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Session 88
Poster Abstracts Mechanisms of Drug Resistance and Increased Susceptibility Tuesday, 1:30 - 3:30 pm Poster Hall |
Background: One of 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. Although the resistant variants have been reported previously, the mechanism of the resistance remains to be defined. To elucidate the mechanism in detail, we generated the resistant variants to C34, a peptide derived from gp41 carboxyl terminus heptad repeat (C-HR), in vitro.
Methods: Fusion inhibitors-resistant HIV variants were selected in vitro by propagating HIV-1NL4-3 in the increasing doses of C34. Various recombinant HIV clones (HIV-1NL4-3 background) were generated with site directed mutagenesis. Antiviral activity was determined using HeLa CD4/LTR-b-Gal cells. Replication kinetics of the variants was assessed by competitive HIV replication assay. Peptide binding assay was performed with synthesized peptides. Rev and Rev responsive element (RRE) interaction was examined by gel shift assay.
Results: After 93 passages, C34 resistant variants emerged with 5 amino acids (FNSTW) deletion in the gp120 V4 region and total 7 amino acid substitutions in the gp41 region. The resistant variants showed decreased susceptibility to C34 (83-fold). The binding assay revealed that I37K substitutions in the N-HR impaired the binding of C34, whereas N126K in the C-HR enhanced the binding to mutated N-HR, indicating both mutations were directly implicated in the resistance. On the other hand, nucleotides encoding A30 and D36 that seemed to be a secondary mutation, complementary located in the RRE and were mutated simultaneously to maintain the secondary structure of RRE impaired by mutations at I37. However, little difference (less than 10 %) by the substitutions was observed in the Rev interaction between with WT and mutated RRE.
Conclusions: HIV-acquired resistance against C34 by mutations in N-HR, which interacted with C34, however, since this region also encodes RRE, additional mutations were required to maintain viral replication, suggesting that HIV fusion is one of the attractive targets for HIV chemotherapy.
Keywords: gp41; fusion; Rev responsive element