differentially phosphorylated lipids that facilitate intracellular trafficking by establishing the identity of organelles. Cellular proteins that interact differentially with the different phosphatidylinositides can thus be targeted to specific membranes, enabling spatial distribution of subcellular activities.
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. Interestingly, the inner leaflet of retroviral membranes exhibits a high degree of negative curvature.
Gag molecules assemble at punctate sites on the plasma membrane, and there is considerable evidence that these sites comprise lipid raft microdomains. Lipid rafts contain elevated levels of cholesterol and sphingolipids with saturated fatty acids, and form liquid-ordered membrane structures. Recent studies suggest that PI(4,5)P2 molecules are homogeneously dispersed within the plasma membrane of quiescent cells and that they colocalize with lipid rafts upon stimulation by a mechanism that has yet to be identified. Our finding that the 2'-acyl chain is sequestered by the protein suggests a potential mechanism for the lateral targeting of PI(4,5)P2:Gag complexes to lipid rafts. It is well known that rafts interact preferentially with saturated fatty acids. In fact, substitution of the saturated myristyl group of HIV-1 Gag by unsaturated lipids reduces the affinity of Gag for rafts—but not for membranes in general—and thereby inhibits particle assembly.
Proteins that bind lipid rafts generally contain two saturated acyl chains or are anchored by adapter molecules that contain two saturated chains (for example, glycosylphosphatidylinositol [GPI]-anchored proteins). Since cellular phosphatidylinositides generally contain stearate, an 18-carbon saturated fatty acid, at the 1'-position and arachidonate, a 20-carbon fatty acid with four nonconjugated double bonds, at the 2'-position, sequestration of the 2'-chain is likely to reduce the affinity of PI(4,5)P2 for fluid regions of the membrane and promote its association with rafts. Differential sequestration of the acyl chains could serve as a general mechanism for the lateral retargeting of phosphatidylinositides within the membrane.
Grants from the National Institutes of Health provided support for these projects.
Last updated: January 29, 2007
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