CROI 2015 Program and Abstracts

Abstract Listing

Poster Abstracts

586 Structural Basis and Distal Effects of Gag Substrate Coevolution in Drug Resistance to HIV-1 Protease Kuan-Hung Lin ; Celia A. Schiffer University of Massachusetts Medical School, Worcester, MA, US

Background: HIV-1 protease is a key antiviral drug target due to its essential function of processing viral polyproteins. Among primary protease mutations, I50V is commonly observed in patients failing therapy with PIs APV and DRV. In addition to conferring resistance to PIs, I50V mutation also impairs substrate processing. Mutations in the p1-p6 cleavage site are statistically associated with I50V protease mutation in the viral sequences retrieved from patients. However, the molecular basis of how compensatory mutations restore substrate recognition in drug resistance is not clear. In this study, we report the structural basis for the co-evolution of I50V/A71V protease with the p1-p6 substrate. Methods: By applying crystallography method and molecular dynamics simulation, we investigate the structural basis of I50V/A71V protease and p1-p6 substrate co-evolution. Results: The LP1’F substrate has more vdW contacts with I50V/A71V protease compared to those in either I50V/A71V WT or WT LP1’F . This is also the case for the PP5’L substrate. In addition, the LP1’F mutation causes a distal change at the substrate P5’ proline that is in an alternative position in the WT complex. This change increases the P5’ proline’s vdW contacts. The RP4’S substrate forms an additional hydrogen bond with both WT and I50V/A71V protease through the P4’ serine side chain. This extra hydrogen bond may compensate for the loss of vdW contacts due to the smaller size of serine in these two complexes In LP1’F substrate mutation, the peptide bond between Gly51 and Gly52 in the I50V/A71V PP5’L structure is flipped compared to the other structures, and this flipped peptide bond pushes the 50s loop towards the substrate, causing increased vdW contacts. In the dynamic conformational ensemble of the WT WT structure, the distance between 80-80 loops is around 17.5 Å, and expands to 19–19.5 Å with mutations in either the protease or substrate, the co-evolution brings this distance back to 17.5–18.0 Å, similar to the WT inter-loop distance. Conclusions: Coevolving mutations in the substrate enhance substrate–protease interactions through a variety of molecular mechanisms. The effects of substrate mutations are not local, but propagate to distal parts of both the substrate and the protease. Drug resistance mutations in the protease or the substrate disturbed the active site dynamics, which was restored in all co-evolved complexes bearing complementary mutations in both the protease and the substrate. 587 Influence of Codon Pair Usage in the Evolvability of HIV-1 Maria Nevot ; Cristina Andrés; Mariona Parera; Glòria Martrus; Miguel Ángel Martínez IrsiCaixa Institute for AIDS Research, Badalona, Spain Background: The extraordinarily large number of possible encodings in natural genes is to some extent restricted by two encoding biases referred to as codon bias and codon pair bias. An unexplored aspect of the genetic architecture of HIV-1 is how codon choice influences population diversity and evolvability. Here we compared the development of HIV-1 resistance to protease inhibitors (PIs) of WT virus and a synthetic virus (MAX) carrying a codon-pair re-engineered protease sequence with 38 (13%) synonymous mutations. Methods: The re-coded protease gene segment (MAX) was synthesized de novo and recombined to an HXB2 HIV-1 infectious clone. By serial culture passages WT and MAX viruses were subjected to the selective pressure of PIs [atazanavir (ATV) and darunavir (DRV)]. Results: WT and MAX virus replicated indistinguishably in MT-4 cells or PBMCs. An initial sequence analysis of individual clones demonstrated that after one passage in MT-4 cells WT protease quasispecies diversity (p-distance) was significantly higher than that of the virus carrying the MAX protease either at the nucleotide level (0.0016 ± 0.0001 vs 0.0014 ± 0.0000, p=0.0033, unpaired t test) or at the amino acid level (0.0034 ± 0.0001 vs 0.0023 ± 0.0001, p< 0.00001). This result indicated that WT and MAX proteases may occupy different sequence spaces. To explore the evolvability of the codon pair re-coded protease, WT and MAX viruses were growth under the selective pressure of ATV and DRV. After the same number of serial passages in MT-4 cells in the presence of PIs, WT and MAX viruses developed phenotypic resistance to PIs (IC50 14.63 ± 5.39 nM and 21.26 ± 8.67 nM, for ATV; and IC50 5.69 ± 1.01 m M and 9.35 ± 1.89 for DRV, respectively). Sequence clonal analysis showed the presence, in both viruses, of resistance mutations to ATV and DRV. However, a different resistance variant repertoire appeared in the MAX virus protease. Specifically, the G16E substitution was only observed in the WT protease. In addition, L10F, L33F, K45I, G48L and L89I substitutions were only detected in the re-coded MAX protease population. Conclusions: The differences in the mutation pattern that emerged after PIs treatment suggested again that WT and MAX virus proteases occupy different sequence spaces although both virus proteases were able to develop PIs resistance. Further studies will required to elucidate whether HIV-1 sequences have evolved to optimize not only the protein coding sequence but also the DNA/RNA sequences. 588 Four Amino Acid Changes in HIV-2 Protease Confer Class-Wide PI Susceptibility Dana N. Raugi 1 ; Robert A. Smith 1 ; Matthew Coyne 1 ; Julia Olson 1 ; Kara Parker 1 ; Selly Ba 2 ; Papa Salif Sow 2 ; Moussa Seydi 2 ; Geoffrey S. Gottlieb 1 University ofWashington-Dakar HIV-2 Study Group 1 University of Washington School of Medicine, Seattle, WA, US; 2 Centre Hospitalier National Universitaire de Fann, Universite Cheikh Anta Diop de Dakar, Dakar, Senegal Background: Protease inhibitor (PI)- based regimens are the mainstay of antiretroviral therapy for HIV-2. However, HIV-2 exhibits some degree of intrinsic resistance to the majority of FDA-approved PI, only retaining clinically-useful susceptibility to lopinavir, darunavir, and saquinavir. The mechanisms for this resistance remain largely unknown; although HIV-1 and HIV-2 proteases share only 40-50% sequence identity, structural studies indicate that the ligand-binding sites differ by just four amino acids. In the current study, we examined the contributions of these four residues to intrinsic PI resistance in HIV-2. Methods: We used site-directed mutagenesis to construct an HIV-2 ROD9 molecular clone in which protease codons 32, 47, 76, and 82 were substituted to encode the amino acids found in wild-type HIV-1 (clone PR Δ 4: I32V+V47I+M76L+I82V), as well clones containing each single substitution. We used single-cycle assays to quantify the phenotypic sensitivity of the five mutant clones, as well as wild-type (WT) HIV-1 NL4-3 and HIV-2 ROD9 , to nine FDA-approved PI. EC 50 values were calculated from dose-response data using sigmoidal regression and tested for statistically significant differences by ANOVA. Results: Relative to WT HIV-2, HIV-2 clones containing single amino acid substitutions I32V, V47I, M76L, or I82V conferred effects ranging from no change to a 13-fold decrease in EC 50 , depending on the PI tested. Clone PR Δ 4 displayed significant reductions in EC 50 (3.6 to 60-fold) to all PI except saquinavir. EC 50 values for PR Δ 4 vs. WT were as follows: saquinavir 12 vs. 31 nM, ritonavir 160 vs. 580 nM, lopinavir 21 vs. 105 nM, tipranavir 120 vs. >1000 nM, indinavir 16 vs. 150 nM, nelfinavir 47 vs. 490 nM, darunavir 2.2 vs. 58 nM, amprenavir 23 vs. >1000 nM, and atazanavir 1.1 vs. 66 nM. EC 50 values for PR Δ 4 were lower than, or equivalent to, those for WT HIV-1. Conclusions: Taken together, our data show that four amino acid changes in HIV-2 protease are sufficient to confer a pattern of PI susceptibility comparable to that of HIV-1. These findings enhance our overall understanding of the genetic basis of PI susceptibility and show that active site residues in protease are the primary determinants of intrinsic PI resistance in HIV-2.

Poster Abstracts

375

CROI 2015