Retrovirus capsids are unusual in that they are produced 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 a spherical shell of the Gag polyprotein. After the provirion has budded off, maturation ensues whereby the viral protease dissects Gag into its matrix (MA), capsid (CA), and nucleocapsid (NC) components. Of these, CA assembly into the viral capsid, housing the RNA, NC, and the replicase. Evidence has suggested that a correctly formed core is essential for infectivity. There are three ways in which interference with maturation can inhibit the virus. One is Protease Inhibitors which were the first drugs to be used successfully against HIV. More recently, maturation inhibitors (MI) have been discovered that act by blocking the protease from access to its cleavage site in the SP1 spacer peptide that connects CA to NC. See (i) below. The third is a class of compounds that sabotage maturation through their effects on the viral integrase. See (ii) below. We have been using cry-electron tomography to investigate the modes of action of the latter two inhibitors. (i) The class member MI is a compound called Beviramat (BVM). We found that virions isolated from HIV-infected cells after BVM treatment mostly lack capsids but have an incomplete shell of protein underlying the viral envelope, with a honeycomb substructure similar to the Gag lattice of immature HIV but lacking the innermost layer that is associated with NC/RNA. These findings were published in 2011. Next, we investigatedhow the biconical capsids inside mature HIV virions are generated from spherical shells of Gag molecules in immature virions. A priori, the majority of evidence suggested that capsids assemble de novo inside maturing virions from capsid (CA) protein shed from the processed Gag shell, but the possibility persisted of a displacive pathway. Our and other related observations have shown that BVM-mediated Inhibition of the cleavage between CA and spacer peptide SP1 blocks the production of mature capsids. Virions produced in the presence of a second MI. called PF-46396, resemble BVM-treated visions although this MI is less stringent in both CA-SP1 cleavage inhibition and in blocking infectivity. This compound has a similar mode of action to BVM although it is chemically quite distinct. Inhibitor-treated have a shell that resembles the CA layer of the immature Gag shell but is less complete. Some CA protein is generated but usually not enough for a mature core to assemble. We concluded that inhibitors like PF-46396 bind to the Gag lattice where they deny the protease access to the CA-SP1 cleavage site and prevent the release of CA. These studies were published in YEAR. Our most recent work with MIs concerns a mutant that phenocopies PF-46396. Propagating HIV-1 in the presence of PF-46396 selected for compound-dependent mutants. These mutants turned out to have amino acid substitutions in the major homology region (MHR) of CA. Propagation of these mutants in the absence of PF-46396 resulted in the acquisition of second-site compensatory mutations. One such mutant had a Thr-to-Ile substitution at position 8 in SP1. This mutant (T8I) turned out is impaired for CA-SP1 processing, i.e. in this respect it phenocopies PF-46396 treatment in terms of its ability to impede CA-SP1 processing. In a paper recently submitted for publication, we present cryo-ET data that show that, like MIs, the SP1-T8I mutation stabilizes the immature-like CA-SP1 lattice. These results have important implications for the mechanism of action of HIV-1 MIs. (ii) HIV-1 CA protein assembles into a conical core containing the viral ribonucleoprotein (vRNP) complex, thought to consist mainly of the viral RNA and nucleocapsid protein (NC). After infection, the viral RNA is reverse-transcribed into double-stranded DNA, which is then incorporated into host chromosomes by integrase (IN) catalysis. Certain IN mutations (class II) and antiviral drugs (allosteric IN inhibitors, ALLINIs) adversely affect maturation, resulting in virions that contain eccentric condensates, electron-dense aggregates located outside seemingly empty capsids. Our data have demonstrated that in addition to this mislocalization of electron density, a class II IN mutation and ALLINIs both have the effect of increasing the fraction of virions with malformed capsids (from 12% to 53%). We showed that eccentric condensates have a high NC content by tomo-bubblegram imaging, a novel labeling technique that exploits NC's susceptibility to radiation damage. Tomo-bubblegrams also localized NC inside wild-type cores and lining the spherical Gag shell in immature virions. We concluded that eccentric condensates represent non-packaged vRNPs and that either genetic or pharmacological inhibition of IN can impair vRNP incorporation into mature cores. The ability of ALLINIs to induce eccentric condensate formation required both IN and viral RNA. Based on these observations, we propose a role for IN in initiating core morphogenesis and vRNP incorporation into the mature core during HIV-1 maturation. 2) 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. This complex engages with the nuclear export machinery of the host cell. 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 thwarted 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. These results were published in FY 11. Our continuing research - with the broad goal of characterizing intermolecular interactions that Rev engages in in physiologically relevant complexes - has involved further exploitation of the properties of this antibody. In particular, we constructed a with a single-chain version of the same Fab (scFv) and found that it also co-crystallized with Rev and these crystals diffracted to significantly higher resolution. The crystals came in four different space groups. All were solved and revealed essentially the same structure of the monomer, although the crossing angle of the Rev dimer varies widely from 90 to 140 degrees. We also performed cryo-EM studies of helical tubes that Rev assembles into in vitro. They exhibited polymorphism, with the tube diameter varying between 11 nm and 13 nm. These variations in tube width correlated with the variations in crossing-angle seen in the crystals. Our data also revealed a third interface between Revs which offers an explanation for how the arrangement of Rev subunits is matched to the A-shaped architecture of the RRE in export-active complexes. The paper describing this work is at an advanced stage of preparation.

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8
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2015
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Arthritis, Musculoskeletal, Skin Dis
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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. (2015) 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
Uetrecht, Charlotte; Watts, Norman R; Stahl, Stephen J et al. (2010) Subunit exchange rates in Hepatitis B virus capsids are geometry- and temperature-dependent. Phys Chem Chem Phys 12:13368-71
Stahl, Stephen J; Watts, Norman R; Rader, Christoph et al. (2010) Generation and characterization of a chimeric rabbit/human Fab for co-crystallization of HIV-1 Rev. J Mol Biol 397:697-708

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