Effects of human TRIM5alpha polymorphisms on antiretroviral function and susceptibility to human immunodeficiency virus infection.

TRIM5alpha acts on several retroviruses, including human immunodeficiency virus (HIV-1), to restrict cross-species transmission. Using natural history cohorts and tissue culture systems, we examined the effect of polymorphism in human TRIM5alpha on HIV-1 infection. In African Americans, the frequencies of two non-coding SNP variant alleles in exon 1 and intron 1 of TRIM5 were elevated in HIV-1-infected persons compared with uninfected subjects.

Enthalpy measurement using calorimetry shows a significant difference in potential energy between the active and latent conformations of PAI-1.

A central feature of the serpin inhibition mechanism is insertion of the reactive center loop into the central beta-sheet (beta-sheet A). This insertion also occurs when the reactive center loop is cleaved without protease inhibition. Using this effect, we have measured the enthalpy (DeltaH) of loop cleavage and insertion for plasminogen activator inhibitor 1 (PAI-1) as -38 kcal/mol.

Increased ubiquitination and other covariant phenotypes attributed to a strain- and temperature-dependent defect of reovirus core protein mu2.

Reovirus replication and assembly are thought to occur within cytoplasmic inclusion bodies, which we call viral factories. A strain-dependent difference in the morphology of these structures reflects more effective microtubule association by the mu2 core proteins of some viral strains, which form filamentous factories, than by those of others, which form globular factories. For this report, we identified and characterized another strain-dependent attribute of the factories, namely, the extent to which they colocalized with conjugated ubiquitin (cUb).

TRIM5alpha mediates the postentry block to N-tropic murine leukemia viruses in human cells.

Murine leukemia viruses (MLVs) have been classified as N-tropic (N-MLV) or B-tropic (B-MLV), depending on their ability to infect particular mouse strains. The early phase of N-MLV infection is blocked in the cells of several mammalian species, including humans. This block is mediated by a dominant host factor that targets the viral capsid soon after virus entry into the cell has been achieved.

19F NMR studies of plasminogen activator inhibitor-1.

Plasminogen activator inhibitor-1 (PAI-1) is a 43 kDa protein involved in the regulation of fibrinolysis. PAI-1 is the principal inhibitor of tissue-type plasminogen activator (t-PA), trapping the proteinase as an acyl-enzyme covalent complex (approximately 105 kDa). Four single tryptophan mutants of PAI-1 have been constructed in which three of the four tryptophan residues (Trp86, Trp139, Trp175, and Trp262) were replaced with phenylalanine.

Mutation of the highly conserved tryptophan in the serpin breach region alters the inhibitory mechanism of plasminogen activator inhibitor-1.

We have demonstrated that interactions within the conserved serpin breach region play a direct role in the critical step of the serpin reaction in which the acyl-enzyme intermediate must first be exposed to hydrolyzing water and aqueous deacylation. Substitution of the breach tryptophan in PAI-1 (Trp175), a residue found in virtually all known serpins, with phenylalanine altered the kinetics of the reaction mechanism and impeded the ability of PAI-1 to spontaneously become latent without compromising the inherent rate of cleaved loop insertion or partitioning between the final inhibited serpin-proteinase complex and hydrolyzed serpin. Kinetic dissection of the PAI-1 inhibitory mechanism using multiple target proteinases made possible the identification of a single rate-limiting intermediate step coupled to the molecular interactions within the breach region.

Distortion of the catalytic domain of tissue-type plasminogen activator by plasminogen activator inhibitor-1 coincides with the formation of stable serpin-proteinase complexes.

Plasminogen activator inhibitor-1 (PAI-1) is a typical member of the serpin family that kinetically traps its target proteinase as a covalent complex by distortion of the proteinase domain. Incorporation of the fluorescently silent 4-fluorotryptophan analog into PAI-1 permitted us to observe changes in the intrinsic tryptophan fluorescence of two-chain tissue-type plasminogen activator (tPA) and the proteinase domain of tPA during the inhibition reaction. We demonstrated three distinct conformational changes of the proteinase that occur during complex formation and distortion.

A concerted structural transition in the plasminogen activator inhibitor-1 mechanism of inhibition.

The inhibition mechanism of serpins requires a change in structure to entrap the target proteinase as a stable acyl-enzyme complex. Although it has generally been assumed that reactive center loop insertion and associated conformational change proceeds in a concerted manner, this has not been demonstrated directly. Through the substitution of tryptophan with 7-azatryptophan and an analysis of transient reaction kinetics, we have described the formation of an inhibited serpin-proteinase complex as a single concerted transition of the serpin structure.