Treatment with the fusion inhibitor enfuvirtide influences the appearance of mutations in the human immunodeficiency virus type 1 regulatory protein rev

Abstract

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 Rev-binding 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.

FIG. 1.

Schematic view of HIV-1 genome and transcripts produced during HIV-1 replication cycle (modified from reference with permission). The ENF target region encompassing the amino acids 36 to 45 in gp41 is also shown. During the HIV-1 replication cycle, three classes of viral RNAs are produced: (i) the 9-kb unspliced mRNAs that are packaged into progeny virions as genomic RNA and can also serve for the expression of Gag/Pol genes; (ii) the singly spliced mRNAs encoding Vif, Vpr, Vpu, and env; and (iii) doubly spliced 2-kb transcripts encoding Tat, Rev, and Nef (29). The doubly spliced mRNAs encoding Tat, Rev, and Nef are the first produced and are transported into the cytoplasm by the ordinary cell machinery. When regulatory protein Rev is produced, it returns into the nucleus and binds the RRE, thus allowing the shuttling of unspliced and singly spliced mRNAs from the nucleus to the cytoplasm of HIV-1-infected cells.

FIG. 2.

Prevalence and localization of Rev mutations associated with ENF treatment. (A) The frequency of mutations was calculated in isolates from 83 ENF-näive patients and 88 patients who experienced virological failure to ENF. Statistically significant differences were assessed by chi-square tests of independence (based on a 2-by-2 contingency table). (B) Localization of Rev mutations in the schematic structure of HIV-1 Rev protein. The HIV-1 Rev is composed of several domains harboring distinct functions: (i) an NLS that mediates Rev import in the nucleus and the RRE binding; (ii) two oligomerization domains, flanking the NLS, that are implicated in the oligomerization of Rev along the RRE; (iii) an NES that acts as an NES and contains also another NLS.

FIG. 3.

Covariation analysis among gp41 and Rev mutations. The dendrogram was obtained from average linkage hierarchical agglomerative clustering, showing significant clusters among Rev and gp41 mutations. The length of branches reflects distances between mutations in the original distance matrix. Bootstrap values, indicating the significance of clusters, are reported in the boxes.

FIG. 4.

Putative secondary structure of the HIV-1 RRE. HIV-1 RRE wild-type stem-loops III, IV, and V, together with the single strand of three guanosines important for the binding with Rev, are shown in panel A. The HIV-1 RRE with a nucleotide substitution at 40 position is shown in panel B and with nucleotide substitutions at positions 40 and 45 is shown in panel C. The putative secondary structure of the HIV-1 RRE with a nucleotide substitution only at position 45 is not shown since it is superimposable on the structure shown in panel B.