TABLE 2.

E. coli expression screen and 11 clinically identified resistance-conferring mutations^a

Amino acid position contributing to clinical resistance to indinavir	Variation found in screen of E. coli expression library	Decreased susceptibility by site-directed mutagenesis
10		Yes
20		NDb
24	Yes
46		No
54	Yes
63	Yes
64	Yes
71		ND
82	Yes
84		Yes
90	Yes

^aThe 11 positions at which amino acid substitutions were identified to contribute to clinical resistance to indinavir are indicated (5). All high-level-resistant protease variants contain substitutions at either of two positions, position 82 or 90 (5). The amino acid positions which were found to be altered by using the reverse transcriptase assay to screen 12,000 E. coli library colonies and which match the amino acid substitutions in the resistant clinical isolates are also indicated. The results of site-directed mutagenesis experiments to construct protease variant genes which contain specific amino acid substitutions are also presented. These altered HIV protease genes were inserted into the E. coli expression vector pL124.23 to enable us to report protease activity as a function of reverse transcriptase activity. Decreased susceptibility was detected by the reverse transcriptase ELISA. By the E. coli expression assay, the screen identified 6 of the 11 previously identified positions which are known to be associated with clinical resistance to indinavir. Substitutions at 2 more of the 11 positions, positions 84 and 10, also show lowered susceptibility using the E. coli based assay. A substitution at position 46 does not show altered susceptibility by the E. coli-based screen. 

^bND, not done.