BACKGROUND
molecular evolutionary models are often applied to detect and quantify the type and the intensity of selective pressures acting on genes and on specific sites within them.
sites playing an essential role, such as those composing the active site of an enzyme, are unlikely to undergo amino-acid replacements over evolutionary time.
this constraint reflects a purifying selection force, which prevents the fixation of mutations that otherwise would disturb protein structure or function  <cit> .
the higher the intensity of purifying selection acting on a specific nucleotide or amino-acid site, the more conserved the site is expected to be.
hence, under the neutral theory of molecular evolution, conserved sites are assumed to indicate functional or structural importance, while variable sites may be regarded as evolving free from, or under weak, selective constraints.
some sites, however, are very fast evolving, yet are of great functional significance.
such fast evolution may reflect positive darwinian selection.
for example, the influenza virus hemagglutinin  gene encodes a major surface antigen, targeted by neutralizing antibodies during infection.
sequence analysis of this gene suggested a few sites to be positively selected  <cit> , and these were shown to be responsible for evading the host immune system.
taken together, sites that are under strong purifying or positive selection are likely to be functionally or structurally important and their identification is often a critical first step towards understanding and manipulating a protein’s function [reviewed in  <cit> .
in protein-coding genes, the selection type and intensity are usually inferred by contrasting the rate of nonsynonymous  to the rate of synonymous  nucleotide substitutions  <cit> .
the most currently-used models assume that the selective forces acting on protein-coding genes operate at the protein level only, while synonymous substitutions are free from selection and reflect the neutral rate of evolution  <cit> .
in such a case, ks is constant across codon positions, ka is heterogeneous, and the inference of site-specific ka/ks values is based solely on ka variation.
codon sites showing a ka/ks ratio significantly lower than  <dig> are regarded as undergoing purifying selection, suggesting that they are functionally or structurally important.
codon sites with ka/ks ratios significantly higher than  <dig> are indicative of positive darwinian selection, suggesting adaptive evolution.
finally, sites inferred to evolve under a ka/ks ratio not significantly different from  <dig> are regarded as free from selection at the protein level  <cit> .
a large number of studies have used this methodology to infer adaptive evolution in human immunodeficiency virus type  <dig> , resulting from immune pressure or drug treatment [e.g.,  <cit> .
the assumption that ks reflects the neutral rate of evolution suggests that synonymous substitutions should be fixed at a constant rate along the sequence.
however, it has been shown that selection regularly operates on synonymous sites in different organisms [e.g.,  <cit> .
for example, purifying selection on synonymous sites was found in  <dig> % of all yeast genes and  <dig> % in all worm genes using a likelihood based model that distinguishes between synonymous substitutions between preferred and unpreferred codons  <cit> .
purifying selection on synonymous sites was suggested to result from considerations such as mrna stability  <cit> , splicing regulatory elements [e.g.,  <cit> , cis regulatory elements, and overlapping genes  <cit> .
furthermore, in bacteria and yeast, codon bias in highly expressed genes is the most documented type of selection on synonymous sites [e.g.,  <cit> .
additionally, the choice among synonymous codons was shown to affect protein structure  <cit> .
in addition, conservation of “optimal” and “non-optimal” codons was shown to reflect selection for cotranslational folding in yeast.
thus, it is becoming recognized that the evolutionary selective forces acting on synonymous sites reflect various biological phenomena [recently reviewed in  <cit> .
the “ks equal neutrality” assumption is particularly problematic in viruses since, due to their compact genome, regulatory elements are abundant within their protein coding regions.
for example, there are well known rna secondary structures along the hiv- <dig> genome, such as the rev-response element , which plays important functional roles during the virus life cycle  <cit> .
another obvious example is the existence of overlapping genes, in which strong selection against both synonymous and nonsynonymous substitutions is expected.
however, since the commonly used codon models fail to account for ks variation, such regions are usually excluded from the analysis, despite their high biological interest.
for example, to avoid overlapping regions, yang et al.
<cit>  excluded the entire tat, rev, and vpu genes and analyzed the evolutionary selective forces in the rest of the hiv- <dig> genome.
thus, ignoring ks variability across sites is biologically unrealistic, problematic and may bias inference of selective forces across protein coding genes  <cit> .
furthermore, the ability to correctly model ks variability across sites may reveal novel insights into the functional constraints acting on the dna and rna levels, which cannot be inferred using standard codon models.
ngandu et al.
<cit>  have previously applied a sliding-window approach to detect consecutive codons with low synonymous substitution rates within the hiv- <dig> genome.
one of the regions discovered in that study was a 12-base-pair long region that resides within env for which no functional role had been previously described.
mutagenesis experiments in which synonymous mutations were introduced in this region, led to no observed difference between wild type and mutated clones in cell lines.
kosokovski-pond et al.
<cit>  have also applied models allowing for ks variation to hiv- <dig> and other rna viruses.
however, their goal was to find shared evolutionary rate patterns across alignments rather than to detect conserved ks regions.
in this study we constructed a genome-wide map of the selective forces operating against the fixation of synonymous substitutions across the hiv- <dig> genome.
our method relies on a model that explicitly accounts for dependency of synonymous rates among adjacent sites, thus removing stochastic noise from site specific ks estimates  <cit> .
specifically, we developed the strechfinder methodology that detects statistically significant consecutive synonymous-conserved sites.
this methodology overcomes the inherent statistical problematic aspects of sliding window approaches for detecting evolutionary rate variation among sites  <cit> .
we detected twenty-one short linear stretches within the genome, which display significantly lower levels of ks than expected under neutrality.
the forces leading to lowered ks in these regions were explored.
the majority of the identified regions were mapped to previously known regulatory functional regions, or to overlapping open reading frames , validating our methodology as a general tool for predicting functional regions at the nucleotide level nested within protein-coding regions.
furthermore, we detected a number of ks conserved regions that could not be assigned to a known function, and are thus predicted to be under selection due to a yet unknown reason.
one such region is located within the pol gene.
mutagenesis experiments of this region were conducted to reveal its function but showed no clear effect on viral replication in cell culture.
the implications of these results are discussed.
RESULTS
selection intensity on synonymous sites varies within and across hiv orfs
a large dataset of  <dig> hiv- <dig> sequences spanning eight subtypes showed that variability in the synonymous substitution rate is present in each of the nine hiv- <dig> orfs.
the two evolutionary codon models that account for ks variability  exhibited a significantly better fit to the data, compared to the commonly used kav-ksc model that assumes a single ks rate for all sequence sites .
furthermore, for all orfs other than vpr, significant support was found for the model accounting for dependency in evolutionary rates between adjacent sequence sites.
the kad-ksd model differs from the kav-ksc model in two attributes: the dependence between adjacent sites and the variability in ks.
by comparing the differences between kav-ksc and kav-ksv and between kad-ksd and kav-ksv , it is clear that assuming ks variability leads to the dominant factor in the improved fit of the kad-ksd model to the hiv- <dig> sequences.
a comparison of the kad-ksd versus the kav-ksc model.
b comparison of the kad-ksd versus the kav-ksv model.
having established that kad-ksd is the best fitting model, it was next used to infer site-specific ka and ks scores for each site in each of the orfs.
the posterior distributions of inferred ka and ks values were then examined .
there is a large difference between the two distributions: on the one hand, most of the ka distribution surrounds very low values, with an average of  <dig> , supporting the notion that extensive purifying selection is acting on the coding sequences.
on the other hand, the mass of the ks distribution is centered around  <dig> , suggesting that most synonymous sites are under neutral selection.
indeed, the coefficient of variation  of the ka distribution is much higher than that of the ks distribution  suggesting higher level of variation of ka compared to ks values.
identification of ks-conserved stretches across the hiv- <dig> genome
the stretchfinder algorithm  was used to scan the estimated ks values for extended regions exhibiting a statistically significant reduction of ks.
a stretch was defined as a consecutive region spanning between  <dig> and  <dig> codons.
assigning a p-value allowed the distinction between a region with a few randomly dispersed ks-conserved sites and an extended region with low ks values, which may be indicative of a functionally important region.
this analysis yielded  <dig> stretches with significantly low values of ks, following correction for multiple testing .
notably, these stretches were very similar to those obtained when stretchfinder was applied to site specific ks rates inferred using the kav-ksv model.
however, in a few cases the stretches obtained by the model assuming site dependency were slightly longer .
this can be explained by the smoothing effect induced by models assuming site dependencies.
regions which could not be fully accounted for  are marked in bold.
seven of the conserved stretches were mapped to regions previously reported to have a functional role.
the most statistically significant ks conserved stretch along the hiv- <dig> genome region is located inside the pol orf and spans positions  <dig> to  <dig>  this region spans a few functional nucleic acid elements: the central polypurine tract   <cit> , the central termination sequence , and the dna flap region  <cit> .
this stretch resides near a second significant stretch within pol, which spans positions  <dig> to  <dig> .
the conservation in this stretch may be partially explained by the presence of the end of the cts within it.
indeed, this region within pol was previously identified using a manual scan for ks conserved regions  <cit> .
the second polypurine tract  in hiv- <dig> is located inside the nef gene and is also part of a stretch with significantly low ks values .
a third known functional element that was detected as a significantly conserved stretch is sl <dig>  <cit> , which serves as part of the rna packaging signal and is located in gag .
three out of five conserved stretches found in env  are mapped to the rre region  <cit> .
additional twelve significantly conserved stretches are mapped to regions of overlapping genes.
indeed, in almost all of these cases the overlapped region was frame-shifted from the first stretch, and the low ks values could be explained by selective constraints on the overlapped gene, i.e.
by low ka values in the other gene.
of the  <dig> stretches in table  <dig>  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– <dig> within pol.
visual examination of the ka and ks values in this stretch  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– <dig> 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– <dig> region .
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  <cit> , the reason for the conservation remains unknown.
we next tested whether secondary structure motifs within this stretch may explain the apparent ks conservation.
to determine the possible functional role of this region, an rna secondary structure prediction was performed, and was compared to the experimental values obtained for the whole hiv- <dig> genome  <cit> .
the predicted rna secondary structure  suggested that the pol 82– <dig> region is mostly unstructured, apart from a short stem and loop at the beginning of the stretch.
this prediction matches the secondary structure model based on a high-throughput rna analysis performed on the entire rna genome of hiv- <dig>  <cit> .
taken together, these analyses reinforce the hypothesis that the low ks of the pol 82– <dig> stretch is not related to its participation in rna secondary structures and is most likely also unrelated to translational efficiency; thus, it may have a regulatory role, such as binding an unknown factor.
experimental testing
we next proceeded towards revealing the biological role of the above conserved stretch comprising codon positions 82– <dig> of pol by mutagenesis experiments in molecular clones of hiv- <dig>  we hypothesized that if these synonymous sites are indeed under selective constraints, artificially mutating these sites would affect the hiv- <dig> replication cycle.
to this end, we introduced multiple synonymous mutations into the above pol region in the context of hiv- <dig> molecular clone nlr+gfp, generating the mutated nlr+gfppolmut clone .
both the wild type and the mutated clones contain the entire hiv- <dig> genome sequence except the env and nef genes.
in addition, the clones encode for the green fluorescent protein .
we then used these clones to test for possible effects of the introduced synonymous mutations on both early  and late  stages of the hiv- <dig> replication cycle, in human embryonic kidney 293t cells .
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- <dig> genome into the host chromosomes.
late events include expression of the hiv- <dig> genes, assembly of the virion, its release from the infected cell, and maturation into fully infectious particles.
disruption of early stages should result in decreased levels of integrated hiv- <dig> proviruses.
to detect such a reduction, we measured the overall expression of genes  transcribed from the hiv- <dig> provirus.
searching for a possible effect of the mutations on late events, we quantified virion release from the cells by measuring capsid levels in the context of assembled virions.
in addition, the infectivity of produced virions was monitored using single cycle infection assays.
two identical  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- <dig> 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 .
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- <dig> particles of the tested clones .
overall, these analyses suggest that the mutations in pol did not hamper the late stages  of the hiv- <dig> replication cycle.
the above results cannot rule out possible effects of the mutations on viral infectivity.
to further investigate this option, naïve hek293t cells were infected with equal amounts of wild type and mutant virions, normalized by reverse transcriptase activity [as in  <cit> .
to measure infection levels, infected cells were tested for either intracellular gag or gfp levels, quantified by western blot or flow cytometry analyses, respectively.
both gag  and gfp levels  were not significantly different among the wild type and mutated clones, suggesting that the mutations did not hamper infectivity.
finally, we tested whether hiv- <dig> particle production originating from infected cells  varied between the tested clones, and here too, no significant difference was observed .
overall, these results indicate that in the current experimental settings, the functional role of this conserved stretch has no  effect on the hiv- <dig> replication cycle.
possible reasons for the disagreement between the strong evolutionary conservation signal and the experimental results are discussed below.
CONCLUSIONS
this study provides and explores a comprehensive map of the evolutionary selective forces operating on hiv- <dig>  previous studies inferring natural selection in hiv did not account for selection on synonymous substitutions, which may have possibly inflated estimates of positive selection  <cit> .
specifically, overlapping regions of genes are often considered problematic and are removed from analyses  <cit> .
here we show that complex inter-related selective forces may operate on all protein coding genes within hiv- <dig>
this analysis underscores the association between synonymously conserved regions and functional regions at the dna or rna levels.
hiv- <dig> represents one of the most studied biological systems and thus provides us with the opportunity to correlate between the inferred ks conserved stretches and known functional regions residing within coding regions.
it is noteworthy that nearly all well studied regulatory regions residing within the orfs of hiv- <dig> were also detected using our methodology.
nevertheless, four synonymously conserved regions were detected with no previously reported function.
an important aspect of this work was the effort to experimentally elucidate the significance of the computational predictions derived from the evolutionary models.
however, the mutagenesis assay performed here on one of the stretches  resulted in insignificant differences between the wild type and mutated constructs.
insignificant differences between wild type and mutated clones were also obtained by ngandu et al.
<cit> , who experimentally tested the role for a synonymous conserved region within env.
notably, these experiments were performed in cell cultures, which do not necessarily mimic natural infections of cd4+ cells.
for instance, interactions with salient host proteins  are often dependent on the specific cell-line used  <cit> .
in addition, it is possible that these synonymous mutations reduce viral fitness, but this reduction is not significant enough to be revealed after only a single infection cycle.
finally, since this stretch may have an important role only in the context of the virus interaction with the host adaptive immune response, it was not discovered in the artificial cell culture experiments conducted here.
hence, future work is needed to determine the role of the novel conserved stretches detected here.
nevertheless, the methodology presented here is generic, and can be used to investigate other less-studied viral datasets to predict novel functional regions, as well as to explore the breadth of synonymous conservation in both cells and viruses.
moreover, the identification of conserved sites in viral populations should enhance pcr diagnostics of a variety of viral strains that otherwise significantly differ from each other, like in the case of hiv- <dig>

 molecular evolutionary models are often applied to detect and quantify the type and the intensity of selective pressures acting on genes and on specific sites within them. sites playing an essential role, such as those composing the active site of an enzyme, are unlikely to undergo amino-acid replacements over evolutionary time. this constraint reflects a purifying selection force, which prevents the fixation of mutations that otherwise would disturb protein structure or function. the higher the intensity of purifying selection acting on a specific nucleotide or amino-acid site, the more conserved the site is expected to be. hence, under the neutral theory of molecular evolution, conserved sites are assumed to indicate functional or structural importance, while variable sites may be regarded as evolving free from, or under weak, selective constraints. some sites, however, are very fast evolving, yet are of great functional significance. such fast evolution may reflect positive darwinian selection. for example, the influenza virus hemagglutinin gene encodes a major surface antigen, targeted by neutralizing antibodies during infection. sequence analysis of this gene suggested a few sites to be positively selected, and these were shown to be responsible for evading the host immune system. taken together, sites that are under strong purifying or positive selection are likely to be functionally or structurally important and their identification is often a critical first step towards understanding and manipulating a protein’s function [reviewed in. in protein-coding genes, the selection type and intensity are usually inferred by contrasting the rate of nonsynonymous to the rate of synonymous nucleotide substitutions. the most currently-used models assume that the selective forces acting on protein-coding genes operate at the protein level only, while synonymous substitutions are free from selection and reflect the neutral rate of evolution. in such a case, ks is constant across codon positions, ka is heterogeneous, and the inference of site-specific ka/ks values is based solely on ka variation. codon sites showing a ka/ks ratio significantly lower than are regarded as undergoing purifying selection, suggesting that they are functionally or structurally important. codon sites with ka/ks ratios significantly higher than are indicative of positive darwinian selection, suggesting adaptive evolution. finally, sites inferred to evolve under a ka/ks ratio not significantly different from are regarded as free from selection at the protein level. a large number of studies have used this methodology to infer adaptive evolution in human immunodeficiency virus type, resulting from immune pressure or drug treatment [e.g.,. the assumption that ks reflects the neutral rate of evolution suggests that synonymous substitutions should be fixed at a constant rate along the sequence. however, it has been shown that selection regularly operates on synonymous sites in different organisms [e.g.,. for example, purifying selection on synonymous sites was found in % of all yeast genes and % in all worm genes using a likelihood based model that distinguishes between synonymous substitutions between preferred and unpreferred codons. purifying selection on synonymous sites was suggested to result from considerations such as mrna stability, splicing regulatory elements [e.g.,, cis regulatory elements, and overlapping genes. furthermore, in bacteria and yeast, codon bias in highly expressed genes is the most documented type of selection on synonymous sites [e.g.,. additionally, the choice among synonymous codons was shown to affect protein structure. in addition, conservation of “optimal” and “non-optimal” codons was shown to reflect selection for cotranslational folding in yeast. thus, it is becoming recognized that the evolutionary selective forces acting on synonymous sites reflect various biological phenomena [recently reviewed in. the “ks equal neutrality” assumption is particularly problematic in viruses since, due to their compact genome, regulatory elements are abundant within their protein coding regions. for example, there are well known rna secondary structures along the hiv- genome, such as the rev-response element, which plays important functional roles during the virus life cycle. another obvious example is the existence of overlapping genes, in which strong selection against both synonymous and nonsynonymous substitutions is expected. however, since the commonly used codon models fail to account for ks variation, such regions are usually excluded from the analysis, despite their high biological interest. for example, to avoid overlapping regions, yang et al. excluded the entire tat, rev, and vpu genes and analyzed the evolutionary selective forces in the rest of the hiv- genome. thus, ignoring ks variability across sites is biologically unrealistic, problematic and may bias inference of selective forces across protein coding genes. furthermore, the ability to correctly model ks variability across sites may reveal novel insights into the functional constraints acting on the dna and rna levels, which cannot be inferred using standard codon models. ngandu et al. have previously applied a sliding-window approach to detect consecutive codons with low synonymous substitution rates within the hiv- genome. one of the regions discovered in that study was a 12-base-pair long region that resides within env for which no functional role had been previously described. mutagenesis experiments in which synonymous mutations were introduced in this region, led to no observed difference between wild type and mutated clones in cell lines. kosokovski-pond et al. have also applied models allowing for ks variation to hiv- and other rna viruses. however, their goal was to find shared evolutionary rate patterns across alignments rather than to detect conserved ks regions. in this study we constructed a genome-wide map of the selective forces operating against the fixation of synonymous substitutions across the hiv- genome. our method relies on a model that explicitly accounts for dependency of synonymous rates among adjacent sites, thus removing stochastic noise from site specific ks estimates. specifically, we developed the strechfinder methodology that detects statistically significant consecutive synonymous-conserved sites. this methodology overcomes the inherent statistical problematic aspects of sliding window approaches for detecting evolutionary rate variation among sites. we detected twenty-one short linear stretches within the genome, which display significantly lower levels of ks than expected under neutrality. the forces leading to lowered ks in these regions were explored. the majority of the identified regions were mapped to previously known regulatory functional regions, or to overlapping open reading frames, validating our methodology as a general tool for predicting functional regions at the nucleotide level nested within protein-coding regions. furthermore, we detected a number of ks conserved regions that could not be assigned to a known function, and are thus predicted to be under selection due to a yet unknown reason. one such region is located within the pol gene. mutagenesis experiments of this region were conducted to reveal its function but showed no clear effect on viral replication in cell culture. the implications of these results are discussed.  selection intensity on synonymous sites varies within and across hiv orfs a large dataset of hiv- sequences spanning eight subtypes showed that variability in the synonymous substitution rate is present in each of the nine hiv- orfs. the two evolutionary codon models that account for ks variability exhibited a significantly better fit to the data, compared to the commonly used kav-ksc model that assumes a single ks rate for all sequence sites. furthermore, for all orfs other than vpr, significant support was found for the model accounting for dependency in evolutionary rates between adjacent sequence sites. the kad-ksd model differs from the kav-ksc model in two attributes: the dependence between adjacent sites and the variability in ks. by comparing the differences between kav-ksc and kav-ksv and between kad-ksd and kav-ksv, it is clear that assuming ks variability leads to the dominant factor in the improved fit of the kad-ksd model to the hiv- sequences. a comparison of the kad-ksd versus the kav-ksc model. b comparison of the kad-ksd versus the kav-ksv model. having established that kad-ksd is the best fitting model, it was next used to infer site-specific ka and ks scores for each site in each of the orfs. the posterior distributions of inferred ka and ks values were then examined. there is a large difference between the two distributions: on the one hand, most of the ka distribution surrounds very low values, with an average of, supporting the notion that extensive purifying selection is acting on the coding sequences. on the other hand, the mass of the ks distribution is centered around, suggesting that most synonymous sites are under neutral selection. indeed, the coefficient of variation of the ka distribution is much higher than that of the ks distribution suggesting higher level of variation of ka compared to ks values. identification of ks-conserved stretches across the hiv- genome the stretchfinder algorithm was used to scan the estimated ks values for extended regions exhibiting a statistically significant reduction of ks. a stretch was defined as a consecutive region spanning between and codons. assigning a p-value allowed the distinction between a region with a few randomly dispersed ks-conserved sites and an extended region with low ks values, which may be indicative of a functionally important region. this analysis yielded stretches with significantly low values of ks, following correction for multiple testing. notably, these stretches were very similar to those obtained when stretchfinder was applied to site specific ks rates inferred using the kav-ksv model. however, in a few cases the stretches obtained by the model assuming site dependency were slightly longer. this can be explained by the smoothing effect induced by models assuming site dependencies. regions which could not be fully accounted for are marked in bold. seven of the conserved stretches were mapped to regions previously reported to have a functional role. the most statistically significant ks conserved stretch along the hiv- genome region is located inside the pol orf and spans positions to this region spans a few functional nucleic acid elements: the central polypurine tract, the central termination sequence, and the dna flap region. this stretch resides near a second significant stretch within pol, which spans positions to. the conservation in this stretch may be partially explained by the presence of the end of the cts within it. indeed, this region within pol was previously identified using a manual scan for ks conserved regions. the second polypurine tract in hiv- is located inside the nef gene and is also part of a stretch with significantly low ks values. a third known functional element that was detected as a significantly conserved stretch is sl, which serves as part of the rna packaging signal and is located in gag. three out of five conserved stretches found in env are mapped to the rre region. additional twelve significantly conserved stretches are mapped to regions of overlapping genes. indeed, in almost all of these cases the overlapped region was frame-shifted from the first stretch, and the low ks values could be explained by selective constraints on the overlapped gene, i.e. by low ka values in the other gene. of the stretches in table  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– within pol. visual examination of the ka and ks values in this stretch 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– 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– region. 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, the reason for the conservation remains unknown. we next tested whether secondary structure motifs within this stretch may explain the apparent ks conservation. to determine the possible functional role of this region, an rna secondary structure prediction was performed, and was compared to the experimental values obtained for the whole hiv- genome. the predicted rna secondary structure suggested that the pol 82– region is mostly unstructured, apart from a short stem and loop at the beginning of the stretch. this prediction matches the secondary structure model based on a high-throughput rna analysis performed on the entire rna genome of hiv-. taken together, these analyses reinforce the hypothesis that the low ks of the pol 82– stretch is not related to its participation in rna secondary structures and is most likely also unrelated to translational efficiency; thus, it may have a regulatory role, such as binding an unknown factor. experimental testing we next proceeded towards revealing the biological role of the above conserved stretch comprising codon positions 82– of pol by mutagenesis experiments in molecular clones of hiv- we hypothesized that if these synonymous sites are indeed under selective constraints, artificially mutating these sites would affect the hiv- replication cycle. to this end, we introduced multiple synonymous mutations into the above pol region in the context of hiv- molecular clone nlr+gfp, generating the mutated nlr+gfppolmut clone. both the wild type and the mutated clones contain the entire hiv- genome sequence except the env and nef genes. in addition, the clones encode for the green fluorescent protein. we then used these clones to test for possible effects of the introduced synonymous mutations on both early and late stages of the hiv- replication cycle, in human embryonic kidney 293t cells. 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- genome into the host chromosomes. late events include expression of the hiv- genes, assembly of the virion, its release from the infected cell, and maturation into fully infectious particles. disruption of early stages should result in decreased levels of integrated hiv- proviruses. to detect such a reduction, we measured the overall expression of genes transcribed from the hiv- provirus. searching for a possible effect of the mutations on late events, we quantified virion release from the cells by measuring capsid levels in the context of assembled virions. in addition, the infectivity of produced virions was monitored using single cycle infection assays. two identical 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- 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. 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- particles of the tested clones. overall, these analyses suggest that the mutations in pol did not hamper the late stages of the hiv- replication cycle. the above results cannot rule out possible effects of the mutations on viral infectivity. to further investigate this option, naïve hek293t cells were infected with equal amounts of wild type and mutant virions, normalized by reverse transcriptase activity [as in. to measure infection levels, infected cells were tested for either intracellular gag or gfp levels, quantified by western blot or flow cytometry analyses, respectively. both gag and gfp levels were not significantly different among the wild type and mutated clones, suggesting that the mutations did not hamper infectivity. finally, we tested whether hiv- particle production originating from infected cells varied between the tested clones, and here too, no significant difference was observed. overall, these results indicate that in the current experimental settings, the functional role of this conserved stretch has no effect on the hiv- replication cycle. possible reasons for the disagreement between the strong evolutionary conservation signal and the experimental results are discussed below.  this study provides and explores a comprehensive map of the evolutionary selective forces operating on hiv- previous studies inferring natural selection in hiv did not account for selection on synonymous substitutions, which may have possibly inflated estimates of positive selection. specifically, overlapping regions of genes are often considered problematic and are removed from analyses. here we show that complex inter-related selective forces may operate on all protein coding genes within hiv- this analysis underscores the association between synonymously conserved regions and functional regions at the dna or rna levels. hiv- represents one of the most studied biological systems and thus provides us with the opportunity to correlate between the inferred ks conserved stretches and known functional regions residing within coding regions. it is noteworthy that nearly all well studied regulatory regions residing within the orfs of hiv- were also detected using our methodology. nevertheless, four synonymously conserved regions were detected with no previously reported function. an important aspect of this work was the effort to experimentally elucidate the significance of the computational predictions derived from the evolutionary models. however, the mutagenesis assay performed here on one of the stretches resulted in insignificant differences between the wild type and mutated constructs. insignificant differences between wild type and mutated clones were also obtained by ngandu et al., who experimentally tested the role for a synonymous conserved region within env. notably, these experiments were performed in cell cultures, which do not necessarily mimic natural infections of cd4+ cells. for instance, interactions with salient host proteins are often dependent on the specific cell-line used. in addition, it is possible that these synonymous mutations reduce viral fitness, but this reduction is not significant enough to be revealed after only a single infection cycle. finally, since this stretch may have an important role only in the context of the virus interaction with the host adaptive immune response, it was not discovered in the artificial cell culture experiments conducted here. hence, future work is needed to determine the role of the novel conserved stretches detected here. nevertheless, the methodology presented here is generic, and can be used to investigate other less-studied viral datasets to predict novel functional regions, as well as to explore the breadth of synonymous conservation in both cells and viruses. moreover, the identification of conserved sites in viral populations should enhance pcr diagnostics of a variety of viral strains that otherwise significantly differ from each other, like in the case of hiv- 
we used a combined computational-experimental approach to detect and characterize regions exhibiting strong purifying selection against synonymous substitutions along the hiv- genome. this superimposition of functions leads to complex selective forces acting on all levels of the genome and proteome. 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. these models are combined with a randomization procedure to automatically identify the most statistically significant regions of conserved synonymous sites along the genome. 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. such observations suggest the existence of stretches of codon sites that are evolutionary conserved at both dna-rna and protein levels. our methodology is based on advanced probabilistic evolutionary models that explicitly account for synonymous rate variation among sites and rate dependencies among adjacent sites. such a system does not account for the complexity of hiv- infections in vivo, which involves multiple infection cycles and interaction with the host immune system. considering the constraints on hiv- to maintain such a highly compact genome, we hypothesized that stretches of synonymous conservation would be common within its genome. such stretches may point to functionally important regions within protein coding sequences not necessarily reflecting functional constraints on the amino-acid sequence.
 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- 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- to maintain such a highly compact genome, we hypothesized that stretches of synonymous conservation would be common within its genome.  we used a combined computational-experimental approach to detect and characterize regions exhibiting strong purifying selection against synonymous substitutions along the hiv- 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 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.  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- infections in vivo, which involves multiple infection cycles and interaction with the host immune system. codon modelshiv-1synonymous substitutionssilent substitutionska/ks 