Human immunodeficiency virus type 1 N-terminal capsid mutants that exhibit aberrant core morphology and are blocked in initiation of reverse transcription in infected cells.

A group of conserved hydrophobic residues faces the interior of the coiled-coil-like structure within the N-terminal domain of the human immunodeficiency virus type 1 (HIV-1) capsid protein (CA). It has been suggested that these residues are important for maintaining stable structure and functional activity. To investigate this possibility, we constructed two HIV-1 clones, in which Trp23 or Phe40 was changed to Ala. We also constructed a third mutant, D51A, which has a mutation that destroys a salt bridge between Pro1 and Asp51. All three mutants are replication defective but produce virus particles. Mutant virions contain all of the viral proteins, although the amount and stability of CA are decreased and levels of virion-associated integrase are reduced. The mutations do not affect endogenous reverse transcriptase activity; however, the mutants are blocked in their ability to initiate reverse transcription in infected cells and no minus-strand strong-stop DNA is detected. The defect in reverse transcription is associated with striking defects in the morphology of mutant virus cores, as determined by transmission electron microscopy. Our data indicate that the mutations made in this study disrupt CA structure and prevent proper maturation of virus cores. We propose that this results in a defect in core stability or in an early postentry event preceding reverse transcription.

Tsg101, a homologue of ubiquitin-conjugating (E2) enzymes, binds the L domain in HIV type 1 Pr55(Gag).

Ubiquitination appears to be involved in virus particle release from infected cells. Free ubiquitin (Ub), as well as Ub covalently bound to a small fraction of p6 Gag, is detected in mature HIV particles. Here we report that the p6 region in the Pr55(Gag) structural precursor polyprotein binds to Tsg101, a putative Ub regulator that is involved in trafficking of plasma membrane-associated proteins. Tsg101 was found to interact with Gag in (i) a yeast two-hybrid assay, (ii) in vitro coimmunoprecipitation by using purified Pr55(Gag) and rabbit reticulocyte lysate-synthesized Tsg101, and (iii) in vivo in the cytoplasm of COS cells transfected with gag. The PTAPP motif [or late (L) domain] within p6, which is required for release of mature virus from the plasma membrane, was the determinant for binding Pr55(Gag). The N-terminal region in Tsg101, which is homologous to the Ubc4 class of Ub-conjugating (E2) enzymes, was the determinant of interaction with p6. Mutation of Tyr-110 in Tsg101, present in place of the active-site Cys that binds Ub in E2 enzymes, and other residues unique to Tsg101, impaired p6 interaction, indicating that features that distinguish Tsg101 from active E2 enzymes were important for binding the viral protein. The results link L-domain function in HIV to the Ub machinery and a specific component of the cellular trafficking apparatus.

Cyclophilin A-induced alterations of human immunodeficiency virus type 1 CA protein in vitro.

Cyclophilin A (CyP A), a cellular chaperone with cis-trans prolyl isomerase activity, is required for postassembly events in human immunodeficiency virus type 1 (HIV-1) replication. The requirement for CyP A maps to sequences in the capsid (CA) domain of the structural precursor, Gag. To determine the effects of interaction with CyP A on capsid (CA) protein structure, the binding interaction was investigated in vitro, using recombinant HIV-1 CA protein (which forms oligomers in solution) and human CyP A. The CA and CyP A proteins interacted to form a complex which was detected predominantly as a heterodimer on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Complex formation exhibited a pH optimum of 5. The CA protein in the complex was exclusively in a conformation whereby the N terminus was blocked to Edman degradation. This finding was unexpected since CyP A binds to the central region of the CA protein (residues 85 to 93). Examination of CA protein incubated with CyP A for alterations in structure indicated that CyP A preferentially interacted with the subpopulation of trypsin-susceptible subunits in the oligomers and significantly reduced their sensitivity to proteolysis. Like CA-CyP A complex formation, conversion to trypsin resistance also exhibited a pH optimum of 5. Both complex formation and the changes in tryptic susceptibility were only partially inhibited by cyclosporin A (CsA). This appeared to be due to a CA subpopulation able to bind CyP A despite the presence of CsA. Our results identify specific tryptic sites both proximal and distal to the CyP A binding region that are altered by CyP A binding and/or by CyP A's prolyl isomerase activity. Comparison with the X-ray structure of a complex containing CyP A bound to an amino-terminal fragment of the CA protein (CA1-151) (T.R. Gamble et al., Cell 87:1285-1294, 1996) indicates that the tryptic sites that become inaccessible are among the same residues that lose a significant amount of accessible surface area through CA-CA subunit contacts made in the presence of CyP A.

Spectral analysis and tryptic susceptibility as probes of HIV-1 capsid protein structure.

The structures of HIV-1 capsid protein (CA, p24) isolated from mature virions and CA protein autoprocessed from a recombinant Gag-Pol precursor expressed in Escherichia coli were compared using circular dichroic (CD) spectral analysis. The spectra obtained for the intact recombinant and viral proteins were indistinguishable, indicating that the backbone configurations directed by the primary amino acid sequences of the proteins were similar or identical. The structure predictions derived from CD were, in general, inconsistent with a model proposing the eight-stranded beta barrel motif found in several RNA viruses. However, aspects of the model were supported by experiments that identified surface-exposed regions. Biochemical analysis indicated that the recombinant CA protein formed nonrandom higher-ordered structures in vitro. Under physiological conditions, the protein assembled into oligomers containing subunits in two packing arrangements. In one arrangement, the central region near Arg100 was exposed and susceptible to tryptic digestion at low enzyme concentrations (enzyme:substrate ratios = 1:5000 to 1:100). Also, in this arrangement, the proteins were susceptible to crosslinking by the bifunctional agent DTSSP. Proteins in the other arrangement were resistant to proteolysis at low enzyme concentrations. The central region of these resistant molecules was inaccessible to monospecific antibodies that recognized antigenic sites between residues 94 and 107 and these proteins were not crosslinked by DTSSP or EGS. Following incubation with trypsin, both the resistant molecules and the fragments derived from the susceptible proteins in the oligomer migrated as smaller complexes, suggesting that the regions digested by trypsin stabilized the oligomer unit. The results indicate that the central region of the HIV-1 CA protein plays a role in formation of higher-ordered structures. Moreover, the relative stability of the N- and C-terminal partial digestion fragments arising from cleavage at Arg100/Gly101 suggests that this exposed central region separates two structural domains of the protein.

Assembly of recombinant human immunodeficiency virus type 1 capsid protein in vitro.

The capsid protein (CA) (p24) of human immunodeficiency virus (HIV) type 1 expressed in Escherichia coli and purified to greater than 90% homogeneity was used to examine assembly in vitro and to probe the nature of interactions involved in the formation of capsid structures. The protein was detected in dimeric and oligomeric forms as indicated by molecular size measurements by gel filtration column chromatography, sedimentation through sucrose, and nondenaturing gel electrophoresis. Chemical cross-linking of CA molecules was observed with several homobifunctional reagents. Oligomer size was dependent on cross-linker concentration and exhibited a nonrandom pattern in which dimers and tetramers were more abundant than trimers and pentamers. Oligomers as large as dodecamers were detected in native polyacrylamide gels. These were stable in solutions of high ionic strength or in the presence of nonionic detergent, indicating that strong interactions were involved in oligomer stabilization. Limited tryptic digestion converted the putative dodecamers to octamers, suggesting that a region involved in CA protein multimerization was exposed in the structure. This region was mapped to the central portion of the protein. The recombinant CA proteins assembled in vitro into long rodlike structures and were disassembled into small irregular spheres by alterations in ionic strength and pH. The observation that assembly and disassembly of purified HIV type 1 CA protein can be induced in vitro suggests an approach for identifying possible control mechanisms involved in HIV viral core assembly.