Table 2.
Phenotypic characterization of recombinant HCMV pUL54 mutants and predicted enzyme structural changes
| Strain or recombinant mutant (selectiona) | UL54 conserved region | GCV |
CDV |
FOS |
Protein stability analyses |
||||
|---|---|---|---|---|---|---|---|---|---|
| EC50b (μM) | Ratioc | EC50 (μM) | Ratio | EC50 (μM) | Ratio | Predicted ΔΔGd (kcal/mol) | Domain involved | ||
| AD169 | 0.91 (0.72–1.10) | 1.1 | 0.099 (0.079–0.119) | 1.1 | 21.6 (15.1–28.1) | 0.9 | |||
| RvHB5e | 0.81 (0.56–1.07) | 1.0 | 0.085 (0.081–0.088) | 0.9 | 17.8 (10.9–24.6) | 0.7 | |||
| RvHBWTf | 0.82 (0.60–1.04) | 0.094 (0.076–0.112) | 24.3 (16.6–32.1) | ||||||
| Mutations selected with GCV-1 (from a clinical strain) | |||||||||
| 488R (10) | IV-δC | 2.84 (2.45–3.23) | 3.5 | 0.739 (0.622–0.857) | 7.9 | 15.2 (9.8–20.5) | 0.6 | 3.24 (S) | Exo |
| 539R (10) | δC (Exo III) | 2.60 (2.08–3.12) | 3.2 | 1.247 (1.037–1.456) | 13.3 | 15.9 (10.1–21.7) | 0.7 | −3.75 (D) | Exo |
| 595I (25) | δC-II | 1.10 (0.73–1.47) | 1.3 | 0.109 (0.082–0.137) | 1.2 | 47.8 (34.2–61.4) | 2.0 | 5.05 (S) | N terminal |
| 787L (1,000) | VI | 1.50 (1.14–1.86) | 1.8 | 0.082 (0.049–0.115) | 0.9 | 58.3 (35.3–81.3) | 2.4 | 2.06 (S) | Pol |
| 862F (1,000) | III-I | 1.40 (1.14–1.65) | 1.7 | 0.080 (0.057–0.104) | 0.9 | 27.2 (23.5–30.9) | 1.1 | NIg | NI |
| 946L (1,000) | I-VII | 0.93 (0.60–1.26) | 1.1 | 0.086 (0.053–0.118) | 0.9 | 57.9 (34.1–81.7) | 2.4 | 2.49 (S) | Pol |
| Mutations selected with GCV-2 (from laboratory strain AD169) | |||||||||
| 545S (10) | δC (Exo III) | 4.13 (3.02–5.24) | 5.0 | 1.113 (0.917–1.308) | 11.9 | 24.3 (16.6–32.0) | 1.0 | 1.5 (S) | Exo |
| 812L (1,000) | III | 2.14 (1.39–2.89) | 2.6 | 0.190 (0.162–0.217) | 2.0 | 20.6 (19.6–21.5) | 0.8 | 0.51 (S) | Pol |
| 829S (300) | III | 1.68 (1.20–2.16) | 2.0 | 0.148 (0.118–0.177) | 1.6 | 27.2 (21.4–33.0) | 1.1 | 1.25 (S) | Pol |
| 879G (1,000) | III-I | 1.02 (0.64–1.39) | 1.2 | 0.086 (0.061–0.110) | 0.9 | 21.7 (16.9–26.5) | 0.9 | NI | NI |
| 545S + 829S | 5.98 (2.82–9.14) | 7.3 | 0.424 (0.350–0.498) | 4.5 | 44.7 (33.4–56.0) | 1.8 | |||
| Mutations selected with FOS-1 (from laboratory strain AD169) | |||||||||
| 408D (800) | IV (Exo II) | 3.33 (2.46–4.19) | 4.0 | 0.392 (0.342–0.442) | 4.2 | 31.5 (18.7–44.2) | 1.3 | 2.31 (S) | Exo |
| 552N (300) | δC | 1.53 (1.14–1.92) | 1.9 | 0.110 (0.072–0.149) | 1.2 | 63.0 (35.2–90.9) | 2.6 | −0.17 (D) | Exo |
| 957F (3,000) | I-VII | 2.20 (1.33–3.06) | 2.7 | 0.128 (0.089–0.166) | 1.4 | 31.8 (20.5–43.1) | 1.3 | −0.2 (D) | Pol |
| 408D + 552N | 3.45 (2.55–4.36) | 4.2 | 0.181 (0.120–0.243) | 1.9 | 97.3 (60.4–134.2) | 4.0 | |||
| 408D + 957F | 4.05 (2.47–5.62) | 4.9 | 0.420 (0.318–0.522) | 4.5 | 81.5 (79.2–83.7) | 3.3 | |||
| 552N + 957F | 1.30 (1.04–1.56) | 1.6 | 0.088 (0.050–0.125) | 0.9 | 134.1 (97.8–170) | 5.5 | |||
| Mutations selected with FOS-2 (from laboratory strain AD169) | |||||||||
| 500N (300) | δC | 2.61 (2.32–2.90) | 3.2 | 0.284 (0.227–0.341) | 3.0 | 29.3 (24.1–34.4) | 1.2 | −0.5 (D) | Exo |
| 585A (300) | δC | 1.24 (0.83–1.66) | 1.5 | 0.133 (0.115–0.150) | 1.4 | 65.1 (50.0–80.3) | 2.7 | −1.03 (D) | Exo |
| 757K (3,000) | II-VI | 0.92 (0.57–1.26) | 1.3 | 0.076 (0.048–0.103) | 0.8 | 32.5 (18.9–46.0) | 1.3 | 1.11 (S) | Pol |
| 802 Mh | VI-III | 1.39 (1.14–1.64) | 1.7 | 0.090 (0.070–0.110) | 1.0 | 65.7 (45.2–86.2) | 2.7 | 0.02 (S) | Pol |
| 802V (800) | VI-III | 1.46 (1.15–1.76) | 1.8 | 0.105 (0.087–0.124) | 1.1 | 22.8 (15.4–30.2) | 0.9 | −0.7 (D) | Pol |
| 926V (3,000) | I-VII | 1.11 (0.94–1.28) | 1.3 | 0.107 (0.066–0.147) | 1.1 | 22.7 (13.9–31.5) | 0.9 | 1.49 (S) | Pol |
| 500N + 585A | 1.95 (1.34–2.56) | 2.4 | 0.227 (0.165–0.288) | 2.4 | 66.5 (44.2–88.8) | 2.7 | |||
Selection refers to the concentration (in μM) of the selection agent at which the mutation was first detected.
EC50s were determined using the LRA previously described (29). Results represent mean values of at least 3 independent experiments. Values in parentheses represent 95% confidence intervals.
Ratios express the EC50 for the recombinant mutant/EC50 for the wild-type recombinant. Ratios indicated in boldface type indicate a significant change (i.e., ratio of at least 1.7 combined with statistical significance).
The predicted effect on domain stability was evaluated using the program CUPSAT (45). Effects on structure are indicated in parentheses, with mutations classified as having either a stabilizing (S) or a destabilizing (D) effect on the domain structure (see Materials and Methods).
RvHB5 is the virus reconstituted from the original (unmodified) HCMV/BAC pHB5 (9).
RvHBWT is the wild-type virus reconstituted using our system (i.e., with the residual FRT motif).
NI, not included in the 3D model.
Mutation L802M was included as a comparison for mutation L802V identified in our studies.