The goal of this study was to explore the presence of integrase strand transfer inhibitor (InSTI) resistance mutations in HIV-1 quasispecies present in InSTI-naïve patients and to evaluate their in vitro effects on phenotypic susceptibility to InSTIs and their replication capacities. The RT-RNase H-IN region was PCR amplified from plasma viral RNA obtained from 49 HIV-1 subtype B-infected patients (21 drug naïve and 28 failing highly active antiretroviral therapy [HAART] not containing InSTIs) and recombined with an HXB2-based backbone with RT and IN deleted. Recombinant viruses were tested against raltegravir and elvitegravir and for replication capacity. Three-hundred forty-four recombinant viruses from 49 patients were successfully analyzed both phenotypically and genotypically. The majority of clones were not phenotypically resistant to InSTIs: 0/344 clones showed raltegravir resistance, and only 3 (0.87%) showed low-level elvitegravir resistance. No primary resistance mutations for raltegravir and elvitegravir were found as major or minor species. The majority of secondary mutations were also absent or rarely present. Secondary mutations, such as T97A and G140S, found rarely and only as minority quasispecies, were present in the elvitegravir-resistant clones. A novel mutation, E92G, although rarely found in minority quasispecies, showed elvitegravir resistance. Preexisting genotypic and phenotypic raltegravir resistance was extremely rare in InSTI-naïve patients and confined to only a restricted minority of secondary variants. Overall, these results, together with others based on population and ultradeep sequencing, suggest that at this point IN genotyping in all patients before raltegravir treatment may not be cost-effective and should not be recommended until evidence of transmitted drug resistance to InSTIs or the clinical relevance of IN minor variants/polymorphisms is determined.

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The gp41-encoding sequence of the env gene contains in two separate regions the Rev-responsive elements (RRE) and the alternative open reading frame of the second exon of the regulatory protein Rev. The binding of Rev to the RRE allows the transport of unspliced/singly spliced viral mRNAs out of the nucleus, an essential step in the life cycle of human immunodeficiency virus type 1 (HIV-1). In this study, we have investigated whether the fusion-inhibitor enfuvirtide (ENF) can induce mutations in Rev and if these mutations correlate with the classical ENF resistance gp41 mutations and with viremia and CD4 cell count. Specific Rev mutations were positively associated with ENF treatment and significantly correlated with classical ENF resistance gp41 mutations. In particular, a cluster was observed for the Rev mutations E57A (E57A rev ) and N86S rev with the ENF resistance gp41 mutations Q40H (Q40H gp41 ) and L45M gp41 . In addition, the presence at week 48 of the E57A rev correlates with a significant viremia increase from baseline to week 48 and with a CD4 cell count loss from baseline to week 48. By modeling the RRE structure, we found that the Q40 gp41 and L45 gp41 codons form complementary base pairs in a region of the RRE involved in Rev binding. The conformation of this Revbinding site is disrupted when Q40H gp41 and L45M gp41 occur alone while it is restored when both mutations are present. In conclusion, our study shows that ENF pressure may also affect both Rev and RRE structures and can provide an excellent example of compensatory evolution. This highlights the multiple roles of ENF (and perhaps other entry inhibitors) in modulating the correct interplay between the different HIV-1 genes and proteins during the HIV-1 life cycle.Retroviruses, such as human immunodeficiency virus (HIV), employ a variety of different overlapping reading frames and splicing events to express a large array of messenger RNAs (mRNAs) (over 30) and proteins (at least 15) from a single primary transcript (5). The HIV type 1 (HIV-1) RNA sequence contains at least four different 5Ј splice sites and eight different 3Ј splice sites that are used alternatively (28, 32, 34). During HIV-1 replication cycle, three groups of viral mRNAs are produced (Fig. 1). One group includes the doubly spliced 2-kb transcripts that encode the regulatory proteins Tat, Rev, and Nef (32). Another group includes the singly spliced mRNAs of approximately 4 kb that serve for the production of Vif, Vpr, Vpu, and Env proteins (gp120 and gp41). The last group includes the 9-kb unspliced mRNAs that encode the Gag and Gag-Pol polyprotein products and serve as genomic RNA for packaging into virions (32, 34). The doubly spliced mRNAs are produced early and are transported out of the nucleus into the cytoplasm by the ordinary cell machinery. In contrast, the export of the singly spliced and unspliced viral mRNAs from the nucleus to the cytoplasm is mediated by the interaction between the regulatory protein Rev and a nucleotide RNA sequence named Rev-responsive elemen...

To date, very little information is available regarding the evolution of drug resistance mutations during treatment interruption (TI). Using a survival analysis approach, we investigated the dynamics of mutations associated with resistance to nucleoside analogue reverse transcriptase inhibitors (NRTIs) during TI. Analyzing 132 patients having at least two consecutive genotypes, one at last NRTI-containing regimen failure, and at least one during TI, we observed that the NRTI resistance mutations disappear at different rates during TI and are lost independently of each other in the majority of patients. The disappearance of the K65R and M184I/V mutations occurred in the majority of patients, was rapid, and was associated with the reemergence of wild-type virus, thus showing their negative impact on viral fitness. Overall, it seems that the loss of NRTI drug resistance mutations during TI is not an ordered process, and in the majority of patients occurs without specific interaction among mutations.