BACKGROUND
phylogenetic studies have shown that hiv emerged in humans through at least eleven cross-species transmission events of simian immunodeficiency virus  from non-human african primates.
three transmissions of chimpanzee siv  from the central african chimpanzee subspecies  gave rise to the hiv- <dig> groups m, n and o  <cit> , with the group m causing the aids pandemic.
other transmissions of sooty mangabey  siv  gave rise to hiv- <dig> groups a-h  <cit> .
siv infection of african non-human primate host species  is non-pathogenic despite high levels of viremia  <cit> .
different levels of pathogenicity of immunodeficiency viruses in their host species  <cit>  as well as the lack of adaptation to their non-natural species  <cit>  show how interspecies differences can impact viral infectivity and drive adaptation.
host genetic differences between individuals also affect the dynamics of disease progression  <cit> .
there is a growing list of genes and alleles for which there is evidence of a positive or negative effect on infection and disease progression.
among them, several recently identified host factors block or restrict retroviral infections in primates: trim5α, a tripartite motif  family protein  <cit> ; apolipoprotein b editing catalytic polypeptide , a member of the family of cytidine deaminases  <cit>  and tetherin   <cit> .
these restriction factors constitute defense mechanisms of the host acting in a species-specific manner  <cit>  blocking the viruses from replication in their non-natural host species and thus being potential agents of anti-hiv defense.
one feature of pathogenic hiv- <dig> infection that distinguishes it from non-pathogenic siv infections is the high level of chronic immune activation associated with accelerated t-cell turnover rates and apoptosis  <cit> .
the basis for this difference in pathogenicity is not understood, however deciphering which viral and host factors are responsible for the nonpathogenic course of natural siv infections could prove useful in developing more effective treatments and prevention strategies for aids.
positive selection, demonstrated in part by the rapidly evolving immune system genes  <cit> , reflects the evolution of the host defense against various infections.
several hiv restriction factors have been shown to be under positive selection throughout primate evolution  <cit> .
due to the relatively long generation times of primate species with slow rate of genetic evolution in contrast to the short generation times of viruses with high rates of genetic evolution and the potentially recent introduction of siv into primates  <cit> , the impact of the selection pressures solely from siv on the host species is likely to be negligible.
however, genetic polymorphisms in genes interacting with the virus can influence traits relevant for the susceptibility to lentiviral infection and point to a potential role of a gene in infection and its contribution to disease.
comparative genomics can offer insights into disease mechanisms by correlating molecular differences that arose during primate evolution with the variation in disease susceptibility.
there is ample scientific knowledge on hiv- <dig> human protein interactions.
the hiv- <dig> human protein interaction database is a catalogue of over  <dig> human proteins that participate in approximately  <dig> unique hiv-1-to-human protein interactions reported in peer reviewed scientific literature  <cit> .
the size and scope of this database allow for large-scale analyses of hiv-host molecular interactions.
together with several fully sequenced primate genomes it allows for a systematic search for host factors under positive selection that might be relevant for infection and merit further investigation.
previous studies of positive selection in the hiv host factors focused on individual examples  <cit> , as well as on a set of  <dig> proteins compiled from the literature  <cit> .
here we analyze all  <dig> genes available in the hiv- <dig> human protein interaction database.
we explore genetic differences of hiv-interacting genes among primates.
we perform a comparative genomics analysis of the hiv-interacting proteins in search of positively selected genes in four different primate species.
we characterize the positively selected genes in terms of their biological function, role in the protein-protein interaction networks and interactions with the virus.
we then analyze the relationship between the strength of positive selection in the host proteins with the evolutionary rates of the interacting proteins of five immunodeficiency viruses in search of patterns in the evolution of host-pathogen interactions.
RESULTS
positive selection
in order to find which of the  <dig> host factors reported in the hiv-1-human protein interaction database are under positive selection we extracted and aligned their human genetic sequences with the respective homologs in three monkey species: chimpanzee, orangutan and rhesus macaque.
we applied a measure of positive selection, based on the likelihood ratio test  for the presence of sites under positive selection.
we considered a significant lrt to be an indicator of a gene being under positive selection.
the number of positively selected sites in each gene sequence constitutes a site-based score of positive selection.
in order to assess the robustness of, and provide additional support for, the site-based score we compared it with a sliding window score based on dn/ds ratio  in sliding windows along the gene sequence.
in case of a discrepancy between the two positive selection scores we used the more stringent lrt significance as the indicator of positive selection, the sliding window measure was used as a supporting score.
we used both tests to search for positive selection in all four primate species and subsequently in the human and chimpanzee gene sequences separately.
the site-based search for genes under positive selection returned  <dig> genes having sites under positive selection among all four primate species and in  <dig> genes having sites under positive selection in the human-chimp comparison with an overlap of  <dig> genes.
the full list of the analyzed genes together with all scores is provided in the additional file  <dig>  the genes were next ranked according to their site-based and sliding window scores.
the ranks of genes obtained with the sliding window score correlate with the site-based score ranks with a correlation coefficient of  <dig>  in all species and  <dig>  in the human-chimp comparison .
this positive correlation of ranks obtained with two different methods, together with the high scores assigned to proteins reported to be under positive selection in other studies  suggests that the ranks used in further analyses are robust with respect to the scoring method.
the recently identified host restriction factor tetherin   <cit>  was not included in the hiv- <dig> human interaction database.
we separately extracted, aligned and performed the positive selection tests on the tetherin sequences of the four primate species.
even though positive selection in the primate tetherins has been reported before  <cit> , the site-based approach did not result in significant lrt.
however the sliding window test showed this protein to be under positive selection with a rank of  <dig> among the full list of  <dig> genes .
in order to inspect the distribution of positive selection scores in subsets of the hiv-interacting genes we established an interaction grouping based on with which viral protein the host proteins interact.
the interaction grouping showed variation in the distributions of site-based scores as well as in the ratio of positively selected host proteins among groups .
permutation tests revealed a significantly lower mean of the ranks based on site-based scores of host genes interacting with gag protein and a comparatively higher mean for integrase , protease , vpr and rev proteins .
the ranks based on sliding window scores were additionally significantly lower for the envelope -, gp120-, gp41- and capsid -interacting genes and higher for vif- matrix - and nucleocapsid -interacting genes.
the site-based ranking was limited to the  <dig> genes under positive selection.
the discrepancies of the significance of mean ranks of gene groups between the two scorings were due to the differing numbers of genes included in both rankings.
the mean group ranks in both scorings are positively correlated with r =  <dig>  .
if we consider only the  <dig> genes under positive selection the correlation increases substantially to r =  <dig>  .
positively selected host genes are inferred from the significance of the lrt.
signs in parentheses indicate significantly high  and low  mean normalized rank of interacting host genes under positive selection.
ranks in both scorings are normalized to  <cit>  range to facilitate comparison.
in order to additionally estimate positive selection that acted along the more recent timescale after the primate interspecies split we searched for single nucleotide polymorphisms  in the human gene sequences.
we found  <dig> genes containing positively selected snps with a maximum number of  <dig> snps in a gene  and the majority of genes having only one positively selected snp .
among genes found to be under positive selection using site-based method in the four primates and in the human-chimp comparison,  <dig> and  <dig> genes, respectively, were found to contain positively selected snps.
we found no significant correlation between the presence of snps and positive selection in primates.
gene ontology
subsequently, we searched for gene ontology   <cit>  terms enriched among the positively selected hiv-interacting genes.
go separates biological roles performed by genes of different organisms into three separate ontologies: biological process, molecular function and cellular component, each organized in a hierarchical manner with more general terms preceding more specific terms in the go graph.
we applied two statistical tests, fisher's exact test  and kolmogorov-smirnov test , to test for the overrepresentation of terms in the three groups of positively selected genes: in all four primates, in human-chimp comparison and human only as inferred from the snps analysis resulting six enrichment tests in total.
the fet and ks test for two different aspects of enrichment - enrichment of genes under positive selection and enrichment among high-ranking genes based on a positive selection score.
the go annotation of the genes from the hiv- <dig> human protein interaction database has been previously reported  <cit> , our goal was to find which terms were specific for the three groups of genes being under positive selection as compared to the full set of hiv-interacting genes.
the detailed results of all tests are provided as additional file  <dig>  here we list selected terms that were significantly enriched  in at least three out of the six tests.
in the biological process ontology several terms related to immune response were found to be enriched among the positively selected genes  and several immune response terms were enriched among the high-ranking genes in all three groups of genes .
among the terms of the cellular component ontology we found evidence of genes related to the cellular membrane being under positive selection.
eight out of  <dig> terms of this ontology that were overrepresented in at least four out of six enrichment tests were associated with the cellular membrane .
the mhc protein complex group of terms in this gene ontology also appeared to be enriched among positively selected hiv-interacting genes.
in the molecular function ontology the terms enriched in the positively selected genes are predominantly related to receptor or signaling activity, e.g.
"receptor activity", "mhc class i and ii activity", "transmembrane receptor activity", "signal transducer activity".
given the high enrichment of the terms "membrane" and "innate immune response" we further analyzed genes in those two functional groups.
to identify the first group we extracted all genes annotated with the go term go: <dig>  and its subterms.
to identify the innate immune response genes we performed a two step procedure.
first, we extracted all genes annotated with the go term go: <dig>  and its subterms and then we searched our dataset for all proteins known to participate in the innate immune response  <cit> .
we found the membrane-related proteins to be overrepresented among the positively selected genes in all four primates  and innate immune response to be overrepresented in all three groups of positively selected genes .
protein-protein interaction networks
to characterize the importance of the hiv-interacting proteins for the host and those among them being under positive selection, we analyzed their position in the network of human protein-protein interactions  and human-pathogen ppis, based on the analysis of human-pathogen interactions for  <dig> pathogens, both bacterial and viral, merged into  <dig> pathogen groups based on taxonomic similarity  <cit> .
dyer et al.
<cit>  reported that both viral and bacterial proteins preferentially interact with human proteins that are either hubs - i.e., those involved in many interactions, or bottlenecks - i.e., those central to many human ppi network pathways suggesting that pathogens may have evolved to interact with proteins controlling critical processes in the host cell as a mechanism of disrupting the key elements of the host cellular machinery.
we inspected the distribution of the local connectivity, centrality, number of interacting pathogens, and number of pathogen groups for the host proteins in the interaction grouping.
local connectivity of a protein is defined as the number of human ppis in which it participates; centrality is the fraction of shortest paths in human ppi network between all protein pairs that pass through the given protein.
high centrality is characteristic of a bottleneck in an interaction network, high connectivity - of a hub.
we found that in, p <dig>  pr, rev, reverse transcriptase , vif, vpr interact with host proteins of a significantly high local connectivity.
p <dig> and rev interact with proteins that are also highly central .
in contrast, gp <dig>  env, ca and gag seem to interact with host proteins of a significantly lower local connectivity.
p-values of the significance of local connectivity, centrality, number of interacting pathogens and pathogen groups are indicated with a  sign for high values and a  sign for low values.
given the low local connectivity of envelope-interacting host proteins, we investigated the distributions of the local connectivity, centrality, number of interacting pathogens and pathogen groups of the hiv-interacting proteins annotated with membrane-related go terms.
these proteins were of  <dig> -fold lower degree , interact with  <dig> -fold lower number of pathogens  and  <dig> -fold lower number pathogen groups .
all of the positively selected genes in the four primates tend to be less connected  and less centrally located  in the human ppi than the genes that show no positive selection.
no clear patterns were observed in the number of interacting pathogens and pathogen groups or in the positively selected genes in the human-chimp comparison.
virus evolutionary rates
in order to further characterize the host proteins interacting with the virus, we devised a measure of genetic variability in the viral genes of immunodeficiency virus species corresponding to the positive selection measure in the primate genes.
we aligned  <dig> genomes of five species of hivs and sivs and the genomes of the hiv- <dig> and sivcpz only .
viral gene sequences extracted from the alignments were ranked according to their relative evolutionary rates , a measure of genetic variability in the genes based on their phylogenetic distances.
in order to determine how this ranking depended on the distance measure, we recalculated the relative evolutionary rates based on hamming distances.
the ranks based on distances inferred from phylogenetic trees correlated with the rates based on hamming distance .
the main goal of the ranking was to compare the relative variability of the viral genes in order to assess the correlation with the positive selection of the interacting host genes.
regardless of the distance measure used in both of the alignments, env and its two subproteins together with vpu and rev were among the top  <dig> in the protein ranking.
polymerase  and pol-encoded proteins  together with ca were among the  <dig> lowest ranked proteins.
the only discordance among the distance measures and alignments were observed among the middle-ranked genes .
gene rank correlation
to investigate the relative rates of evolution among the interacting host and virus genes we next calculated the correlation between the strength of positive selection acting on the host factors and the rate of evolution of the interacting viral genes.
we calculated the correlation of the host factor ranks assigned according to the site-based and the sliding window scores and the viral gene ranks assigned according to the relative evolutionary rate measure.
the ranks of interacting host and viral proteins showed only minor but significant  positive correlation .
in the human-chimp comparison the sliding window ranking produced the only significant correlation .
given the small but significant correlation of the ranks of interacting host and virus proteins we devised an interaction binning test to investigate the proximate relationships between interacting gene ranks.
bins were defined on the viral gene ranking, the ranks of host genes within one bin were averaged.
<dig> bin sizes were tested containing all possible number of genes adjacent in the viral gene ranking.
the test showed a markedly increased correlation of approximate ranks of interacting viral and host genes .
for example, binning interactions over a bin of size  <dig> , that averages host gene ranks interacting with  <dig> viral genes neighboring in the viral gene ranking increases substantially the correlation to r =  <dig>  in the site-based and r =  <dig>  in the sliding window scoring .
permutation tests showed the significant correlations  in 14/ <dig> bin sizes in the site-based ranking and in 13/ <dig> in the sliding window ranking.
in the human-chimp comparison only the correlation based on the sliding window score was significant for 16/ <dig> bin sizes, with the correlation of  <dig>  on average and of  <dig>  for the  <dig>  bin size.
the high correlations obtained after binning suggest that the viral genes of different evolutionary rates tend to interact with host factors under commensurate levels of positive selection.
discussion and
CONCLUSIONS
understanding the genome-wide selection pressure on hiv-interacting proteins can provide insights into the evolutionary dynamics of host factors, the genetic basis of differences between nonpathogenic and pathogenic lentivirus infection and the roles of individual genes in host-pathogen interaction and immunopathogenesis.
only two of the host species analyzed in this study are naturally infected with hiv/siv  moreover because these viruses have been recently introduced into their host species  <cit>  the selection pressures we observe are not driven by modern lentiviruses.
even though the interaction data used is mainly human and hiv- <dig> specific, the majority of interactions are shared with sivs and with many other viruses.
many of the host factors found to be under positive selection interact with multiple pathogens so the selection pressures on the host factors are likely to be driven by those pathogens.
however the comprehensive hiv interaction data offers opportunity for a broad study of the evolution of host-pathogen interactions.
the interactions in the hiv- <dig> human protein interaction database are catalogued manually based on a literature screen and cannot be considered as fully validated.
nevertheless our screen for positive selection points to a narrow set of potentially interesting interactions that can be examined and validated individually.
in our analysis we identify ~10% of the  <dig> hiv-interacting genes as being under positive selection based on lrt of sites under positive selection in four primate species.
ortiz et al.
<cit>  reported a similar fraction of genes as being under positive selection based on the analysis of  <dig> genes.
among the  <dig> proteins analyzed in both this study and by ortiz et al.
<dig> are identified as being under positive selection in the ortiz et al.
study.
among these,  <dig> are confirmed in our study either by showing significant lrt or being within the upper quintile of the sliding window scores.
the reasons for discrepancies between the two studies might lie in different criteria of which genes to analyze and an expanded number of species for the analysis of individual genes in the ortiz et al.
study.
three screens using small interfering rna  have been reported  <cit>  that search human genes having effect on hiv infection - so called hiv dependency factors .
the overlap among the hdfs identified in the three studies and the proteins in the hiv interaction database is known to be small  <cit> .
we scanned the results of one of the sirna screening studies  <cit>  for the presence of genes under positive selection detected in our analysis.
among  <dig> genes common between ours and brass et al.
studies only one protein  appeared to be under positive selection.
several host factors not identified as being under positive selection in the lrt showed an elevated dn/ds ratio in the sliding window test.
for example, tetherin, previously reported to be under positive selection  <cit> , did not show significant lrt but ranked among the top 5% of all hiv-interacting proteins in the sliding window test.
however, in order to be able to identify genes with small number of polymorphic sites in relatively long and conserved sequences we relied on the criterion of the significance of the lrt to assess positive selection.
our functional analysis of the hiv-interacting host factors under positive selection pointed to two functional groups showing evidence of positive selection: membrane-related proteins and innate immune response proteins.
these functional groups were overrepresented among the subsets of genes positively selected in all four primates, in human-chimp comparison and human only as inferred from the snps analysis .
the interaction network of the virus proteins and the positively selected host proteins discussed below is visualized in figure  <dig>
among the membrane-related hiv-interacting proteins under positive selection we observe several that are known to interact with other pathogens.
c-type lectin  a transmembrane receptor expressed on the surface of dendritic cells and macrophages, known to bind gp <dig>  <cit> , is a part of signaling pathways induced by other pathogens such as mycobacterium tuberculosis  <cit> , hepatitis c virus  <cit>  and ebola virus  <cit> .
intercellular adhesion molecule  <dig> , a cell surface glycoprotein expressed in endothelial cells and cells of the immune system involved in a range of interactions with the hiv  <cit>  is a receptor used by the rhinovirus  <cit> .
chemokine  receptor  <dig> , a g-protein-coupled receptor expressed in activated t cells, nk cells, and dendritic cells, suggested to interact with nef  <cit>  and tat  <cit> , participates in the signaling cascade of the t-cell activation in genital herpes simplex virus type  <dig>  <cit>  and hepatitis c virus  <cit>  infections.
binding to the surface receptors of the host cell and internalization into that cell are the first steps of the viral infection; the genetic variability of the proteins expressed on the cell surface therefore represents a potential host defense mechanism to infection, preventing viral recognition and efficient cell entry.
the genetic variation in membrane genes has been previously reported  <cit> .
this variation might have resulted from previous infections and can have impact on modern lentivirus restriction and primate species susceptibility to infection.
we detected a substantial amount of genetic variation in the membrane gene cd3e , coding for a part of the t-cell receptor cd <dig> complex .
the highest concentration of positively selected residues was found in the protein extracellular and transmembrane domains.
the cd3-tcr complex is known to participate in the mechanism responsible for different levels of immune activation and t-cell apoptosis in pathogenic hiv-human and nonpathogenic siv-monkey infections  <cit> .
this and other examples of membrane proteins being under positive selection suggests that other proteins in this functional group might be relevant for differences in hiv pathogenesis.
similar to the membrane-related genes, we find the innate immune response gene group to be significantly enriched among the positively selected genes.
the innate immune response is the first line of defense against viral infection detecting virus particles, modulating signaling pathways leading to an increased interferon  production and inhibition of virus spread to limit pathogenesis.
there is growing evidence that the variability in the innate immune response proteins among primate species might contribute to different levels of immune activation in immunodeficiency virus infections  <cit> .
we found interferon regulatory factor  <dig>  to be under positive selection both in the test of four primate species and in the human-chimp comparison.
irf <dig> is a transcriptional factor involved in signaling triggered by the toll-like receptor  tlr <dig> and tlr <dig> in response to the siv infection  <cit> .
previous studies have shown that the genetic change in the primate irf <dig> is correlated with the immune activation in siv infection of primates  <cit> .
mutations in irf <dig> might therefore be responsible for an altered tlr <dig> and tlr <dig> signaling leading to different levels of immune activation between pathogenic and nonpathogenic infections.
caspase  <dig> , a member of cysteine-aspartic acid protease family is an example of innate immune response gene under positive selection in primates  that is also located on a positively selected allele in humans.
casp <dig> is known to play a role in cellular apoptosis induced by hiv- <dig> vpr  <cit>  and gp <dig>  <cit> .
caspase activation has also been shown to participate in the cellular defense against cowpox  <cit> , baculovirus  <cit> , and dengue virus  <cit> .
hiv- <dig> interaction with the caspase-regulated apoptosis might play a role in limiting the host immune response to the virus and facilitating viral persistence.
among the high-scoring innate immune response genes under positive selection we also found fas  , a member of the tumor necrosis factor  receptor family.
the fas-mediated pathway plays an important role in hiv- <dig> immunopathogenesis.
fas has been suggested to contribute to the loss of cd4+ t-cells in progression to aids as a part of the tcr-cd <dig> signaling pathway  <cit> .
high levels of positive selection in fas among primates and its regulatory relationship with tcr-cd <dig> point to this molecular pathway being possibly differently regulated among primates and partially responsible for immunopathogenesis.
hyperactivation of the immune system in response to a pathogen recently introduced from another species can result in severe pathogenesis .
disentangling the interaction of the hiv with the innate immune response and the host-specific differences in primate species might thus be of therapeutic interest.
the examples of innate immune response genes under positive selection relevant for diverse immune activation in hiv infection suggest that other genes reported by our analysis might merit further study.
to gain further insight into the positively selected genes we grouped them according to their interacting viral genes and characterized each group with the use of the positive selection score, gene roles in the ppi network and the evolutionary rate of the corresponding viral gene.
among the gene groups, genes interacting with the viral envelope and its subunits show elevated scores of positive selection.
in contrast, we found limited evidence of positive selection in the genes interacting with viral pol-encoded genes, with pr and in in particular.
in the context of their role in the ppi network and interactions with other pathogens the envelope and gp41-interacting genes revealed a significantly low local connectivity, centrality and number of interacting pathogens and pathogen groups.
the same pattern was observed for the group of membrane-related genes.
this less crucial role in the ppi and high specificity for the interacting pathogens might be key factors allowing for the accelerated evolution of the membrane and envelope-interacting genes.
the less critical role of these proteins in the host biology makes them potential drug targets, for example, the chemokine receptor ccr <dig> is targeted by the maraviroc antagonist  <cit> .
in contrast to the host proteins interacting with the viral envelope and its subunits we found significantly higher local connectivity and centrality in human ppis of the host genes interacting with viral pol-encoded genes.
together with their higher conservation this suggests that the crucial role of these highly connected genes in host cellular biology could be an evolutionary constraint.
pathogens might therefore interact preferentially with highly connected human proteins not only as a strategy to control critical host molecular processes  <cit>  but also because the lack of variation makes them a static target for virus-host interaction.
the high variability of the viral envelope gene and its subunits gp <dig> and gp <dig>  <cit>  is in accordance with the high positive selection score of envelope-interacting and host membrane genes in general.
envelope proteins that participate in the crucial steps of binding to the cell receptors and coreceptors and membrane fusion  <cit> , are located on the surface of the virion and contain recognition sites for various adaptive immune responses.
thus changes in optimal host cell receptor affinity and evasion of host immune responses create selection pressure on the env gene.
together with the positive selection acting on the host membrane genes it points to the viral recognition and host cell receptor affinity as processes in which both the viral and host genes undergo accelerated evolution driven by viral evasion and host suppression.
conversely, pol-encoded proteins showed the least genetic variation among viral proteins.
these proteins perform essential enzymatic functions common to all retroviruses, such as rna retrotranscription, dna integration and protein maturation and are unsurprisingly among the most conserved hiv/siv proteins.
host factors interacting with the pol-encoded proteins also show less genetic variation among the hiv-interacting genes, suggesting that these interactions tend to be the conserved parts of the host-pathogen interface.
the significant correlation of the viral and host gene ranks based on the rates of gene evolution further supports the hypothesis of reciprocal evolutionary effects between interacting host and pathogen proteins  <cit> .
highly conserved processes tend to be those crucial for viral replication; those less conserved might not be essential for the virus survival but involve accessory proteins and might contribute to pathogenesis.
the highest rates of genetic change tend to be in the processes acting on the viral envelope and host cell membrane as they involve viral evasion and host pathogen immune recognition.
we identified and characterized host defense factors under positive selection potentially involved in different responses to immunodeficiency virus infections in primates.
identifying genetic differences in the interacting proteins can open the way to biological testing of hypotheses regarding their role in various siv/hiv infection phenotypes with different levels of immune activation.
in addition to providing new insights into viral pathogenesis and host immunity, the approach presented here provides the potential for discovering new targets for antiviral therapies based on the knowledge of crucial elements of the host-pathogen interface and the pace of their evolution.

 phylogenetic studies have shown that hiv emerged in humans through at least eleven cross-species transmission events of simian immunodeficiency virus from non-human african primates. three transmissions of chimpanzee siv from the central african chimpanzee subspecies gave rise to the hiv- groups m, n and o, with the group m causing the aids pandemic. other transmissions of sooty mangabey siv gave rise to hiv- groups a-h. siv infection of african non-human primate host species is non-pathogenic despite high levels of viremia. different levels of pathogenicity of immunodeficiency viruses in their host species as well as the lack of adaptation to their non-natural species show how interspecies differences can impact viral infectivity and drive adaptation. host genetic differences between individuals also affect the dynamics of disease progression. there is a growing list of genes and alleles for which there is evidence of a positive or negative effect on infection and disease progression. among them, several recently identified host factors block or restrict retroviral infections in primates: trim5α, a tripartite motif family protein; apolipoprotein b editing catalytic polypeptide, a member of the family of cytidine deaminases and tetherin. these restriction factors constitute defense mechanisms of the host acting in a species-specific manner blocking the viruses from replication in their non-natural host species and thus being potential agents of anti-hiv defense. one feature of pathogenic hiv- infection that distinguishes it from non-pathogenic siv infections is the high level of chronic immune activation associated with accelerated t-cell turnover rates and apoptosis. the basis for this difference in pathogenicity is not understood, however deciphering which viral and host factors are responsible for the nonpathogenic course of natural siv infections could prove useful in developing more effective treatments and prevention strategies for aids. positive selection, demonstrated in part by the rapidly evolving immune system genes, reflects the evolution of the host defense against various infections. several hiv restriction factors have been shown to be under positive selection throughout primate evolution. due to the relatively long generation times of primate species with slow rate of genetic evolution in contrast to the short generation times of viruses with high rates of genetic evolution and the potentially recent introduction of siv into primates, the impact of the selection pressures solely from siv on the host species is likely to be negligible. however, genetic polymorphisms in genes interacting with the virus can influence traits relevant for the susceptibility to lentiviral infection and point to a potential role of a gene in infection and its contribution to disease. comparative genomics can offer insights into disease mechanisms by correlating molecular differences that arose during primate evolution with the variation in disease susceptibility. there is ample scientific knowledge on hiv- human protein interactions. the hiv- human protein interaction database is a catalogue of over human proteins that participate in approximately unique hiv-1-to-human protein interactions reported in peer reviewed scientific literature. the size and scope of this database allow for large-scale analyses of hiv-host molecular interactions. together with several fully sequenced primate genomes it allows for a systematic search for host factors under positive selection that might be relevant for infection and merit further investigation. previous studies of positive selection in the hiv host factors focused on individual examples, as well as on a set of proteins compiled from the literature. here we analyze all genes available in the hiv- human protein interaction database. we explore genetic differences of hiv-interacting genes among primates. we perform a comparative genomics analysis of the hiv-interacting proteins in search of positively selected genes in four different primate species. we characterize the positively selected genes in terms of their biological function, role in the protein-protein interaction networks and interactions with the virus. we then analyze the relationship between the strength of positive selection in the host proteins with the evolutionary rates of the interacting proteins of five immunodeficiency viruses in search of patterns in the evolution of host-pathogen interactions.  positive selection in order to find which of the host factors reported in the hiv-1-human protein interaction database are under positive selection we extracted and aligned their human genetic sequences with the respective homologs in three monkey species: chimpanzee, orangutan and rhesus macaque. we applied a measure of positive selection, based on the likelihood ratio test for the presence of sites under positive selection. we considered a significant lrt to be an indicator of a gene being under positive selection. the number of positively selected sites in each gene sequence constitutes a site-based score of positive selection. in order to assess the robustness of, and provide additional support for, the site-based score we compared it with a sliding window score based on dn/ds ratio in sliding windows along the gene sequence. in case of a discrepancy between the two positive selection scores we used the more stringent lrt significance as the indicator of positive selection, the sliding window measure was used as a supporting score. we used both tests to search for positive selection in all four primate species and subsequently in the human and chimpanzee gene sequences separately. the site-based search for genes under positive selection returned genes having sites under positive selection among all four primate species and in genes having sites under positive selection in the human-chimp comparison with an overlap of genes. the full list of the analyzed genes together with all scores is provided in the additional file the genes were next ranked according to their site-based and sliding window scores. the ranks of genes obtained with the sliding window score correlate with the site-based score ranks with a correlation coefficient of in all species and in the human-chimp comparison. this positive correlation of ranks obtained with two different methods, together with the high scores assigned to proteins reported to be under positive selection in other studies suggests that the ranks used in further analyses are robust with respect to the scoring method. the recently identified host restriction factor tetherin was not included in the hiv- human interaction database. we separately extracted, aligned and performed the positive selection tests on the tetherin sequences of the four primate species. even though positive selection in the primate tetherins has been reported before, the site-based approach did not result in significant lrt. however the sliding window test showed this protein to be under positive selection with a rank of among the full list of genes. in order to inspect the distribution of positive selection scores in subsets of the hiv-interacting genes we established an interaction grouping based on with which viral protein the host proteins interact. the interaction grouping showed variation in the distributions of site-based scores as well as in the ratio of positively selected host proteins among groups. permutation tests revealed a significantly lower mean of the ranks based on site-based scores of host genes interacting with gag protein and a comparatively higher mean for integrase, protease, vpr and rev proteins. the ranks based on sliding window scores were additionally significantly lower for the envelope -, gp120-, gp41- and capsid -interacting genes and higher for vif- matrix - and nucleocapsid -interacting genes. the site-based ranking was limited to the genes under positive selection. the discrepancies of the significance of mean ranks of gene groups between the two scorings were due to the differing numbers of genes included in both rankings. the mean group ranks in both scorings are positively correlated with r =. if we consider only the genes under positive selection the correlation increases substantially to r =. positively selected host genes are inferred from the significance of the lrt. signs in parentheses indicate significantly high and low mean normalized rank of interacting host genes under positive selection. ranks in both scorings are normalized to range to facilitate comparison. in order to additionally estimate positive selection that acted along the more recent timescale after the primate interspecies split we searched for single nucleotide polymorphisms in the human gene sequences. we found genes containing positively selected snps with a maximum number of snps in a gene and the majority of genes having only one positively selected snp. among genes found to be under positive selection using site-based method in the four primates and in the human-chimp comparison, and genes, respectively, were found to contain positively selected snps. we found no significant correlation between the presence of snps and positive selection in primates. gene ontology subsequently, we searched for gene ontology terms enriched among the positively selected hiv-interacting genes. go separates biological roles performed by genes of different organisms into three separate ontologies: biological process, molecular function and cellular component, each organized in a hierarchical manner with more general terms preceding more specific terms in the go graph. we applied two statistical tests, fisher's exact test and kolmogorov-smirnov test, to test for the overrepresentation of terms in the three groups of positively selected genes: in all four primates, in human-chimp comparison and human only as inferred from the snps analysis resulting six enrichment tests in total. the fet and ks test for two different aspects of enrichment - enrichment of genes under positive selection and enrichment among high-ranking genes based on a positive selection score. the go annotation of the genes from the hiv- human protein interaction database has been previously reported, our goal was to find which terms were specific for the three groups of genes being under positive selection as compared to the full set of hiv-interacting genes. the detailed results of all tests are provided as additional file here we list selected terms that were significantly enriched in at least three out of the six tests. in the biological process ontology several terms related to immune response were found to be enriched among the positively selected genes and several immune response terms were enriched among the high-ranking genes in all three groups of genes. among the terms of the cellular component ontology we found evidence of genes related to the cellular membrane being under positive selection. eight out of terms of this ontology that were overrepresented in at least four out of six enrichment tests were associated with the cellular membrane. the mhc protein complex group of terms in this gene ontology also appeared to be enriched among positively selected hiv-interacting genes. in the molecular function ontology the terms enriched in the positively selected genes are predominantly related to receptor or signaling activity, e.g. "receptor activity", "mhc class i and ii activity", "transmembrane receptor activity", "signal transducer activity". given the high enrichment of the terms "membrane" and "innate immune response" we further analyzed genes in those two functional groups. to identify the first group we extracted all genes annotated with the go term go: and its subterms. to identify the innate immune response genes we performed a two step procedure. first, we extracted all genes annotated with the go term go: and its subterms and then we searched our dataset for all proteins known to participate in the innate immune response. we found the membrane-related proteins to be overrepresented among the positively selected genes in all four primates and innate immune response to be overrepresented in all three groups of positively selected genes. protein-protein interaction networks to characterize the importance of the hiv-interacting proteins for the host and those among them being under positive selection, we analyzed their position in the network of human protein-protein interactions and human-pathogen ppis, based on the analysis of human-pathogen interactions for pathogens, both bacterial and viral, merged into pathogen groups based on taxonomic similarity. dyer et al. reported that both viral and bacterial proteins preferentially interact with human proteins that are either hubs - i.e., those involved in many interactions, or bottlenecks - i.e., those central to many human ppi network pathways suggesting that pathogens may have evolved to interact with proteins controlling critical processes in the host cell as a mechanism of disrupting the key elements of the host cellular machinery. we inspected the distribution of the local connectivity, centrality, number of interacting pathogens, and number of pathogen groups for the host proteins in the interaction grouping. local connectivity of a protein is defined as the number of human ppis in which it participates; centrality is the fraction of shortest paths in human ppi network between all protein pairs that pass through the given protein. high centrality is characteristic of a bottleneck in an interaction network, high connectivity - of a hub. we found that in, p pr, rev, reverse transcriptase, vif, vpr interact with host proteins of a significantly high local connectivity. p and rev interact with proteins that are also highly central. in contrast, gp env, ca and gag seem to interact with host proteins of a significantly lower local connectivity. p-values of the significance of local connectivity, centrality, number of interacting pathogens and pathogen groups are indicated with a sign for high values and a sign for low values. given the low local connectivity of envelope-interacting host proteins, we investigated the distributions of the local connectivity, centrality, number of interacting pathogens and pathogen groups of the hiv-interacting proteins annotated with membrane-related go terms. these proteins were of -fold lower degree, interact with -fold lower number of pathogens and -fold lower number pathogen groups. all of the positively selected genes in the four primates tend to be less connected and less centrally located in the human ppi than the genes that show no positive selection. no clear patterns were observed in the number of interacting pathogens and pathogen groups or in the positively selected genes in the human-chimp comparison. virus evolutionary rates in order to further characterize the host proteins interacting with the virus, we devised a measure of genetic variability in the viral genes of immunodeficiency virus species corresponding to the positive selection measure in the primate genes. we aligned genomes of five species of hivs and sivs and the genomes of the hiv- and sivcpz only. viral gene sequences extracted from the alignments were ranked according to their relative evolutionary rates, a measure of genetic variability in the genes based on their phylogenetic distances. in order to determine how this ranking depended on the distance measure, we recalculated the relative evolutionary rates based on hamming distances. the ranks based on distances inferred from phylogenetic trees correlated with the rates based on hamming distance. the main goal of the ranking was to compare the relative variability of the viral genes in order to assess the correlation with the positive selection of the interacting host genes. regardless of the distance measure used in both of the alignments, env and its two subproteins together with vpu and rev were among the top in the protein ranking. polymerase and pol-encoded proteins together with ca were among the lowest ranked proteins. the only discordance among the distance measures and alignments were observed among the middle-ranked genes. gene rank correlation to investigate the relative rates of evolution among the interacting host and virus genes we next calculated the correlation between the strength of positive selection acting on the host factors and the rate of evolution of the interacting viral genes. we calculated the correlation of the host factor ranks assigned according to the site-based and the sliding window scores and the viral gene ranks assigned according to the relative evolutionary rate measure. the ranks of interacting host and viral proteins showed only minor but significant positive correlation. in the human-chimp comparison the sliding window ranking produced the only significant correlation. given the small but significant correlation of the ranks of interacting host and virus proteins we devised an interaction binning test to investigate the proximate relationships between interacting gene ranks. bins were defined on the viral gene ranking, the ranks of host genes within one bin were averaged. bin sizes were tested containing all possible number of genes adjacent in the viral gene ranking. the test showed a markedly increased correlation of approximate ranks of interacting viral and host genes. for example, binning interactions over a bin of size, that averages host gene ranks interacting with viral genes neighboring in the viral gene ranking increases substantially the correlation to r = in the site-based and r = in the sliding window scoring. permutation tests showed the significant correlations in 14/ bin sizes in the site-based ranking and in 13/ in the sliding window ranking. in the human-chimp comparison only the correlation based on the sliding window score was significant for 16/ bin sizes, with the correlation of on average and of for the bin size. the high correlations obtained after binning suggest that the viral genes of different evolutionary rates tend to interact with host factors under commensurate levels of positive selection. discussion and  understanding the genome-wide selection pressure on hiv-interacting proteins can provide insights into the evolutionary dynamics of host factors, the genetic basis of differences between nonpathogenic and pathogenic lentivirus infection and the roles of individual genes in host-pathogen interaction and immunopathogenesis. only two of the host species analyzed in this study are naturally infected with hiv/siv moreover because these viruses have been recently introduced into their host species the selection pressures we observe are not driven by modern lentiviruses. even though the interaction data used is mainly human and hiv- specific, the majority of interactions are shared with sivs and with many other viruses. many of the host factors found to be under positive selection interact with multiple pathogens so the selection pressures on the host factors are likely to be driven by those pathogens. however the comprehensive hiv interaction data offers opportunity for a broad study of the evolution of host-pathogen interactions. the interactions in the hiv- human protein interaction database are catalogued manually based on a literature screen and cannot be considered as fully validated. nevertheless our screen for positive selection points to a narrow set of potentially interesting interactions that can be examined and validated individually. in our analysis we identify ~10% of the hiv-interacting genes as being under positive selection based on lrt of sites under positive selection in four primate species. ortiz et al. reported a similar fraction of genes as being under positive selection based on the analysis of genes. among the proteins analyzed in both this study and by ortiz et al. are identified as being under positive selection in the ortiz et al. study. among these, are confirmed in our study either by showing significant lrt or being within the upper quintile of the sliding window scores. the reasons for discrepancies between the two studies might lie in different criteria of which genes to analyze and an expanded number of species for the analysis of individual genes in the ortiz et al. study. three screens using small interfering rna have been reported that search human genes having effect on hiv infection - so called hiv dependency factors. the overlap among the hdfs identified in the three studies and the proteins in the hiv interaction database is known to be small. we scanned the results of one of the sirna screening studies for the presence of genes under positive selection detected in our analysis. among genes common between ours and brass et al. studies only one protein appeared to be under positive selection. several host factors not identified as being under positive selection in the lrt showed an elevated dn/ds ratio in the sliding window test. for example, tetherin, previously reported to be under positive selection, did not show significant lrt but ranked among the top 5% of all hiv-interacting proteins in the sliding window test. however, in order to be able to identify genes with small number of polymorphic sites in relatively long and conserved sequences we relied on the criterion of the significance of the lrt to assess positive selection. our functional analysis of the hiv-interacting host factors under positive selection pointed to two functional groups showing evidence of positive selection: membrane-related proteins and innate immune response proteins. these functional groups were overrepresented among the subsets of genes positively selected in all four primates, in human-chimp comparison and human only as inferred from the snps analysis. the interaction network of the virus proteins and the positively selected host proteins discussed below is visualized in figure among the membrane-related hiv-interacting proteins under positive selection we observe several that are known to interact with other pathogens. c-type lectin a transmembrane receptor expressed on the surface of dendritic cells and macrophages, known to bind gp, is a part of signaling pathways induced by other pathogens such as mycobacterium tuberculosis, hepatitis c virus and ebola virus. intercellular adhesion molecule, a cell surface glycoprotein expressed in endothelial cells and cells of the immune system involved in a range of interactions with the hiv is a receptor used by the rhinovirus. chemokine receptor, a g-protein-coupled receptor expressed in activated t cells, nk cells, and dendritic cells, suggested to interact with nef and tat, participates in the signaling cascade of the t-cell activation in genital herpes simplex virus type and hepatitis c virus infections. binding to the surface receptors of the host cell and internalization into that cell are the first steps of the viral infection; the genetic variability of the proteins expressed on the cell surface therefore represents a potential host defense mechanism to infection, preventing viral recognition and efficient cell entry. the genetic variation in membrane genes has been previously reported. this variation might have resulted from previous infections and can have impact on modern lentivirus restriction and primate species susceptibility to infection. we detected a substantial amount of genetic variation in the membrane gene cd3e, coding for a part of the t-cell receptor cd complex. the highest concentration of positively selected residues was found in the protein extracellular and transmembrane domains. the cd3-tcr complex is known to participate in the mechanism responsible for different levels of immune activation and t-cell apoptosis in pathogenic hiv-human and nonpathogenic siv-monkey infections. this and other examples of membrane proteins being under positive selection suggests that other proteins in this functional group might be relevant for differences in hiv pathogenesis. similar to the membrane-related genes, we find the innate immune response gene group to be significantly enriched among the positively selected genes. the innate immune response is the first line of defense against viral infection detecting virus particles, modulating signaling pathways leading to an increased interferon production and inhibition of virus spread to limit pathogenesis. there is growing evidence that the variability in the innate immune response proteins among primate species might contribute to different levels of immune activation in immunodeficiency virus infections. we found interferon regulatory factor to be under positive selection both in the test of four primate species and in the human-chimp comparison. irf is a transcriptional factor involved in signaling triggered by the toll-like receptor tlr and tlr in response to the siv infection. previous studies have shown that the genetic change in the primate irf is correlated with the immune activation in siv infection of primates. mutations in irf might therefore be responsible for an altered tlr and tlr signaling leading to different levels of immune activation between pathogenic and nonpathogenic infections. caspase, a member of cysteine-aspartic acid protease family is an example of innate immune response gene under positive selection in primates that is also located on a positively selected allele in humans. casp is known to play a role in cellular apoptosis induced by hiv- vpr and gp. caspase activation has also been shown to participate in the cellular defense against cowpox, baculovirus, and dengue virus. hiv- interaction with the caspase-regulated apoptosis might play a role in limiting the host immune response to the virus and facilitating viral persistence. among the high-scoring innate immune response genes under positive selection we also found fas, a member of the tumor necrosis factor receptor family. the fas-mediated pathway plays an important role in hiv- immunopathogenesis. fas has been suggested to contribute to the loss of cd4+ t-cells in progression to aids as a part of the tcr-cd signaling pathway. high levels of positive selection in fas among primates and its regulatory relationship with tcr-cd point to this molecular pathway being possibly differently regulated among primates and partially responsible for immunopathogenesis. hyperactivation of the immune system in response to a pathogen recently introduced from another species can result in severe pathogenesis. disentangling the interaction of the hiv with the innate immune response and the host-specific differences in primate species might thus be of therapeutic interest. the examples of innate immune response genes under positive selection relevant for diverse immune activation in hiv infection suggest that other genes reported by our analysis might merit further study. to gain further insight into the positively selected genes we grouped them according to their interacting viral genes and characterized each group with the use of the positive selection score, gene roles in the ppi network and the evolutionary rate of the corresponding viral gene. among the gene groups, genes interacting with the viral envelope and its subunits show elevated scores of positive selection. in contrast, we found limited evidence of positive selection in the genes interacting with viral pol-encoded genes, with pr and in in particular. in the context of their role in the ppi network and interactions with other pathogens the envelope and gp41-interacting genes revealed a significantly low local connectivity, centrality and number of interacting pathogens and pathogen groups. the same pattern was observed for the group of membrane-related genes. this less crucial role in the ppi and high specificity for the interacting pathogens might be key factors allowing for the accelerated evolution of the membrane and envelope-interacting genes. the less critical role of these proteins in the host biology makes them potential drug targets, for example, the chemokine receptor ccr is targeted by the maraviroc antagonist. in contrast to the host proteins interacting with the viral envelope and its subunits we found significantly higher local connectivity and centrality in human ppis of the host genes interacting with viral pol-encoded genes. together with their higher conservation this suggests that the crucial role of these highly connected genes in host cellular biology could be an evolutionary constraint. pathogens might therefore interact preferentially with highly connected human proteins not only as a strategy to control critical host molecular processes but also because the lack of variation makes them a static target for virus-host interaction. the high variability of the viral envelope gene and its subunits gp and gp is in accordance with the high positive selection score of envelope-interacting and host membrane genes in general. envelope proteins that participate in the crucial steps of binding to the cell receptors and coreceptors and membrane fusion, are located on the surface of the virion and contain recognition sites for various adaptive immune responses. thus changes in optimal host cell receptor affinity and evasion of host immune responses create selection pressure on the env gene. together with the positive selection acting on the host membrane genes it points to the viral recognition and host cell receptor affinity as processes in which both the viral and host genes undergo accelerated evolution driven by viral evasion and host suppression. conversely, pol-encoded proteins showed the least genetic variation among viral proteins. these proteins perform essential enzymatic functions common to all retroviruses, such as rna retrotranscription, dna integration and protein maturation and are unsurprisingly among the most conserved hiv/siv proteins. host factors interacting with the pol-encoded proteins also show less genetic variation among the hiv-interacting genes, suggesting that these interactions tend to be the conserved parts of the host-pathogen interface. the significant correlation of the viral and host gene ranks based on the rates of gene evolution further supports the hypothesis of reciprocal evolutionary effects between interacting host and pathogen proteins. highly conserved processes tend to be those crucial for viral replication; those less conserved might not be essential for the virus survival but involve accessory proteins and might contribute to pathogenesis. the highest rates of genetic change tend to be in the processes acting on the viral envelope and host cell membrane as they involve viral evasion and host pathogen immune recognition. we identified and characterized host defense factors under positive selection potentially involved in different responses to immunodeficiency virus infections in primates. identifying genetic differences in the interacting proteins can open the way to biological testing of hypotheses regarding their role in various siv/hiv infection phenotypes with different levels of immune activation. in addition to providing new insights into viral pathogenesis and host immunity, the approach presented here provides the potential for discovering new targets for antiviral therapies based on the knowledge of crucial elements of the host-pathogen interface and the pace of their evolution. 
we additionally detect the innate immune response as a gene functional group harboring large differences among primates that could potentially account for the different levels of immune activation in the hiv/siv primate infection. we find a significant correlation between the evolutionary rates of interacting host and viral proteins pointing to processes of the host-pathogen biology that are relatively conserved among species and to those undergoing accelerated genetic evolution. positive selection of host proteins that interact with pathogens can indicate factors relevant for infection and potentially be a measure of pathogen driven evolution. these results indicate specific host factors and their functional groups experiencing pathogen driven evolutionary selection pressures. our analysis of primate genes and lentivirus genomes points to specific host factors of high genetic variability that could account for differences in susceptibility to disease and indicate specific mechanisms of host defense and pathogen adaptation.
 positive selection of host proteins that interact with pathogens can indicate factors relevant for infection and potentially be a measure of pathogen driven evolution.  our analysis of primate genes and lentivirus genomes points to specific host factors of high genetic variability that could account for differences in susceptibility to disease and indicate specific mechanisms of host defense and pathogen adaptation. we find that the largest amount of genetic change occurs in genes coding for cellular membrane proteins of the host as well as in the viral envelope genes suggesting cell entry and immune evasion as the primary evolutionary interface between host and pathogen. we additionally detect the innate immune response as a gene functional group harboring large differences among primates that could potentially account for the different levels of immune activation in the hiv/siv primate infection. we find a significant correlation between the evolutionary rates of interacting host and viral proteins pointing to processes of the host-pathogen biology that are relatively conserved among species and to those undergoing accelerated genetic evolution.  these results indicate specific host factors and their functional groups experiencing pathogen driven evolutionary selection pressures. individual host factors pointed to by our analysis might merit further study as potential targets of antiretroviral therapies. 