1) Retrovirus capsids are unusual in that they are produced inside the maturing virion, not in the cytoplasm or nucleus of the infected cell. Capsid protein is incorporated into the provirion as part 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) domains and two spacer peptides, SP1 and SP2. CA released from the Gag shell assembles into the viral capsid, housing the RNA and NC. Evidence suggests 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 (PI), 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. The class member MI is a compound called Beviramat (BVM). We used cryo-electron tomography to show 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 structure resembling the Gag lattice of immature HIV but lacking the innermost layer that is associated with NC/RNA. These findings were published in 2011. In subsequent work, we recorded data that strongly support the view that the biconical capsids of wild-type HIV virions are assembled de novo inside maturing virions and not by a displacive transition. Virions produced in the presence of a second MI, PF-46396, resemble BVM-treated virions although this MI is less stringent in both CA-SP1 cleavage inhibition and in blocking infectivity. MI-treated virions have a shell that resembles the CA layer of the immature Gag shell but is less complete. We concluded that inhibitors like PF-46396 and BVM bind to the partially processed Gag lattice where they deny the protease access to the CA-SP1 cleavage site and prevent the release of CA. These findings were published in 2013. Next, we Investigated the mode of action of integrase (IN) inhibitors (ALLINIs allosteric IN inhibitors) and certain mutants in the integrase gene that they phenocopy. Both kinds of virions were found to contain eccentric condensates located outside empty and often malformed capsids. We were able to show 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. Based on these observations, we have proposed a role for IN in initiating core assembly and the incorporation of vRNP into the mature core. These findings were published in 2015. In a continuation of our collaboration with E. Freed (NCI), we have been investigating the role of a conserved Pro-Pro-Ile-Pro (PPIP) motif (CA residues 122-125) in the loop connecting helices 6 and 7 (H6-H7 loop) of CA in HIV-1 assembly. Data suggest that the mutations P122A and I124A impair release, infectivity, and replication, and the T58S/T107I/P122A mutant reverts to wild type (WT)-like infectivity. We have used cryo-ET and subtomogram averaging to assess how these mutations affect assembly of the immature (PR-) virion. Our results suggest that the P122A and I124A mutations impair Gag lattice coordination and that T58S/T107I/P122A produces a WT-like lattice. The PPIP motif, then, has an important role in coordinating the immature Gag lattice during virus assembly. PR+ virions are now being examined to ascertain the role of the PPIP motif in virus maturation. 2) HIV Rev is a small regulatory protein that mediates the nuclear export of genomic 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 (RRE). This complex engages with the nuclear export machinery of the host cell. Detailed structural information on this interaction is essential for the purpose of designing Rev-inhibiting antiviral drugs. 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 0.32 nm resolution. These results were published in FY 11. Our research on HIV Rev continued with further exploitation of this antibody. In particular, we constructed a single-chain version (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 that explains how the arrangement of Rev subunits can be matched to the A-shaped architecture of the RRE in export-active complexes. The paper describing this work was published late in the last reporting period: DiMattia et al., Structure, 24:1068-80 2016. 3) Over the current reporting period, our collaboration with the NIAMS Protein Expression Laboratory (P. T. Wingfield, Chief) has shifted to crystallographic studies of full-length or partial Rev constructs complexed with interaction partners. Our emphasis has been on expressing proteins of interest and setting up crystallization trials. Promising crystals have already been obtained for several Rev-containing complexes.
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