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 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...

HIV-2 A-subtype gp125C2-V3-C3 mutations and their association with CCR5 and CXCR4 tropism

“…Because pairwise analysis suggested that most mutations were associated, we performed average linkage hierarchical agglomerative cluster analysis [59][60][61] to investigate this hypothesis in more detail. By using this analytical method, we found for the first time specific and statistically significant correlations between C2, V3 and C3 mutations.…”

“…A positive and statistically significant correlation between mutations at two specific positions (0 \ u \ 1; P \ 0.05) indicates that the latter mutates in a correlated manner in order to confer an advantage in terms of co-receptor selection and that the co-occurrence of these mutations is not due to chance. Moreover, to analyze the covariation structure of mutations in more detail, average linkage hierarchical agglomerative clustering was performed as described elsewhere [59][60][61]. Mann-Whitney U tests were used to assess statistically significant differences among all the pairwise-associated mutations.…”

Analysis of the Relationship between the Structure and Function of the HIV-1 Rev Response Element (RRE)

“…Secondary mutations in the RRE were found to restore the RRE structure predicted to be disrupted by the primary mutations. Such "structure conservation mutations" were observed in stem loop IIC (163) and III (223), which underscores the importance of structural integrity of regions of the RRE outside the primary Rev binding site. Secondary mutations in Rev were also found in both ARM and oligomerization domains (223).…”

“…Such "structure conservation mutations" were observed in stem loop IIC (163) and III (223), which underscores the importance of structural integrity of regions of the RRE outside the primary Rev binding site. Secondary mutations in Rev were also found in both ARM and oligomerization domains (223). Those in the ARM domain accompanied mutation in the primary Rev binding site of RRE, suggesting that these Rev mutations were selected to correct impaired Rev-RRE binding.…”

“…Although there is evidence (77,137,224) that stem loops III, IV, V of the RRE contribute to Rev-RRE function, the precise functional role of these regions is not well understood. In this thesis, we have re-examined the secondary structure of the RRE and compared the functional importance of the 5 stem-loop and the 4 stem-loop RRE structures.…”