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Synonymous site conservation in the HIV-1 genome.

Mayrose I, Stern A, Burdelova EO, Sabo Y, Laham-Karam N, Zamostiano R, Bacharach E, Pupko T - BMC Evol. Biol. (2013)

Bottom Line: In our assays aiming to quantify viral fitness in both early and late stages of the replication cycle, no differences were observed between the mutated and the wild type virus following the introduction of synonymous mutations.The contradiction between the inferred purifying selective forces and the lack of effect of these mutations on viral replication may be explained by the fact that the phenotype was measured in single-cycle infection assays in cell culture.Such a system does not account for the complexity of HIV-1 infections in vivo, which involves multiple infection cycles and interaction with the host immune system.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biology and Ecology of Plants, Tel Aviv University, Tel-Aviv 69978, Israel. itaymay@post.tau.ac.il

ABSTRACT

Background: Synonymous or silent mutations are usually thought to evolve neutrally. However, accumulating recent evidence has demonstrated that silent mutations may destabilize RNA structures or disrupt cis regulatory motifs superimposed on coding sequences. Such observations suggest the existence of stretches of codon sites that are evolutionary conserved at both DNA-RNA and protein levels. Such stretches may point to functionally important regions within protein coding sequences not necessarily reflecting functional constraints on the amino-acid sequence. The HIV-1 genome is highly compact, and often harbors overlapping functional elements at the protein, RNA, and DNA levels. This superimposition of functions leads to complex selective forces acting on all levels of the genome and proteome. Considering the constraints on HIV-1 to maintain such a highly compact genome, we hypothesized that stretches of synonymous conservation would be common within its genome.

Results: We used a combined computational-experimental approach to detect and characterize regions exhibiting strong purifying selection against synonymous substitutions along the HIV-1 genome. Our methodology is based on advanced probabilistic evolutionary models that explicitly account for synonymous rate variation among sites and rate dependencies among adjacent sites. These models are combined with a randomization procedure to automatically identify the most statistically significant regions of conserved synonymous sites along the genome. Using this procedure we identified 21 conserved regions. Twelve of these are mapped to regions within overlapping genes, seven correlate with known functional elements, while the functions of the remaining four are yet unknown. Among these four regions, we chose the one that deviates most from synonymous rate homogeneity for in-depth computational and experimental characterization. In our assays aiming to quantify viral fitness in both early and late stages of the replication cycle, no differences were observed between the mutated and the wild type virus following the introduction of synonymous mutations.

Conclusions: The contradiction between the inferred purifying selective forces and the lack of effect of these mutations on viral replication may be explained by the fact that the phenotype was measured in single-cycle infection assays in cell culture. Such a system does not account for the complexity of HIV-1 infections in vivo, which involves multiple infection cycles and interaction with the host immune system.

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Analysis of the pol 82–90 Ks-conserved region. (A) Ka and Ks values for each position in the protein. The pol 82–90 region is boxed. (B) Codon adaptation index analysis based on the human codon usage in highly expressed genes (see Methods). The pol 82–90 region is boxed. (C) RNA secondary structure prediction, including the flanking region (see text for details). Red ovals encircling triplets of nucleotides mark the beginning and end of pol 82–90 within the predicted region.
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Figure 2: Analysis of the pol 82–90 Ks-conserved region. (A) Ka and Ks values for each position in the protein. The pol 82–90 region is boxed. (B) Codon adaptation index analysis based on the human codon usage in highly expressed genes (see Methods). The pol 82–90 region is boxed. (C) RNA secondary structure prediction, including the flanking region (see text for details). Red ovals encircling triplets of nucleotides mark the beginning and end of pol 82–90 within the predicted region.

Mentions: Of the 21 stretches in Table 2, the conservation of four stretches or regions within stretches could not be explained by a known regulatory domain or because of an overlap with other protein coding sequence. Among these four regions, we chose the stretch showing the most significant deviation from Ks homogeneity for further characterization. This stretch encompasses positions 82–107 within pol. Visual examination of the Ka and Ks values in this stretch (Figure 2A) revealed that the stretch is composed of two regions of decreased Ks, separated by a short peak. This led us to focus on the first region, spanning positions 82–90 of pol. We first tested whether the significant Ks conservation signal may be explained by codon bias, possibly affecting translation efficiency. The result of this analysis showed a slight decrease in the codon usage values in the pol 82–90 region (Figure 2B). Such a reduction is expected if indeed there are selective forces at the DNA/RNA level, as such forces do not allow substitutions toward optimal codons. As it has been shown that suboptimal codon usage in HIV is usually uncorrelated with translational efficiency [36], the reason for the conservation remains unknown.


Synonymous site conservation in the HIV-1 genome.

Mayrose I, Stern A, Burdelova EO, Sabo Y, Laham-Karam N, Zamostiano R, Bacharach E, Pupko T - BMC Evol. Biol. (2013)

Analysis of the pol 82–90 Ks-conserved region. (A) Ka and Ks values for each position in the protein. The pol 82–90 region is boxed. (B) Codon adaptation index analysis based on the human codon usage in highly expressed genes (see Methods). The pol 82–90 region is boxed. (C) RNA secondary structure prediction, including the flanking region (see text for details). Red ovals encircling triplets of nucleotides mark the beginning and end of pol 82–90 within the predicted region.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750384&req=5

Figure 2: Analysis of the pol 82–90 Ks-conserved region. (A) Ka and Ks values for each position in the protein. The pol 82–90 region is boxed. (B) Codon adaptation index analysis based on the human codon usage in highly expressed genes (see Methods). The pol 82–90 region is boxed. (C) RNA secondary structure prediction, including the flanking region (see text for details). Red ovals encircling triplets of nucleotides mark the beginning and end of pol 82–90 within the predicted region.
Mentions: Of the 21 stretches in Table 2, the conservation of four stretches or regions within stretches could not be explained by a known regulatory domain or because of an overlap with other protein coding sequence. Among these four regions, we chose the stretch showing the most significant deviation from Ks homogeneity for further characterization. This stretch encompasses positions 82–107 within pol. Visual examination of the Ka and Ks values in this stretch (Figure 2A) revealed that the stretch is composed of two regions of decreased Ks, separated by a short peak. This led us to focus on the first region, spanning positions 82–90 of pol. We first tested whether the significant Ks conservation signal may be explained by codon bias, possibly affecting translation efficiency. The result of this analysis showed a slight decrease in the codon usage values in the pol 82–90 region (Figure 2B). Such a reduction is expected if indeed there are selective forces at the DNA/RNA level, as such forces do not allow substitutions toward optimal codons. As it has been shown that suboptimal codon usage in HIV is usually uncorrelated with translational efficiency [36], the reason for the conservation remains unknown.

Bottom Line: In our assays aiming to quantify viral fitness in both early and late stages of the replication cycle, no differences were observed between the mutated and the wild type virus following the introduction of synonymous mutations.The contradiction between the inferred purifying selective forces and the lack of effect of these mutations on viral replication may be explained by the fact that the phenotype was measured in single-cycle infection assays in cell culture.Such a system does not account for the complexity of HIV-1 infections in vivo, which involves multiple infection cycles and interaction with the host immune system.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biology and Ecology of Plants, Tel Aviv University, Tel-Aviv 69978, Israel. itaymay@post.tau.ac.il

ABSTRACT

Background: Synonymous or silent mutations are usually thought to evolve neutrally. However, accumulating recent evidence has demonstrated that silent mutations may destabilize RNA structures or disrupt cis regulatory motifs superimposed on coding sequences. Such observations suggest the existence of stretches of codon sites that are evolutionary conserved at both DNA-RNA and protein levels. Such stretches may point to functionally important regions within protein coding sequences not necessarily reflecting functional constraints on the amino-acid sequence. The HIV-1 genome is highly compact, and often harbors overlapping functional elements at the protein, RNA, and DNA levels. This superimposition of functions leads to complex selective forces acting on all levels of the genome and proteome. Considering the constraints on HIV-1 to maintain such a highly compact genome, we hypothesized that stretches of synonymous conservation would be common within its genome.

Results: We used a combined computational-experimental approach to detect and characterize regions exhibiting strong purifying selection against synonymous substitutions along the HIV-1 genome. Our methodology is based on advanced probabilistic evolutionary models that explicitly account for synonymous rate variation among sites and rate dependencies among adjacent sites. These models are combined with a randomization procedure to automatically identify the most statistically significant regions of conserved synonymous sites along the genome. Using this procedure we identified 21 conserved regions. Twelve of these are mapped to regions within overlapping genes, seven correlate with known functional elements, while the functions of the remaining four are yet unknown. Among these four regions, we chose the one that deviates most from synonymous rate homogeneity for in-depth computational and experimental characterization. In our assays aiming to quantify viral fitness in both early and late stages of the replication cycle, no differences were observed between the mutated and the wild type virus following the introduction of synonymous mutations.

Conclusions: The contradiction between the inferred purifying selective forces and the lack of effect of these mutations on viral replication may be explained by the fact that the phenotype was measured in single-cycle infection assays in cell culture. Such a system does not account for the complexity of HIV-1 infections in vivo, which involves multiple infection cycles and interaction with the host immune system.

Show MeSH
Related in: MedlinePlus