During FY10, we focussed mainly on three subprojects. During FY10, we focussed on three sub-projects.
Two of them relate to aspects of capsid assembly and polymorphism and their implications for infectivity. The third addresses the structure and assembly properties of Rev, the HIV transactivator. (1) Retrovirus capsids are unusual in assembling inside the maturing virion, not in the cytoplasm or the nucleus of the infected cell. Capsid protein is incorporated into the provirion as part of the Gag polyprotein which forms a spherical shell. After the provirion has budded off, the viral protease is activated and dissects Gag into its matrix (MA), capsid (CA), and nucleocapsid (NC) components. Of these, CA reassembles to form the virus capsid, housing the RNA, NC, and the replicase. Evidence suggests that a correctly formed core is essential for infectivity;however cores are highly polymorphic. In FY08 we published a study in which cryo-electron tomography was used to visualize mature virions of Rous Sarcoma Virus, a prototypic alpha-retrovirus. Their cores were found to be highly polymorphic. From the tomograms we calculated the number of CA subunits per capsid and, from the virion diameters, their original complements of Gag. We found that RSV virions, like HIV, contain unassembled CA subunits;moreover, the fraction of CA that is assembled correlates with core morphology. These observations implied that initiation of capsid assembly is a critical determinant of core morphology. We have now completed a second stage of this investigation by analyzing a conditionally lethal mutant virus. The primary mutation is a substitution in a residue close to the start of the C-terminal domain (CTD) of CA protein. A search for 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. Of particular note, more of the mutant virions than wild-type have closed polyhedral capsids, which appears to be a requirement for infectivity. However, the mutant has a four-fold lower budding efficiency than wild-type and consequently only about 40% of its net infectivity. This study was published in FY11 (1). (2) Protease inhibitors were the first antiviral drugs to be used successfully against HIV. More recently, another class of antiviral drugs has been identified that inhibit maturation differently. One of them, Beviramat (BVM), also known as PA-457, is an effective and specific drug currently in clinical trials. We have investigated its mode of action 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. These findings were published in FY11 (3). We have gone on to carry out similar analyses with a second maturation inhibitor, PF-46396, that is chemically unrelated to BVM. We find that, as with BVM, PF-46396 treatment results in particles containing a partial shell beneath the viral membrane, suggesting similar stabilization of the immature lattice. However, unlike BVM, a certain fraction of PF-46396-inhibited particles still contain a well-formed conical core structure, suggesting a more subtle mature assembly defect than that induced by BVM. (3) 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 structured RNA molecule, the Rev response element. Detailed information on the structure of Rev and on this interaction is essential for the design of antiviral drugs that impede Rev's function. For many years crystallographic studies were hampered by Rev's tendency to aggregate. However, we were able to construct a hybrid monoclonal antibody whose Fab forms a stable complex with Rev, and solve these co-crystals at 3.2 resolution. They revealed a Rev dimer, bound on either side by a Fab, where the ordered portion of Rev (residues 9-65) contains two co-planar alpha-helices arranged in hairpin fashion. The C-terminal 40% is disordered. Subunits dimerize through overlapping of the hairpin prongs. Two papers reporting these studies were published during FY10.

Our continuing research has aimed at improving the resolution of our Rev structure. To do so, we complexed Rev with a single-chain version of the same Fab (scFv). This complex producing crystals in four different space groups that diffracted to a best resolution of 2.3 . They were all solved and all revealed essentially the same structure, implying that it is the solution structure. A paper reporting the scFv-Rev crystal structures and their implications is at an advanced stage of preparation.

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National Institute of Arthritis and Musculoskeletal and Skin Diseases
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Dearborn, Altaira D; Eren, Elif; Watts, Norman R et al. (2018) Structure of an RNA Aptamer that Can Inhibit HIV-1 by Blocking Rev-Cognate RNA (RRE) Binding and Rev-Rev Association. Structure 26:1187-1195.e4
Watts, Norman R; Eren, Elif; Zhuang, Xiaolei et al. (2018) A new HIV-1 Rev structure optimizes interaction with target RNA (RRE) for nuclear export. J Struct Biol 203:102-108
DiMattia, Michael A; Watts, Norman R; Cheng, Naiqian et al. (2016) The Structure of HIV-1 Rev Filaments Suggests a Bilateral Model for Rev-RRE Assembly. Structure 24:1068-80
Fontana, Juan; Keller, Paul W; Urano, Emiko et al. (2016) Identification of an HIV-1 Mutation in Spacer Peptide 1 That Stabilizes the Immature CA-SP1 Lattice. J Virol 90:972-8
Fontana, Juan; Jurado, Kellie A; Cheng, Naiqian et al. (2015) Distribution and Redistribution of HIV-1 Nucleocapsid Protein in Immature, Mature, and Integrase-Inhibited Virions: a Role for Integrase in Maturation. J Virol 89:9765-80
Zhuang, Xiaolei; Stahl, Stephen J; Watts, Norman R et al. (2014) A cell-penetrating antibody fragment against HIV-1 Rev has high antiviral activity: characterization of the paratope. J Biol Chem 289:20222-33
Keller, Paul W; Huang, Rick K; England, Matthew R et al. (2013) A two-pronged structural analysis of retroviral maturation indicates that core formation proceeds by a disassembly-reassembly pathway rather than a displacive transition. J Virol 87:13655-64
Cardone, Giovanni; Brecher, Matthew; Fontana, Juan et al. (2012) Visualization of the two-step fusion process of the retrovirus avian sarcoma/leukosis virus by cryo-electron tomography. J Virol 86:12129-37
Keller, Paul W; Adamson, Catherine S; Heymann, J Bernard et al. (2011) HIV-1 maturation inhibitor bevirimat stabilizes the immature Gag lattice. J Virol 85:1420-8
DiMattia, Michael A; Watts, Norman R; Stahl, Stephen J et al. (2010) Implications of the HIV-1 Rev dimer structure at 3.2 A resolution for multimeric binding to the Rev response element. Proc Natl Acad Sci U S A 107:5810-4

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