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) domains and two spacer peptides, SP1 and SP2. CA released from the Gag shell assembles into the viral capsid, housing the RNA and NC. The capsid may also incorporate reverse transcriptase and the integrase but that is less clear. 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 accomplished by 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. 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 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. 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 an inner 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 studies were published in 2013. 1) Our 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) has turned out to be impaired for CA-SP1 processing, evident in the way that it phenocopies PF-46396. Our most recent results had the form of cryo-ET data that show that, like MIs, the SP1-T8I mutation stabilizes the immature-like CA-SP1 lattice (published by Fontana et al, 2016). These observations have important implications for the mechanism of action of HIV-1 MIs. 2) HIV-1 CA protein assembles into a conical core containing the viral ribonucleoprotein (vRNP) complex. After infection, the viral RNA is reverse-transcribed into double-stranded DNA, which is then incorporated into host chromosomes by the viral integrase (IN). However, recent work has shown that IN also has an important role in capsid assembly and gem packaging. Certain IN mutations (class II) and antiviral drugs (allosteric IN inhibitors, ALLINIs) result in virions that contain eccentric condensates, electron-dense aggregates located outside seemingly empty capsids. We found 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. These findings were published by Fontana et al. (2015). 3) HIV 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 (RRE). This complex engages with the nuclear export machinery of the host cell. Detailed structural information on this interaction is essential for the design of 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 3.2 resolution. These results were published in FY 11. Our research on 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 into which Rev assembles 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 was published a few months ago (DiMattia et al, 2016).
Showing the most recent 10 out of 14 publications
