To study the phenotypic effects of molecular infectious clones with L23I, four pairs of isogenic viruses were created containing either a wild-type or mutant residue at position 23 (Table ). Two pairs of recombinant molecular infectious clones were created from plasma samples that, by direct PCR sequencing, had an electrophoretic mixture at position 23 indicating the presence of a quasispecies containing a mixture of wild-type and mutant viruses. The third pair of isogenic viruses consisted of the common laboratory strain NL43 and a site-directed mutant, NL43+L23I. The fourth pair of isogenic viruses consisted of a recombinant molecular infectious clone (isolate 1277) and a modified version of that clone in which L23I was removed by site-directed mutagenesis, restoring the wild-type residue at this position (1277_23wt).
Drug susceptibility of HIV-1 isolates containing the substrate cleft mutation L23I
To create recombinant molecular infectious clones, the 3′ part of gag
and the complete protease gene amplified from samples were ligated into deleted pNL43 vector with 3′-gag
/protease deleted (pNLPFB digested with ApaI and MscI) (5
). Competent Escherichia coli
cells were transformed with vectors containing infectious HIV-1 recombinants, and colonies were selected for confirmatory sequencing and transfection into CEM cells to create infectious virus stock. Recombinant isolates were tested with the PhenoSense assay (ViroLogic, South San Francisco, Calif.), an assay that has a coefficient of variation for protease inhibitors between 14 and 17% for both wild-type and mutant HIV-1 isolates (9
Table shows the drug susceptibility results (PhenoSense, ViroLogic) of the four pairs of isolates described above and of one uncloned clinical isolate. By itself, L23I had no effect on drug susceptibility when placed in the wild-type NL43 vector. However, in clone 1277, which also contained V82I, L23I contributed to nelfinavir resistance, raising the level of resistance with V82I alone from 3.2-fold to 6.7-fold—a level similar to that observed in the uncloned clinical isolate that also had L23I and V82I (isolate 9752). In both of these comparisons, L23I was associated with decreased replication capacity (from 100% to 36% in the NL43 backbone and from 62% to 10% in clone 1277).
In combination with other major protease inhibitor resistance mutations, L23I did not have a consistent effect on drug susceptibility. For isolate 4485, L23I was associated with increased levels of resistance to amprenavir, nelfinavir, ritonavir, and saquinavir of about 50 to 100%. For isolate 4733, L23I was associated with decreased resistance to indinavir and lopinavir but increased resistance to saquinavir. In both pairs, L23I was associated with an increase in replication capacity.