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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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Gag expression and virion release of the NLR+GFP and NLR+GFPpolmut clones in transfected and infected cells. (A) HEK293T cells were transfected with plasmids expressing the VSV-G envelope, and NLR+GFP (WT) or two identical clones of NLR+GFPpolmut (mut1, mut2). Mock represents control cells transfected with no plasmid DNA. Gag precursor (Pr55gag) was detected in extracts of transfected cells (two days post transfection) by Western blotting using anti-capsid monoclonal antibody. Actin was used to control for protein levels in the samples. (B) Virions were purified from equal volumes of supernatants of cells in (A) and their levels were determined by detecting the capsid protein (p24), using Western blotting as above. (C) Equal amounts of virions from (B), normalized by RT activity, were used to infect naïve 293T cells and the newly infected cells were analyzed two days post infection as in (A). (D) Virions from supernatants of the cells indicated in (C) were analyzed as in (B). For (A-D), one representative Western blot is shown at the top of each panel and bars (bottom part) represent the average densitometry of the bands from three independent experiments.
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Figure 4: Gag expression and virion release of the NLR+GFP and NLR+GFPpolmut clones in transfected and infected cells. (A) HEK293T cells were transfected with plasmids expressing the VSV-G envelope, and NLR+GFP (WT) or two identical clones of NLR+GFPpolmut (mut1, mut2). Mock represents control cells transfected with no plasmid DNA. Gag precursor (Pr55gag) was detected in extracts of transfected cells (two days post transfection) by Western blotting using anti-capsid monoclonal antibody. Actin was used to control for protein levels in the samples. (B) Virions were purified from equal volumes of supernatants of cells in (A) and their levels were determined by detecting the capsid protein (p24), using Western blotting as above. (C) Equal amounts of virions from (B), normalized by RT activity, were used to infect naïve 293T cells and the newly infected cells were analyzed two days post infection as in (A). (D) Virions from supernatants of the cells indicated in (C) were analyzed as in (B). For (A-D), one representative Western blot is shown at the top of each panel and bars (bottom part) represent the average densitometry of the bands from three independent experiments.

Mentions: Two identical (but separately prepared) clones of NLR+GFPpolmut and the wild type clone NLR+GFP were transfected into HEK293T cells and their expression levels were examined by immunoblotting of the transfected cells extracts with a monoclonal antibody against HIV-1 capsid protein. This antibody detects both free capsid and Gag precursor proteins. Similar expression levels of the wild type and mutant clones were observed, as determined by quantifying the levels of the Gag precursor within transfected cells (Figure 4A). Thus, the mutations did not affect viral expression levels. Furthermore, applying the above analysis for the detection of virions purified from the supernatants of transfected cultures, yielded no significant difference between the production levels of HIV-1 particles of the tested clones (as deduced from measuring the levels of capsid in these preparations; Figure 4B). Overall, these analyses suggest that the mutations in pol did not hamper the late stages (expression, assembly and budding) of the HIV-1 replication cycle.


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)

Gag expression and virion release of the NLR+GFP and NLR+GFPpolmut clones in transfected and infected cells. (A) HEK293T cells were transfected with plasmids expressing the VSV-G envelope, and NLR+GFP (WT) or two identical clones of NLR+GFPpolmut (mut1, mut2). Mock represents control cells transfected with no plasmid DNA. Gag precursor (Pr55gag) was detected in extracts of transfected cells (two days post transfection) by Western blotting using anti-capsid monoclonal antibody. Actin was used to control for protein levels in the samples. (B) Virions were purified from equal volumes of supernatants of cells in (A) and their levels were determined by detecting the capsid protein (p24), using Western blotting as above. (C) Equal amounts of virions from (B), normalized by RT activity, were used to infect naïve 293T cells and the newly infected cells were analyzed two days post infection as in (A). (D) Virions from supernatants of the cells indicated in (C) were analyzed as in (B). For (A-D), one representative Western blot is shown at the top of each panel and bars (bottom part) represent the average densitometry of the bands from three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Gag expression and virion release of the NLR+GFP and NLR+GFPpolmut clones in transfected and infected cells. (A) HEK293T cells were transfected with plasmids expressing the VSV-G envelope, and NLR+GFP (WT) or two identical clones of NLR+GFPpolmut (mut1, mut2). Mock represents control cells transfected with no plasmid DNA. Gag precursor (Pr55gag) was detected in extracts of transfected cells (two days post transfection) by Western blotting using anti-capsid monoclonal antibody. Actin was used to control for protein levels in the samples. (B) Virions were purified from equal volumes of supernatants of cells in (A) and their levels were determined by detecting the capsid protein (p24), using Western blotting as above. (C) Equal amounts of virions from (B), normalized by RT activity, were used to infect naïve 293T cells and the newly infected cells were analyzed two days post infection as in (A). (D) Virions from supernatants of the cells indicated in (C) were analyzed as in (B). For (A-D), one representative Western blot is shown at the top of each panel and bars (bottom part) represent the average densitometry of the bands from three independent experiments.
Mentions: Two identical (but separately prepared) clones of NLR+GFPpolmut and the wild type clone NLR+GFP were transfected into HEK293T cells and their expression levels were examined by immunoblotting of the transfected cells extracts with a monoclonal antibody against HIV-1 capsid protein. This antibody detects both free capsid and Gag precursor proteins. Similar expression levels of the wild type and mutant clones were observed, as determined by quantifying the levels of the Gag precursor within transfected cells (Figure 4A). Thus, the mutations did not affect viral expression levels. Furthermore, applying the above analysis for the detection of virions purified from the supernatants of transfected cultures, yielded no significant difference between the production levels of HIV-1 particles of the tested clones (as deduced from measuring the levels of capsid in these preparations; Figure 4B). Overall, these analyses suggest that the mutations in pol did not hamper the late stages (expression, assembly and budding) of the HIV-1 replication cycle.

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