Processing of the human immunodeficiency virus type 1 (HIV-1) Gag precursor is highly regulated, with differential rates of cleavage at the five major processing sites to give characteristic processing intermediates. We examined the role of the P1 amino acid in determining the rate of cleavage at each of these five sites by using libraries of mutants generated by site-directed mutagenesis. Between 12 and 17 substitution mutants were tested at each P1 position in Gag, using recombinant HIV-1 protease (PR) in an in vitro processing reaction of radiolabeled Gag substrate. There were three sites in Gag (MA/CA, CA/p2, NC/p1) where one or more substitutions mediated enhanced rates of cleavage, with an enhancement greater than 60-fold in the case of NC/p1. For the other two sites (p2/NC, p1/p6), the wild-type amino acid conferred optimal cleavage. The order of the relative rates of cleavage with the P1 amino acids Tyr, Met, and Leu suggests that processing sites can be placed into two groups and that the two groups are defined by the size of the P1 amino acid. These results point to a trans effect between the P1 and P1 amino acids that is likely to be a major determinant of the rate of cleavage at the individual sites and therefore also a determinant of the ordered cleavage of the Gag precursor.

Sequence variability associated with human immunodeficiency virus type 1 (HIV-1) is useful for inferring structural and/or functional constraints at specific residues within the viral protease. Positions that are invariant even in the presence of drug selection define critically important residues for protease function. While the importance of conserved active-site residues is easily understood, the role of other invariant residues is not. This work focuses on invariant Thr80 at the apex of the P1 loop of HIV-1, HIV-2, and simian immunodeficiency virus protease. In a previous study, we postulated, on the basis of a molecular dynamics simulation of the unliganded protease, that Thr80 may play a role in the mobility of the flaps of protease. In the present study, both experimental and computational methods were used to study the role of Thr80 in HIV protease. Three protease variants (T80V, T80N, and T80S) were examined for changes in structure, dynamics, enzymatic activity, affinity for protease inhibitors, and viral infectivity. While all three variants were structurally similar to the wild type, only T80S was functionally similar. Both T80V and T80N had decreased the affinity for saquinavir. T80V significantly decreased the ability of the enzyme to cleave a peptide substrate but maintained infectivity, while T80N abolished both activity and viral infectivity. Additionally, T80N decreased the conformational flexibility of the flap region, as observed by simulations of molecular dynamics. Taken together, these data indicate that HIV-1 protease functions best when residue 80 is a small polar residue and that mutations to other amino acids significantly impair enzyme function, possibly by affecting the flexibility of the flap domain.

Background Faculty have traditionally evaluated resident physician professionalism and interpersonal skills without input from patients, family members, nurses, or the residents themselves. The objective of our study was to use “360-degree evaluations,” as suggested by the Accreditation Council for Graduate Medical Education (ACGME), to determine if nonfaculty ratings of resident professionalism and interpersonal skills differ from faculty ratings. Methods Pediatrics residents were enrolled in a hospital-based resident continuity clinic during a 5-week period. Patient/families (P/Fs), faculty (MD [doctor of medicine]), nurses (RNs [registered nurses]), and residents themselves (self) completed evaluator-specific evaluations after each clinic session by using a validated 10-item questionnaire with a 5-point Likert scale. The average Likert score was tallied for each questionnaire. Mean Likert scale scores for each type of rater were compared by using analysis of variance, text with pair-wise comparisons when appropriate. Agreement between rater types was measured by using the Pearson correlation. Results A total of 823 evaluations were completed for 66 residents (total eligible residents, 69; 95% participation). All evaluators scored residents highly (mean Likert score range, 4.4 to 4.9). However, MDs and RNs scored residents higher than did P/Fs (mean scores: MD, 4.77, SD [standard deviation], 0.32; RN, 4.85, SD, 0.30; P/F, 4.53, SD, 0.96; P < .0001). MD and RN scores also were higher than residents' self-evaluation scores, but there was no difference between self-scores and P/F scores (average resident self-score, 4.44, SD, 0.43; P < .0001 compared to MD and RN; P  =  .19 compared to P/F). Correlation coefficients between all combinations of raters ranged from −0.21 to 0.21 and none were statistically significant. Conclusion Our study found high ratings for resident professionalism and interpersonal skills. However, different members of the health care team rated residents differently, and ratings are not correlated. Our results provide evidence for the potential value of 360-degree evaluations.