During FY10, we focussed mainly on three subprojects. (1) Capsid assembly and polymorphism of retroviruses and their implications for infectivity. Retroviruses capsids are unusual in that they are assembled inside the maturing virion, not in the cytoplasm or the nucleus of the infected cell. The capsid protein is incorporated into the provirion as part of the Gag polyprotein which forms a spherical shell lining the membrane of the provirion. After the provirion has budded off, the maturational protease is activated and dissects Gag into its matrix (MA), capsid (CA) and nucleocapsid (NC) moieties. Protease inhibitors were the first antiviral drugs to be used successfully against HIV. Of the Gag fragments, CA reassembles to form the shell of the virus core, housing the viral RNA and replication enzymes. Evidence suggests that a correctly formed core is essential for infectivity;however cores are highly polymorphic. In FY08 we published our study in which cryo-electron tomography was used to visualize mature virions of Rous Sarcoma Virus, the prototypic alpha-retrovirus. The virions ranged from 105 to 175 nm in diameter and their cores were highly polymorphic. We observed angular cores in the form of irregular polyhedra;cores with continuous curvature including a few fullerene cones;and tubular cores. From the tomograms we estimated the number of CA subunits per assembled capsid and, from the virion diameters, their original complements of Gag. We found that RSV virions, like HIV, contain unassembled CA subunits and the fraction of CA that is assembled correlates with core type. These observations implied that initiation of capsid assembly is a critical determinant of core morphology. In FY10, we completed a second stage of this investigation by similarly analyzing a conditionally lethal mutant affected in capsid assembly. The primary mutation is a substitution in the major homology region (MHR), a 20-residue tract at the start of the C-terminal domain (CTD) of CA protein. A search for second-site suppressors identified a mutation in the N-terminal domain (NTD), that restores infectivity despite rendering the virus temperature-sensitive. In mutant virions produced at the non-permissive temperature, capsid assembly is obviated. At the permissive temperature, capsid assembly is restored, albeit with an altered range of polymorphism. Fewer tubular capsidsare assembled than in wild-type and more virions have closed irregular polyhedral capsids, and these are 30% larger than in wild-type. Gag processing appears to proceed normally. In FY10 we completed and published this study. Its principal finding was that although the double mutant at the permissive temperature is more efficient than the wild-type virus in assembling closed RNA-containing capsids, it is approximately 4-fold slower In the budding of immature virions and therefoer, overall, about 50% less efficient in the production of progeny virions. (2) HIV-1 Rev is a small regulatory protein that mediates the nuclear export of viral mRNAs, an essential step in the HIV replication cycle. In this process, Rev oligomerizes in association with a highly structured RNA molecule, the Rev response element. Detailed information on the structure of Rev and on this interaction will be essential for the design of antiviral drugs that act by impeding Rev's function. To date, despite intensive efforts, crystallographic studies of Rev structure have been hampered by the protein's tendency to aggregate. However, we were able to construct a hybrid monoclonal antibody whose Fab fragment forms a stable solution complex, and determined the structure of this complex at 0.32 nm resolution. It reveals a molecular dimer of Rev, bound on either side by a Fab, where the ordered portion of each Rev monomer (residues 9-65) contains two co-planar alpha-helices arranged in hairpin fashion. Subunits dimerize through overlapping of the hairpin prongs. Mating of hydrophobic patches on the outer surface of the dimer are likely to promote higher order interactions. The structure further suggests a model for Rev oligomerization onto the viral RNA. Two papers reporting these studies were published during FY10. (3) Beviramat (BVM), also known as PA-457, is an effective and specific anti-HIV drug currently in clinical trials. Prior investigations have suggested that BVM may act by blocking maturation of newly assembled virions by inhibiting the final step of proteolytic processing of the precursor Gag polyprotein. We have investigated its mode of action further by using cryo-electron tomography to determine the three-dimensional structure of virions isolated from HIV-infected cells after BVM treatment and comparing them with control mature and immature virions. We find that BVM-treated virions contain an incomplete shell of protein underlying the viral envelope, with a hexagonal honeycomb structure similar to the Gag lattice of immature HIV but lacking the innermost layer that is associated with nucleocapsid (NC) protein. We infer that this shell represents a remnant of the immature Gag lattice that has been processed, except at the CA-SP1 sites, but has remained largely intact. We also compared BVM-treated particles with virions formed by the mutant CA5, in which cleavage between CA and SP1 is also blocked. Here, we find a thinner CA-related shell with no evidence of honeycomb organization, a difference that further suggests that BVM binding stabilizes the immature lattice. In both cases, the observed failure to assemble mature nucleocapsids is consistent with the loss of infectivity.
Showing the most recent 10 out of 14 publications
