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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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Schematic presentation of the NLR+GFP clone with wild type and mutated sequence in the pol gene. Shown are the HIV-1 reading frames. Black and gray rectangles represent the long terminal repeats (LTRs) and the out-of-frame env gene, respectively. GFP represents the GFP-expression cassette under the control of an internal CMV promoter. The mutated nucleotides are underlined. The indicated mutations do not change the amino acid sequence of the encoded protein.
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Figure 3: Schematic presentation of the NLR+GFP clone with wild type and mutated sequence in the pol gene. Shown are the HIV-1 reading frames. Black and gray rectangles represent the long terminal repeats (LTRs) and the out-of-frame env gene, respectively. GFP represents the GFP-expression cassette under the control of an internal CMV promoter. The mutated nucleotides are underlined. The indicated mutations do not change the amino acid sequence of the encoded protein.

Mentions: We next proceeded towards revealing the biological role of the above conserved stretch comprising codon positions 82–90 of pol by mutagenesis experiments in molecular clones of HIV-1. We hypothesized that if these synonymous sites are indeed under selective constraints, artificially mutating these sites would affect the HIV-1 replication cycle. To this end, we introduced multiple synonymous mutations into the above pol region in the context of HIV-1 molecular clone NLR+GFP, generating the mutated NLR+GFPpolmut clone (Figure 3 and Methods). Both the wild type and the mutated clones contain the entire HIV-1 genome sequence except the env and nef genes. In addition, the clones encode for the green fluorescent protein (GFP). We then used these clones to test for possible effects of the introduced synonymous mutations on both early (infectivity) and late (production) stages of the HIV-1 replication cycle, in human embryonic kidney 293T cells (HEK293T). Specifically, we aimed to determine whether these mutations affect early and/or late events of the virus replication cycle. Early events include virus entry into the host cells, reverse transcription, and integration of the HIV-1 genome into the host chromosomes. Late events include expression of the HIV-1 genes, assembly of the virion, its release from the infected cell, and maturation into fully infectious particles.


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)

Schematic presentation of the NLR+GFP clone with wild type and mutated sequence in the pol gene. Shown are the HIV-1 reading frames. Black and gray rectangles represent the long terminal repeats (LTRs) and the out-of-frame env gene, respectively. GFP represents the GFP-expression cassette under the control of an internal CMV promoter. The mutated nucleotides are underlined. The indicated mutations do not change the amino acid sequence of the encoded protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Schematic presentation of the NLR+GFP clone with wild type and mutated sequence in the pol gene. Shown are the HIV-1 reading frames. Black and gray rectangles represent the long terminal repeats (LTRs) and the out-of-frame env gene, respectively. GFP represents the GFP-expression cassette under the control of an internal CMV promoter. The mutated nucleotides are underlined. The indicated mutations do not change the amino acid sequence of the encoded protein.
Mentions: We next proceeded towards revealing the biological role of the above conserved stretch comprising codon positions 82–90 of pol by mutagenesis experiments in molecular clones of HIV-1. We hypothesized that if these synonymous sites are indeed under selective constraints, artificially mutating these sites would affect the HIV-1 replication cycle. To this end, we introduced multiple synonymous mutations into the above pol region in the context of HIV-1 molecular clone NLR+GFP, generating the mutated NLR+GFPpolmut clone (Figure 3 and Methods). Both the wild type and the mutated clones contain the entire HIV-1 genome sequence except the env and nef genes. In addition, the clones encode for the green fluorescent protein (GFP). We then used these clones to test for possible effects of the introduced synonymous mutations on both early (infectivity) and late (production) stages of the HIV-1 replication cycle, in human embryonic kidney 293T cells (HEK293T). Specifically, we aimed to determine whether these mutations affect early and/or late events of the virus replication cycle. Early events include virus entry into the host cells, reverse transcription, and integration of the HIV-1 genome into the host chromosomes. Late events include expression of the HIV-1 genes, assembly of the virion, its release from the infected cell, and maturation into fully infectious particles.

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