Artikel
Differences in the frameshift-regulating p1-site in treatment-naïve and PI-resistant HIV isolates
Unterschiede in der Frameshift-regulierenden p1-Region in therapie-naiven und PI-resistenten HIV
Suche in Medline nach
Autoren
Veröffentlicht: | 2. Juni 2010 |
---|
Gliederung
Text
Objectives: The HIV replication cycle include a maturation step, in which the viral enzymes are cleaved from the Gag-Pol precursor built due to a frameshift event in p1. Two Gag cleavage-sites (CS) (p7/p1, p1/p6-gag), which were found to be important in PI-resistant HIV, and Pol CS (p7/TFP, TFP/p6-pol) are encoded by a frameshift-regulating site and might contribute to differences in frameshift-efficiencies.
Methods: We analysed the p1 nucleotide sequence of 968 HIV isolates from different patients infected with HIV subtype B of whom 644 patients were treatment-naive and 324 were treatment-experienced patients failing PI therapies. RNA-structure of the p1 hairpin was predicted with m-fold. The frameshift-efficiency was determined with a dual luciferase assay.
Results: Only 21 of 52 nucleotide positions (nt2085-2136) were never subject of sequence variability compared to HxB2 and insertions and deletions were observed. Mutations at five nucleotide positions (ntG2093A, ntA2098G, ntC2108T, ntA2111C, ntC2134T/G) significantly accumulated in PI-resistant HIV. Mainly Gag CS-mutations (G435E, L449F/V) but also non-CS-mutations (S440F, H441Q) were associated with these mutations. Only mutation ntA2098G resulted in CS-mutations in gag (I437V) and pol (05G/05S) reading frame. Interestingly, I437V was correlated with PR mutations (48V, 50V, 54A/V, 82A), whereas 05G only with PR mutation (82A) and 05S with PR mutations (48V, 50V, 54A/V, 82T) in PI-resistant HIV.
The analysis of sequences (n=66) harbouring insertions, deletions, non-CS-mutations (ntC2108T/ntA2111C) and CS-mutations (G435E, 436G/R, I437M, L449P/I/H/V) revealed no significant difference in frameshift-efficiency. However, 449F lead to a significant increase in frameshift-efficiency (HxB2:6.7% vs. 449F:9.6%) due to a second slippery site and I437V in combination with additional polymorphism (I437V:10.6%). Both effects could be reversed by natural polymorphisms either dissolving the second slippery site or the interaction of the proposed lower hairpin. In PI-resistant HIV only 25 of 81 isolates harbouring 449F or 437V had evidence of a moderate increased frameshift-efficiency.
Conclusions: An increase in frameshift-efficiency is no general mechanism of HIV drug-resistance and the predicted free energy of hairpin do not correlate with measured frameshift-efficiencies. In PI-resistant HIV therapy-associated mutations occur at both C-terminal CS and there are evidence that also selection occur at Pol CS TFP/p6-pol.