Previous results showed that the inhibitor and substrate specificities of chimeric FIV/HIV PRs could be altered drastically and made more similar to that of HIV-1 PR by introducing multiple substitutions into the active site of FIV PR (36
). The altered specificity of chimeric FIV mutants was shown both in in vitro
PR assays and in a cell-based Gag-Pol expression system. The results also showed that chimeric mutants could be useful as an alternative to HIV-1 for the screening of compounds for their potential as broad-based inhibitors in vitro
and ex vivo
. We were interested in generating infectious FIV mutants carrying chimeric FIV/HIV PR to enhance the utility of the FIV model system for studies of drug susceptibility and for investigations of the mechanisms involved in the development of resistance against HIV PR inhibitors. Initial attempts to construct infectious mutant FIVs that contained FIV/HIV chimeric PR with multiple substitutions were not successful (38
). Further analysis of FIV Gag polyprotein processing by the FIV/HIV chimeric PRs revealed inefficient cleavage at the NC-p2 and MA-CA junctions of FIV Gag, which was likely responsible for the loss in viral infectivity. In the present study, we identified the individual residues that were responsible for the altered processing patterns of the Gag polyprotein and then generated infectious FIV mutants encoding selected FIV/HIV chimeric PRs. Importantly for the goals of the study, these mutant FIVs demonstrated sensitivity to potent HIV-1 PR inhibitors that failed to inhibit WT FIV.
Methionine (position 56) in the flaps of FIV PR turned out to be an important structural residue that was intolerant to change. This residue resides near the tip of the flap over the S2 subsite, and thus, M5647
I could affect the interactions at the P2/P2′ positions of a substrate. PRs carrying the M5647
I mutation cleaved the FIV NC-p2 site poorly. This might be due to the presence of Gln at P2 and P2′ in the FIV NC-p2 cleavage junction (NQ
A); Gln is not present at P2 in natural HIV PR substrates. It is very likely that M5647
I has drastically changed the P2 selectivity of chimeric PR to give it a more HIV-like character; consequently, it no longer recognizes Gln at P2. Although the mechanism is still unclear, position 56 may require a more flexible and less bulky residue to maintain proper interactions with Gln in the NC-p2 junction. Gln is also found at P2 in the FIV CA-p1 cleavage junction (MQ
L-LAE). This feature is unique to FIV substrates and has never been found in the P2 position of any known HIV-1 cleavage site or any other HIV-1 PR substrate (4
). The possible role of processing at the FIV CA-p1 cleavage junction in viral infectivity is currently under investigation. Interestingly, the processing of the equivalent HIV-1 CA-p2 junction, which is the final cleavage step in the temporal processing of HIV-1 Gag (26
), is blocked by an experimental maturation inhibitor, PA-457 (bevirimat), resulting in the formation of aberrant, noninfectious HIV-1 (1
Leucine at position 97, in the “90s loop” of FIV PR, is also intolerant to change. L9780
is associated with the S1/S2 subsites, and substitutions at this position may influence the preferences at the P1/P2 positions of a substrate (20
). In addition to poor/delayed cleavage at the FIV NC-p2 junction, inefficient processing at the FIV MA-CA cleavage junction by the L9780
T mutant was also observed (Fig. ). The hydrophobic residue L97 most likely makes stronger interactions with the hydrophobic alanine and isoleucine residues at the P2/P2′ positions of the FIV MA-CA cleavage junction (QA
Q) than the polar 97T residue can form. Blockage of the cleavage of the HIV-1 MA-CA site by a mutation in P1 has been shown to strongly inhibit viral maturation, resulting in the generation of an aberrant core and the loss of viral infectivity (30
). These findings underscore the critical nature of substrate processing in the generation of infectious virus particles and are consistent with the phenotype noted with mutations at position 97.
The p2 peptide of the FIV Gag polyprotein is functionally equivalent to the late domain-containing the p6 peptide of HIV-1 Gag polyprotein and contains conserved PSAP and LXXL motifs that are essential for viral assembly and production (39
). The production and release of viral particles in FIV mutant constructs was not affected by PR mutations, despite the processing inefficiency at the NC-p2 junction. This indicates that cleavage at NC-p2 is not required for viral release. However, efficient processing at the NC-p2 junction to produce mature NC and p2 is required for FIV infectivity. The processing efficiency of the equivalent HIV-1 NC-p1-p6 cleavage junctions has been extensively examined, and proper temporal cleavage at these sites correlates with viral fitness, replication capacity, and drug resistance (11
). The findings of the current study reinforce the notion that the cleavage and maturation of NC-p2 in FIV and of the equivalent NC-p1-p6 in HIV-1 offer a new potential therapeutic target.
Two predominant FIV mutants with higher replication rates emerged after transfection with the primary 37V-6s mutant, in which the Pro resulting from the I98P mutation mutated further to either His or Ser after long passage. Some other mutations were noted in a few cloned PCR products, but subsequent generation of isogenic viruses encoding 6s-98H and 6s-98S demonstrated improved replication kinetics for the chimeric 6s FIVs. The results indicate that as with the adjoining residue 9780
, changes at residue 9881
were not well tolerated by FIV, and further change improved viral fitness and infectivity. Interestingly, the two corresponding HIV-1 PR residues, T80 and P81 at the tip of the 80s loop in HIV-1 PR, are conserved, and no drug resistance mutations have been reported at these sites (7
). Our results suggest that these two conserved residues at the tip of the 80s loop in HIV-1 PR (corresponding to the “90s loop” of FIV PR) may be a valuable target for new drug design. If a future inhibitor forms strong interactions with these two conserved residues, it may be difficult for PR to mutate in a way that allows it both to evade the drug and also to process its natural substrates in a manner that maintains infectivity.
The 6s-98H and 6s-98S mutant FIV clones also showed more HIV-1 PR-like inhibitor sensitivity, although they were still less sensitive than HIV-1 PR in vitro
. Earlier studies with the inhibitor TL-3 indicated that FIV PR, which is larger than HIV-1 PR, is sensitive to a longer inhibitor, such as TL-3. Current clinical drugs are much shorter than TL-3, and that might partially explain their lower affinity for FIV PR than for HIV-1 PR. Another reason for lower affinity is that other important substitutions, such as I3530
I, and I5748
G in the active site, were not tolerated in the mutants and thus were not included in these chimeras. Structural studies are in progress to investigate the molecular basis of inhibitor affinity between FIV and HIV-1 PRs. Temporal FIV Gag polyprotein processing by both chimeric FIVs was much more sensitive to the HIV-1 PR-inhibiting drugs DRV and LPV than was that by WT FIV. The chimeric FIV/HIV system demonstrated that HIV-1 PR inhibitors with broad-spectrum properties, such as LPV, DRV, and TL-3, can be distinguished using comparative studies of WT and chimeric FIVs, both in vitro
and ex vivo
. DRV and LPV are new FDA-approved HIV-1 PR inhibitors that are effective against WT HIV-1 and many drug-resistant HIV-1 mutants, and they have high genetic barriers (i.e., they require many mutations for drug resistance to develop) (3
). Conversely, RTV, an older HIV-1 PR inhibitor, was not very potent against the 6s-98S/H PRs; it also displays a lower genetic barrier and is not as effective against many of the common drug-resistant mutants found in HIV-1 PR. Interestingly, LPV has the same core structure, P2 to P1′, as TL-3. DRV, derived from APV, has the same core P1-to-P2′ structure as APV. This suggests (i) similar inhibitor specificities for these two cores against the 6s FIV mutants and (ii) broad inhibitor selectivity for both. In addition, the profile of resistance of HIV-1 PR to LPV and DRV showed that two of the main resistance mutations in the active site, V32I and I50V, are identical to the equivalent residues of WT FIV PR, I3732
, respectively. This suggests that WT FIV PR should be resistant to LPV and DRV, which is consistent with our data. This observation may provide insight regarding the evolutionary pathways that HIV-1 PR uses to escape treatment with LPV and DRV, and it highlights the utility of structure-function studies of chimeric PRs.
HIV/FIV chimeric mutant FIVs offer an alternative cell-based infectivity system to (i) screen for new, broad-spectrum inhibitors and (ii) characterize the development of resistance against FDA-approved HIV PR inhibitors, without the significant biohazards involved in studies of infectious HIV-1 variants. In addition, this new chimeric system allows decoupling of the substrate and drug selectivity profiles to facilitate the definition of structural parameters that dictate PR specificity for drugs, for substrates, or for both. Future studies will aid in the design and development of new classes of broad-based PR inhibitors by defining these structural parameters and by performing high-throughput screens against these new chimeric PRs.