Hidden messages in the nef gene of human immunodeficiency virus type 1 suggest a novel RNA secondary structure

Abstract

The coexistence of multiple codes in the genome of human immunodeficiency virus type 1 (HIV-1) was analyzed. We explored factors constraining the variability of the virus genome primarily in relation to conserved RNA secondary structures overlapping coding sequences, and used a simple combination of algorithms for RNA secondary structure prediction based on the nearest-neighbor thermodynamic rules and a statistical approach. In our previous study, we applied this combination to a non- redundant data set of env nucleotide sequences, confirmed the conservative secondary structure of the rev-responsive element (RRE) and found a new RNA structure in the first conserved (C1) region of the env gene. In this study, we analyzed the variability of putative RNA secondary structures inside the nef gene of HIV-1 by applying these algorithms to a non-redundant data set of 104 nef sequences retrieved from the Los Alamos HIV database, and predicted the existence of a novel functional RNA secondary structure in the beta3/beta4 regions of nef. The predicted RNA fold in the beta3/beta4 region of nef appears in two forms with different loop sizes. The loop of the first fold consists of seven nucleotides (positions 494-500), with consensus UCAAGCU appearing in 79% of sequences. The other has a five-base loop (positions 495-499) with consensus CAAGC. The difference in size between these two loops may reflect the difference between respective counterparts in the hairpin recognition. This may also have an adaptive biological significance.

Figure 1

DNA conservation of nef DNA. To compare the information content of nef DNA sequences with encoded Nef proteins, we used a back-translation technique. The correct back-translation was made by replacing every amino acid by the corresponding codon from the real, known gene sequence; the randomized back-translation was made by replacing every amino acid by one of the corresponding randomly selected codons of its own repertoire (equiprobable within the group). The codon usage-related back-translation was made by using EMBOSS Backtranseq according to the HIV-1 codon usage file: EHuman_immunodeficiency_virus_type_1.cut. The curves showing the information content were smoothed by a running average with a window size equal to six. The green line represents the DNA conservation of the nef sequences retrieved from the original database. The blue line describes the conservation of the codon usage-related back-translated nef sequences, and the orange line describes the conservation of the random back-translation. Conservation of DNA at every position was computed according to Equation 1.

Figure 2

Conservation of predicted RNA folds. RNA secondary structures of the three different data sets of nucleotide sequences mentioned in Figure 1 were predicted by the Vienna package. The outputs of these predictions were aligned and the gaps were inserted according to the alignments of Nef amino acid sequences. Conservation of RNA secondary structure at every position was computed as the information contribution of the stem or loop relative to their expected distribution according to the structure information equation (Equation 2). The colors of the lines refer to the same databases as in Figure 1.

Figure 3

Examples of predicted RNA secondary structures in the β3/β4 region from a few randomly selected nef sequences. The β3/β4 RNA region of nef (positions 469–534 of the multiple alignment notation) was retrieved from 10 different randomly chosen sequences. The retrieved fragments were folded using the Mfold program. An approximate 60 bp bulged hairpin with interior loops appears in all chosen sequences. It is the main feature of the putative common RNA secondary structure. The seven-base hairpin loop appears in (a–g) and the five-base hairpin loop appears in (h–k).

Figure 4

(A and B) Structural RNA logo analysis of the hairpin loop. Two different common loops were detected in position 493–501 in the multiple alignment file: seven bases long (((…….))) and five bases long (((…..))). By composing a correct regular expression pattern for each loop, removing the gaps and including 16–17 bases expanding from each side of the loop, we were able to distinguish between 83 large loop RNA structures and 21 small loop structures. All alignments were analyzed by structural RNA logo analysis. The results are presented in bits, reflecting the information content of each base. The base pair fraction of each position has been calculated and appears below the logo. (A) refers to the large stem–loop structure and (B) refers to the small one. (C and D) Protein logo analysis of the amino acid sequence encoded by the β3/β4 hairpin loop region. The two nucleotide data sets (large and small loop), described in (A) and (B), were translated to amino acid sequences and analyzed by protein logo analysis. The results are presented in bits, reflecting the information content of each amino acid. (C) Refers to the large loop hairpin sequence and (D) refers to the small loop hairpin sequence.