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Discordant Effects of Gag Cleavage Site Mutations on the IC50 of Protease Inhibitors and on HIV Replicative Capacity
Romina Quercia*1, E Dam1,2, D Descamps3, X Duval3, A Hance1, and F Clavel1
1INSERM U552, Paris, France; 2Eurofins-Viralliance, Paris, France; and 3Hosp Bichat-Claude Bernard, Paris, France
Background: Protease (PR) and Gag cleavage-site
mutations occur in vivo and in vitro under selective pressure by
protease inhibitors (PI). Cleavage-site mutations have been described as able
to partially compensate for the loss of replicative capacity of PI-resistant
viruses. However, the extent that these mutations are also involved in changes
in resistance, and the relationship between changes in IC50 and
changes in replicative capacity remain to be determined.
Methods: NL4-3- based molecular clones carrying
sequences from 3 highly resistant patient-derived viruses (ANRS109 Vista study)
were constructed. Recombinants carried either the whole Gag, PR, and reverse
transcriptase (RT) sequences (BS Clones) or PR and RT sequences without Gag (XS
clones). Two of the viruses (VIS13 and VIS18) had selected cleavage site
mutation A431V and one (VIS16) had selected I437V. These naturally selected
mutations were removed from the BS clones and introduced in the XS clones by
site-directed mutagenesis. Drug susceptibility and replication capacity were
determined using a single-cycle assay.
Results: The removal of cleavage site mutations
from BS clones produced strong reductions in IC50 to levels that
were not significantly different from that of the XS clones, which lacked
patient-derived Gag sequences. Correspondingly, the addition of cleavage site
mutations in XS clones restored resistance to levels comparable to that of BS
clones. By contrast, the reduction in replicative capacity produced by removal
of cleavage site mutations from BS clones was only partial, while their
addition in XS clones fully restored replicative capacity to levels comparable
to that of BS clones.
Conclusions: Gag cleavage site mutations not only affect
replicative capacity, but also strongly contribute to resistance.
Interestingly, their removal is sufficient to promote strong changes in IC50,
but not sufficient to promote comparable changes in replicative capacity. These
findings suggest that cleavage site mutations play a direct and important role
in resistance, but that they work in concert with other Gag determinants to
improve viral fitness.
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