In FY19, we continued with two projects already outlined in the Annual Report for FY18. In one, a continuing collaboration with E. Freed (NCI), we analyzed the role of a conserved Pro-Pro-Ile-Pro (PPIP) motif (CA residues 122-125 in the loop connecting helices 6 and 7 of CA) in HIV-1 assembly. Prior data indicated that the mutations P122A and I124A impair release, infectivity, and replication, and the T58S/T107I/P122A mutant restores wild-type-like (WT-like) infectivity. We used cryo-ET with subtomogram averaging to assess how these mutations affect assembly of the immature (protease-minus, PR-) virion. Our data show that the P122A and I124A mutations impair Gag lattice coordination and T58S/T107I/P122A reverts to a WT-like lattice. We concluded that the CA PPIP loop comprises a structural element critical for formation of the immature Gag lattice. A paper reporting this work was published in October 2018. The second project, a collaboration with G. Kalpana (Albert Einstein School of Medicine), addresses the role played by a host protein, INI1, in interacting with the viral integrase to guide capsid assembly. Integrase is known to enter nascent viral particles as the distal component of the Gag-Pol polyprotein. In earlier work (see above), we showed that mutations in integrase or exposure to certain integrase-inhibiting compounds result in a high percentage of malformed capsids. Our more recent In FY19, we continued with two projects already outlined in the Annual Report for FY18. In one, a continuing collaboration with E. Freed (NCI), we analyzed the role of a conserved Pro-Pro-Ile-Pro (PPIP) motif (CA residues 122-125 in the loop connecting helices 6 and 7 of CA) in HIV-1 assembly. Prior data indicated that the mutations P122A and I124A impair release, infectivity, and replication, and the T58S/T107I/P122A mutant restores wild-type-like (WT-like) infectivity. We used cryo-ET with subtomogram averaging to assess how these mutations affect assembly of the immature (protease-minus, PR-) virion. Our data show that the P122A and I124A mutations impair Gag lattice coordination and T58S/T107I/P122A reverts to a WT-like lattice. We concluded that the CA PPIP loop comprises a structural element critical for formation of the immature Gag lattice. A paper reporting this work was published in October 2018. The second project, a collaboration with G. Kalpana (Albert Einstein School of Medicine), addresses the role played by a host protein, INI1, in interacting with the viral integrase to guide capsid assembly. Integrase is known to enter nascent viral particles as the distal component of the Gag-Pol polyprotein. In earlier work (see above), we showed that mutations in integrase or exposure to certain integrase-inhibiting compounds result in a high percentage of malformed capsids. Our more recent studies have detected similar effects involving IN1. These results are part of a paper that has been submitted for publication. 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 the process, Rev oligomerizes in association with a structured RNA molecule, the Rev response element (RRE). The resulting 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 solved these co-crystals at 0.32 nm resolution. These results were published in FY 11. Our research on HIV Rev has continued with further exploitation of this antibody. In particular, a single-chain version (scFv) was found to co-crystallize with Rev, giving crystals that diffracted to significantly higher resolution. These crystals, coming in four different space groups, 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 Over the current reporting period, activity on this projecthas focused on crystallographic studies of Rev constructs alone or complexed with interaction partners. Rev has two domains an alfa-helical hairpin NTD, called the assembly domain, followed by a disordered CTD. We determined a crystal structure for the assembly domain at 2.25 resolution without resort to mutations or chaperones. It reveals a subunit arrangement which suggests how four molecules of Rev can assemble at two interacting sites on the RRE to form a specificity check-point that can be further stabilized by the binding of additional copies of Rev.