Figure 3.

Summary and analysis of HIV-1 RT mutant phenotypes. Phenotypes of single missense mutations in a 109-aa region of the HIV-1 RT. Mutants were isolated from 11 overlapping random substitution libraries designated L, M, N, O, P, Q, R, S, T, U, and V. The region mutagenized in each library is indicated by a horizontal dashed line. The secondary structural elements indicated are from the 3hvt HIV-1 RT crystal structure (24) and not from the 1rtj crystal structure (15), which is shown in Fig. 5. There are minor differences between them with respect to the end points assigned for α helices and β strands. The conservative amino acids are grouped as follows: (A, S, T, G, P); (V, L, I, M); (R, K, H); (D, E, N, Q); (F, Y, W); (C) (25). RT phenotypes are expressed as follows: uppercase = wild-type or positive; italicized uppercase = high intermediate; italicized lowercase = low intermediate; lowercase = negative. In the line labeled “RT sensitivity pattern,” those residues that are mutationally insensitive for RT activity are indicated by X; those residues that have extreme mutational sensitivity for RT activity are indicated by O; those residues that have extreme mutational sensitivity for RT activity with nonconservative substitutions, but not with conservative substitutions, are indicated by Z. The RT and Western sensitivity patterns are defined in Fig. 4. The phenotypes from Western blots of total cell lysates for residues P95 to M164 are expressed as follows: uppercase = wild type; italicized uppercase = US; italicized lowercase = UL; lowercase = Lt. The phenotypes from Western blots of total cell lysates for residues M164 to E203 are expressed as follows: uppercase = wild type; italicized uppercase = intermediate; lowercase = negative. The phenotypes from Western blots of soluble lysates are expressed as follows: red = wild type; green = faint; purple = very faint; cyan = no bands; black = not determined. In the line labeled “Western sensitivity pattern,” those residues that are mutationally insensitive for protein stability are indicated by X; those residues that have extreme mutational sensitivity for protein stability are indicated by O; those residues that have extreme mutational sensitivity for protein stability with nonconservative substitutions, but not with conservative substitutions, are indicated by Z. Roles of key residues are designated as follows: F = functionally important; C = catalytically important; S = important for protein stability (conservative interpretation); s = additional residues important for protein stability from a liberal interpretation; E = external. The roles indicated were those assigned before we made additional substitutions to study residues critical for stability in more detail. These mutants (T107S, T107V, V118T, L120A, L120T, T128V, Y146F, S191T, and H198Q) are underlined, and their phenotypes were not used to establish the sensitivity patterns. A table listing the data used to assemble this figure can be viewed at www.unc.edu/∼clyde. This table includes the actual percent RT activity for each HIV-1 RT mutant.