One of the human immunodeficiency virus (HIV) envelope proteins, gp41, plays a key role in HIV fusion. A gp41-derived peptide, T-20, efficiently inhibits HIV fusion and is currently approved for treatment of HIVinfected individuals. Although resistant variants have been reported, the mechanism of the resistance remains to be defined. To elucidate the mechanism in detail, we generated variants resistant to C34, a peptide derived from the gp41 carboxyl terminus heptad repeat (C-HR) in vitro. The resistant variants had a 5-amino-acid deletion in gp120 and a total of seven amino acid substitutions in gp41. Binding assays revealed that an I37K substitution in the N-terminal heptad repeat (N-HR) impaired the binding of C34, whereas an N126K substitution in the C-HR enhanced the binding to mutated N-HR, indicating that both mutations were directly involved in resistance. On the other hand, substitutions for A30 and D36 seemed to be secondary mutations, located complementary to each other in the Rev-responsive element (RRE), and were mutated simultaneously to maintain the secondary structure of the RRE that was impaired by the mutations at I37. Thus, HIV acquired resistance to C34 by mutations in N-HR, which directly interacted with C34. However, since this region also encoded the RRE, additional mutations were required to maintain viral replication. These results suggest that HIV fusion is one of the attractive targets for HIV chemotherapy.

Several reports have recently shown that drug-resistant human immunodeficiency virus type 1 (HIV-1) is often isolated from treatment-naïve patients. We phenotypically analyzed HIV-1 strains isolated from 44 treatment-naïve individuals and found two strains highly resistant (69- and >310-fold) against nevirapine (NVP). Direct sequencing showed these two isolates had a novel mutation, K238S, in reverse transcriptase (RT), but did not have any reported NVP resistance-associated mutation. A 48-h culture in the presence of NVP, however, selected HIV-1 carrying NVP resistance-associated mutations, V106A, V108I, or both, suggesting that minor viral populations of these two isolates had harbored these mutations. Replication kinetic studies of recombinant HIV-1 clones suggested that K238S conferred a significant resistance against NVP, especially when accompanied with V106A (530-fold) or V108I (56-fold). Our study identified a novel NVP resistance-associated mutation, K238S, which could be persistently detected by genotypic assay longer than V106A and V108I during off-treatment period.