Members of the human APOBEC3 family of editing enzymes can inhibit various mobile genetic elements. APOBEC3A (A3A) can block the retrotransposon LINE-1 and the parvovirus adeno-associated virus type 2 (AAV-2) but does not inhibit retroviruses. In contrast, APOBEC3G (A3G) can block retroviruses but has only limited effects on AAV-2 or LINE-1. What dictates this differential target specificity remains largely undefined. Here, we modeled the structure of A3A based on its homology with the C-terminal domain of A3G and further compared the sequence of human A3A to those of 11 nonhuman primate orthologues. We then used these data to perform a mutational analysis of A3A, examining its ability to restrict LINE-1, AAV-2, and foreign plasmid DNA and to edit a single-stranded DNA substrate. The results revealed an essential functional role for the predicted single-stranded DNA-docking groove located around the A3A catalytic site. Within this region, amino acid differences between A3A and A3G are predicted to affect the shape of the polynucleotide-binding groove. Correspondingly, transferring some of these A3A residues to A3G endows the latter protein with the ability to block LINE-1 and AAV-2. These results suggest that the target specificity of APOBEC3 family members is partly defined by structural features influencing their interaction with polynucleotide substrates.

The current availability of five complete genomes of different primate species allows the analysis of genetic divergence over the last 40 million years of evolution. We hypothesized that the interspecies differences observed in susceptibility to HIV-1 would be influenced by the long-range selective pressures on host genes associated with HIV-1 pathogenesis. We established a list of human genes (n = 140) proposed to be involved in HIV-1 biology and pathogenesis and a control set of 100 random genes. We retrieved the orthologous genes from the genome of humans and of four nonhuman primates (Pan troglodytes, Pongo pygmaeus abeli, Macaca mulatta, and Callithrix jacchus) and analyzed the nucleotide substitution patterns of this data set using codon-based maximum likelihood procedures. In addition, we evaluated whether the candidate genes have been targets of recent positive selection in humans by analyzing HapMap Phase 2 single-nucleotide polymorphisms genotyped in a region centered on each candidate gene. A total of 1,064 sequences were used for the analyses. Similar median K(A)/K(S) values were estimated for the set of genes involved in HIV-1 pathogenesis and for control genes, 0.19 and 0.15, respectively. However, genes of the innate immunity had median values of 0.37 (P value = 0.0001, compared with control genes), and genes of intrinsic cellular defense had K(A)/K(S) values around or greater than 1.0 (P value = 0.0002). Detailed assessment allowed the identification of residues under positive selection in 13 proteins: AKT1, APOBEC3G, APOBEC3H, CD4, DEFB1, GML, IL4, IL8RA, L-SIGN/CLEC4M, PTPRC/CD45, Tetherin/BST2, TLR7, and TRIM5alpha. A number of those residues are relevant for HIV-1 biology. The set of 140 genes involved in HIV-1 pathogenesis did not show a significant enrichment in signals of recent positive selection in humans (intraspecies selection). However, we identified within or near these genes 24 polymorphisms showing strong signatures of recent positive selection. Interestingly, the DEFB1 gene presented signatures of both interspecies positive selection in primates and intraspecies recent positive selection in humans. The systematic assessment of long-acting selective pressures on primate genomes is a useful tool to extend our understanding of genetic variation influencing contemporary susceptibility to HIV-1.

BackgroundAPOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G) has antiretroviral activity associated with the hypermutation of viral DNA through cytosine deamination. APOBEC3G has two cytosine deaminase (CDA) domains; the catalytically inactive amino-terminal domain of APOBEC3G (N-CDA) carries the Vif interaction domain. There is no 3-D structure of APOBEC3G solved by X-ray or nuclear magnetic resonance.Methodology/Principal FindingsWe predicted the structure of human APOBEC3G based on the crystal structure of APOBEC2. To assess the model structure, we evaluated 48 mutants of APOBEC3G N-CDA that identify novel variants altering ΔVif HIV-1 infectivity and packaging of APOBEC3G. Results indicated that the key residue D128 is exposed at the surface of the model, with a negative local electrostatic potential. Mutation D128K changes the sign of that local potential. In addition, two novel functionally relevant residues that result in defective APOBEC3G encapsidation, R122 and W127, cluster at the surface.Conclusions/SignificanceThe structure model identifies a cluster of residues important for packaging of APOBEC3G into virions, and may serve to guide functional analysis of APOBEC3G.

The metabotropic glutamate receptor 4 (mGluR4) is an emerging target for the treatment of Parkinson's disease (PD). However, since the discovery of its therapeutic potential, no ligand has been successfully developed enough to be tested in the clinic. In the present paper, we report for the first time the medicinal chemistry efforts conducted around the pharmacological tool (-)-PHCCC. This work led to the identification of compound 40, a potent and selective mGluR4 positive allosteric modulator (PAM) with good water solubility and demonstrating consistent activity across validated preclinical rodent models of PD motor symptoms after intraperitoneal administration: haloperidol-induced catalepsy in mouse and the rat 6-hydroxydopamine (6-OHDA) lesion model. Moreover, we also describe the identification of compound 60 a close analogue of compound 40 with improved pharmacokinetic profile after oral administration. On the basis of its favorable and unique preclinical profile, compound 60 (PXT002331, now foliglurax) was nominated as a candidate for clinical development.

The CD209 gene family that encodes C-type lectins in primates includes CD209 (DC-SIGN), CD209L (L-SIGN) and CD209L2. Understanding the evolution of these genes can help understand the duplication events generating this family, the process leading to the repeated neck region and identify protein domains under selective pressure. We compiled sequences from 14 primates representing 40 million years of evolution and from three non-primate mammal species. Phylogenetic analyses used Bayesian inference, and nucleotide substitutional patterns were assessed by codon-based maximum likelihood. Analyses suggest that CD209 genes emerged from a first duplication event in the common ancestor of anthropoids, yielding CD209L2 and an ancestral CD209 gene, which, in turn, duplicated in the common Old World primate ancestor, giving rise to CD209L and CD209. K A /K S values averaged over the entire tree were 0.43 (CD209), 0.52 (CD209L) and 0.35 (CD209L2), consistent with overall signatures of purifying selection. We also assessed the Toll-like receptor (TLR) gene family, which shares with CD209 genes a common profile of evolutionary constraint. The general feature of purifying selection of CD209 genes, despite an apparent redundancy (gene absence and gene loss), may reflect the need to faithfully recognize a multiplicity of pathogen motifs, commensals and a number of self-antigens.

Common human variants of TRIM5alpha have no effect or modest effect on HIV-1 disease progression. These variants occur at sites conserved throughout evolution, and are remote from clusters of positive selection in the primate lineage. The evolutionary value of the substitutions remains unclear.

The antiretroviral protein TRIM5␣ is known to have evolved different restriction capacities against various retroviruses, driven by positive Darwinian selection. However, how these different specificities have evolved in the primate lineages is not fully understood. Here we used ancestral protein resurrection to estimate the evolution of antiviral restriction specificities of TRIM5␣ on the primate lineage leading to humans. We used TRIM5␣ coding sequences from 24 primates for the reconstruction of ancestral TRIM5␣ sequences using maximum-likelihood and Bayesian approaches. Ancestral sequences were transduced into HeLa and CRFK cells. Stable cell lines were generated and used to test restriction of a panel of extant retroviruses (human immunodeficiency virus type 1 [HIV-1] and HIV-2, simian immunodeficiency virus [SIV] variants SIV mac and SIV agm , and murine leukemia virus [MLV] variants N-MLV and B-MLV). The resurrected TRIM5␣ variant from the common ancestor of Old World primates (Old World monkeys and apes, ϳ25 million years before present) was effective against present day HIV-1. In contrast to the HIV-1 restriction pattern, we show that the restriction efficacy against other retroviruses, such as a murine oncoretrovirus (N-MLV), is higher for more recent resurrected hominoid variants. Ancestral TRIM5␣ variants have generally limited efficacy against HIV-2, SIV agm , and SIV mac . Our study sheds new light on the evolution of the intrinsic antiviral defense machinery and illustrates the utility of functional evolutionary reconstruction for characterizing recently emerged protein differences.A newly described form of innate immunity, coined "intrinsic immunity," provides a constitutive line of defense, which relies on intracellular obstacles to hinder the replication of pathogens (1). This component of the immune system has gained much attention as a cornerstone of the resistance of mammals against several classes of retroelements and retroviruses (43).Representative components of this cellular defense system include members of the tripartite motif (TRIM) family (21). The best-studied family member, TRIM5␣ (31), restricts retroviral infection by specifically recognizing the viral capsid and promoting its premature disassembly (3,20,32), and, as recently reported, by blocking viral production at a posttranslational stage (23). Human TRIM5␣ has limited efficacy against human immunodeficiency virus type 1 (HIV-1), while proteins encoded by some primate TRIM5␣ orthologs can potently restrict this particular lentivirus (18,28,29,33). Longstanding selective pressures exerted by retroviruses and retroelements may have contributed to the generation of diverse patterns of antiretroviral specificity of TRIM5␣ and other host defense genes (18,28).To better understand the evolution of antiretroviral specificity patterns in primates, in particular along the lineage leading to humans, we utilized a functional evolutionary genomics approach (34). In the present study, we reconstructed ancestral primate TRIM5␣ sequences an...

Retroviruses are both powerful evolutionary forces and dangerous threats to genome integrity. As such, they have imposed strong selective pressure on their hosts, notably triggering the emergence of restriction factors, such as TRIM5␣, that act as potent barriers to their cross-species transmission. TRIM5␣ orthologues from different primates have distinct retroviral restriction patterns, largely dictated by the sequence of their C-terminal PRYSPRY domain, which binds the capsid protein of incoming virions. Here, by combining genetic and functional analyses of human and squirrel monkey TRIM5␣, we demonstrate that the coiled-coil domain of this protein, thus far essentially known for mediating oligomerization, also conditions the spectrum of antiretroviral activity. Furthermore, we identify three coiled-coil residues responsible for this effect, one of which has been under positive selection during primate evolution, notably in New World monkeys. These results indicate that the PRYSPRY and coiled-coil domains cooperate to determine the specificity of TRIM5␣-mediated capture of retroviral capsids, shedding new light on this complex event.The genomes of higher organisms, including primates, contain large numbers of endogenous retroviruses, remnants of past infections established in their germ lines over millions of years (13). Exogenous retroviruses are still an ongoing threat, as illustrated by the human immunodeficiency virus (HIV) pandemic. Retroviruses have thus subjected higher species to formidable selective pressures, leading to the emergence of a number of host-encoded antiviral factors (42). TRIM5␣ is one such factor, which can restrict a range of retroviruses in a virusand species-specific fashion (9,16,31,34,36,40,49). Although the mechanism of TRIM5␣ action is still not fully understood, it is known that it binds to and multimerizes around incoming capsids, inducing their premature uncoating (3,5,30,37). The accelerated disassembly of the capsid impairs reverse transcription (RT), and the viral genome is also prevented from migrating to the cell nucleus (1,16,36,39,43).TRIM5␣ encompasses a RING finger, a B-box type II and a coiled-coil domain, which together form the so-called RBCC or tripartite motif (TRIM), upstream of a C-terminal PRYSPRY domain (24,27). Each of these regions has been demonstrated to participate in the antiviral function of TRIM5␣, and yet a full understanding of their respective contributions is still lacking. A RING domain is often found in proteins that function as E3 ubiquitin ligases and was demonstrated to promote TRIM5␣ auto-ubiquitination and to regulate protein turnover (4, 10, 44). Deletion of the RING domain significantly affects restriction activity, although the exact role of the ubiquitin pathway in restriction is not fully understood (1,3,29,32,43). The B-box II domain plays an essential function in TRIM5␣ activity partly by mediating the higher-order self-assembly of TRIM5␣, which potentiates restriction (6,11,19,29). The coiled-coil region promotes the formation of mu...