PRP51
The specific aims of studying HIV-‐1 drug resistant mutants PRL76V and PRP51 is to investigate the
molecular basis of drug resistance and further assist in the design of new anti-‐viral drugs for AIDS ther-‐ apy. PRL76V and PRP51 are two drug resistant variants with a single mutation and multiple mutations, re-‐
spectively, and thus, it is important to study the effect of a single mutation and multiple mutations in drug resistance. The L76V mutation is a drug resistant mutation selected, interestingly, with decreased susceptibility to LPV, DRV, APV, and IDV but similar or increased susceptibility to ATV, NFV, SQV or TPV (Nijhuis et al., 2009a, Mueller et al., 2004, Vermeiren et al., 2007, Tartaglia et al., 2009, Young et al., 2010). The single L76V mutant has displayed reduced viral replication capacity severely. Therefore, in my study of PRL76V, the stability, activity, structures with inhibitors of RPL76V, and the autocatalytic processing
of the precursor with the single mutation L76V were explored in order to study drug resistance mecha-‐ nism of this mutation (Louis et al., 2011b). DRV and SQV are chosen to co-‐crystallize with PRL76V due to
the opposite effects in susceptibility that L76V mutation produced. The residue 76 is located in a distal region where the residues have no direct contacts with inhibitors or substrates (Figure 1.11). Kinetic as-‐ says of HIV-‐1 PRL76V revealed that PRL76V has similar catalytic activity as wild type PR. The experimental
result from thermal, urea-‐induced denaturation and dimer dissociation indicate that PRL76V has lower
stability than wild type PR. Crystal structures at high resolution of PRL76V with inhibitors, DRV and SQV
show the loss of internal van der Waals contacts of Val76 compared to Leu76 in wild type PR within hy-‐ drophobic core consistent with lower stability, which is considered as a unique molecular mechanism due to absence of contact with inhibitor or other subunit. Analysis of crystal structures showed that PRL76V has lost two hydrogen bonds with DRV in agreement with lower susceptibility to DRV and im-‐
proved interactions with SQV consistent with increased susceptibility and thus helps explain the oppo-‐ site effect of the L76V mutation on PR susceptibility to DRV and SQV. The unique molecular mechanism
for drug resistance provides the insights into the design of new anti-‐viral drugs to combat drug resis-‐ tance. L76V is also studied with new inhibitor GRL02031 designed based on DRV from structural and in-‐ hibition aspects (Chang et al., 2012). Furthermore, the L76V mutation produces a severe defect in auto-‐ processing. The detailed studies including experimental methods and results on PRL76V are described in
Chapter 2 of this dissertation and (Louis et al., 2011b).
PRP51 is an example of drug resistant mutant that contains multiple mutations evolving to give
high level resistance as generally happens when HIV replicates in the presence of drugs (Condra et al., 1999). It is of utility to explore the molecular basis of the multiple mutations and further aid the design of new anti-‐viral drugs. The highly DRV-‐resistant HIV-‐1 variant HIV-‐1MIXP51 (viral population at passage
51) was obtained by the propagation in the laboratory of a mixture of 8 highly DRV-‐susceptible HIV-‐1 clinical isolates. The mixture contains 9 to 14 PI-‐resistant mutations in the presence of DRV and repli-‐ cated well at the concentration of 5 μM DRV (Koh et al., 2010). The HIV-‐1MIXP51 protease (PRP51) has 14
amino acid substitutions (Koh et al., 2010). The studies of physical and biochemical properties of PRP51
showed that the affinity for DRV and SQV was about 7400-‐fold and 135-‐fold weaker, respectively, than the corresponding values for wild-‐type PR. The autoprocessing of the TFR-‐PRP51 precursor was uninhibi-‐
ted by DRV and marginally inhibited by SQV, at 150 μM PI concentration (Louis et al., 2011a). These properties of PRP51 are consistent with the high antiviral resistance to DRV measured for virus bearing
this variant (>300-‐fold increased EC50) relative to wild type (Koh et al., 2010). In my study of PRP51, crys-‐
tallographic study was performed to investigate the DRV-‐bound PRP51 complex and unliganded PRP51 so
as to understand the molecular basis of the high resistance to DRV of this mutant and how the multiple mutations influence the overall structure and changes in specific region of PRP51 and further aid the de-‐
sign of an anti-‐viral drug to target multiple drug resistant mutants. Two crystal structures of PRP51 bear-‐
ing the inactivating mutation D25N to avoid autoproteolysis were determined: a DRV bound structure and a ligand free structure. The crystal structure shows that DRV was bound in an unusual position com-‐
pared to the position of DRV in wild type PR within the active site of the protein parallel to dimer inter-‐ face and also interacts with residues from two symmetry related dimers. The interactions between the variant and DRV mainly depend on van der Waals interaction and fewer hydrogen bonds compared with DRV bound in wild type PR. In both the unliganded PRP51 and DRV bound PRP51 structures, the flaps are
more separated from each other than seen in wild type PR. The multiple mutations effect the structural changes in PRP51/DRV. The structural information suggests that the large separation of flaps may be a
common mechanism for resistance to PIs and the unique binding site for DRV provides the hint for de-‐ signing novel types of antiviral inhibitors that capture the open, inactive conformation of the protease. The description of studies and findings on the crystal structures PRP51 is provided in Chapter 3 of this dis-‐
sertation.