Sooty mangabey genome sequence provides insight into AIDS resistance in a natural SIV host.

In contrast to infections with human immunodeficiency virus (HIV) in humans and simian immunodeficiency virus (SIV) in macaques, SIV infection of a natural host, sooty mangabeys (Cercocebus atys), is non-pathogenic despite high viraemia. Here we sequenced and assembled the genome of a captive sooty mangabey. We conducted genome-wide comparative analyses of transcript assemblies from C. atys and AIDS-susceptible species, such as humans and macaques, to identify candidates for host genetic factors that influence susceptibility. We identified several immune-related genes in the genome of C. atys that show substantial sequence divergence from macaques or humans. One of these sequence divergences, a C-terminal frameshift in the toll-like receptor-4 (TLR4) gene of C. atys, is associated with a blunted in vitro response to TLR-4 ligands. In addition, we found a major structural change in exons 3-4 of the immune-regulatory protein intercellular adhesion molecule 2 (ICAM-2); expression of this variant leads to reduced cell surface expression of ICAM-2. These data provide a resource for comparative genomic studies of HIV and/or SIV pathogenesis and may help to elucidate the mechanisms by which SIV-infected sooty mangabeys avoid AIDS.

Novel MHC class I full-length allele and haplotype characterization in sooty mangabeys.

Sooty mangabeys (Cercocebus atys) are natural SIV hosts and the presumed source of HIV-2 and SIVmac, which makes them a valuable model for HIV/SIV research. However, like other African primates, little is known about their major histocompatibility complex (MHC) genetics. In this study, we used Roche/454 and Illumina MiSeq deep sequencing in order to determine the MHC class I transcripts in a cohort of 165 sooty mangabeys from the Yerkes National Primate Research Center (YNPRC). We have characterized 121 functionally full-length classical (Ceat-A and Ceat-B) and non-classical (Ceat-F and Ceat-I) alleles and have also identified 22 Ceat-A/Ceat-B haplotype chromosomal combinations. We correlated these Ceat-A/Ceat-B haplotype combinations to recently described microsatellite haplotypes from the YNPRC colony. These newly identified alleles and haplotypes establish a resource for studying cellular immunity in sooty mangabeys and provide a framework for rapidly cataloging MHC class I sequences in an understudied, yet important, nonhuman primate species.

Characterization of MHC class I alleles in sooty mangabeys as a tool for evaluating cellular immunity in natural hosts of SIV infection.

Although immune pressure exerted by MHC class I-restricted cytotoxic T lymphocytes (CTL) are an important determinant of outcome in pathogenic HIV and SIV infection, lack of data on MHC class I genes has hampered study of its role in natural hosts with nonpathogenic SIV infection. In this study, we cloned and characterized full-length MHC class I genes derived from the cDNA library of two unrelated naturally infected sooty mangabeys (Cercocebus atys) in whom SIV-specific CTL epitopes were previously mapped. Twenty one full-length MHC class I alleles consisting of five MHC-A (Ceat-A), 13 MHC-B (Ceat-B), and three MHC-E (Ceat-E) alleles were identified. Sequence-specific primers (SSP) for high-throughput screening of genomic DNA by PCR were developed for 16 of the 18 Ceat-A and Ceat-B alleles. Screening of 62 SIV-negative and 123 SIV-infected sooty mangabeys at the Yerkes National Primate Research Center (YNPRC) revealed the presence of up to four MHC-A and eight MHC-B alleles in individual mangabeys, indicating that similar to macaque species, mangabeys have at least two duplications of the MHC-A locus and four duplications of the MHC-B locus in the absence of an MHC-C locus. Using stable transfectants of Ceat MHC Class I alleles in the MHC-null 721.221 cell line, we identified Ceat-B*12:01 as the restricting allele of a previously reported Nef20-28 CTL epitope. Ceat-B*1201/Nef20-28 tetramers identified tetramer-positive CD8+ T lymphocytes in Ceat-B*1201-positive SIV-infected mangabeys. This study has laid the groundwork for comprehensive analysis of CTL and SIV evolution in a natural host of SIV infection.

Immunogenetic characterization of a captive colony of sooty mangabeys (Cercocebus atys) used for SIV research.

BACKGROUND: African non-human primates are SIV natural hosts and do not develop disease following infection. Understanding disease avoidance mechanisms in these species is important for HIV vaccine development. The largest captive population of sooty mangabeys, a SIV natural host species, resides at the Yerkes National Primate Research Center. METHODS: Thirteen primer sets that amplify polymorphic microsatellite loci within the MHC region were used to genotype 144 animals. Immunogenetic Management Software (IMS) was used to identify MHC haplotypes and organize data. RESULTS: Seventy-three haplotypes were identified. Limited haplotype diversity was observed in this population with 88.2% of included animals carrying one of 18 haplotypes. Differences in haplotype frequency were observed between SIV (+) and SIV (-) populations. CONCLUSIONS: We have developed a novel tool for others to use in the analysis of the role of the MHC in a natural host non-human primate model species used for SIV research.

Cloning and analysis of sooty mangabey alternative coreceptors that support simian immunodeficiency virus SIVsmm entry independently of CCR5.

Natural host sooty mangabeys (SM) infected with simian immunodeficiency virus SIVsmm do not develop AIDS despite high viremia. SM and other natural hosts express very low levels of CCR5 on CD4(+) T cells, and we recently showed that SIVsmm infection and robust replication occur in vivo in SM genetically lacking CCR5, indicating the use of additional entry pathways. SIVsmm uses several alternative coreceptors of human origin in vitro, but which molecules of SM origin support entry is unknown. We cloned a panel of putative coreceptors from SM and tested their ability to mediate infection, in conjunction with smCD4, by pseudotypes carrying Envs from multiple SIVsmm subtypes. smCXCR6 supported efficient infection by all SIVsmm isolates with entry levels comparable to those for smCCR5, and smGPR15 enabled entry by all isolates at modest levels. smGPR1 and smAPJ supported low and variable entry, whereas smCCR2b, smCCR3, smCCR4, smCCR8, and smCXCR4 were not used by most isolates. In contrast, SIVsmm from rare infected SM with profound CD4(+) T cell loss, previously reported to have expanded use of human coreceptors, including CXCR4, used smCXCR4, smCXCR6, and smCCR5 efficiently and also exhibited robust entry through smCCR3, smCCR8, smGPR1, smGPR15, and smAPJ. Entry was similar with both known alleles of smCD4. These alternative coreceptors, particularly smCXCR6 and smGPR15, may support virus replication in SM that have restricted CCR5 expression as well as SM genetically lacking CCR5. Defining expression of these molecules on SM CD4(+) subsets may delineate distinct natural host target cell populations capable of supporting SIVsmm replication without CD4(+) T cell loss.

A novel CCR5 mutation common in sooty mangabeys reveals SIVsmm infection of CCR5-null natural hosts and efficient alternative coreceptor use in vivo.

In contrast to HIV infection in humans and SIV in macaques, SIV infection of natural hosts including sooty mangabeys (SM) is non-pathogenic despite robust virus replication. We identified a novel SM CCR5 allele containing a two base pair deletion (Δ2) encoding a truncated molecule that is not expressed on the cell surface and does not support SIV entry in vitro. The allele was present at a 26% frequency in a large SM colony, along with 3% for a CCR5Δ24 deletion allele that also abrogates surface expression. Overall, 8% of animals were homozygous for defective CCR5 alleles and 41% were heterozygous. The mutant allele was also present in wild SM in West Africa. CD8+ and CD4+ T cells displayed a gradient of CCR5 expression across genotype groups, which was highly significant for CD8+ cells. Remarkably, the prevalence of natural SIVsmm infection was not significantly different in animals lacking functional CCR5 compared to heterozygous and homozygous wild-type animals. Furthermore, animals lacking functional CCR5 had robust plasma viral loads, which were only modestly lower than wild-type animals. SIVsmm primary isolates infected both homozygous mutant and wild-type PBMC in a CCR5-independent manner in vitro, and Envs from both CCR5-null and wild-type infected animals used CXCR6, GPR15 and GPR1 in addition to CCR5 in transfected cells. These data clearly indicate that SIVsmm relies on CCR5-independent entry pathways in SM that are homozygous for defective CCR5 alleles and, while the extent of alternative coreceptor use in SM with CCR5 wild type alleles is uncertain, strongly suggest that SIVsmm tropism and host cell targeting in vivo is defined by the distribution and use of alternative entry pathways in addition to CCR5. SIVsmm entry through alternative pathways in vivo raises the possibility of novel CCR5-negative target cells that may be more expendable than CCR5+ cells and enable the virus to replicate efficiently without causing disease in the face of extremely restricted CCR5 expression seen in SM and several other natural host species.

Pathogenic and apathogenic courses of SIV infection are associated with distinct and characteristic regulatory patterns of G1/S and G2/M cell cycle checkpoints in CD4+ T cells.

Dysregulation of both the cell cycle within the CD4(+) T cells and T cell responses is characteristic for pathogenic HIV infection in humans and experimental SIV infection in rhesus macaques. However, SIV infection in sooty mangabeys does not lead to either an AIDS-like disease or such CD4(+) T cell dysregulation. A previous study has highlighted a potential role for cell cycle regulatory proteins in these distinct clinical outcomes. This study was performed to characterize the effect of SIV infection on the expression of cell cycle-related molecules in CD4(+) T cells of rhesus macaques and sooty mangabeys in attempts to define activation-induced gene expression patterns associated with disease resistance or susceptibility. First, T cell receptor (TCR)-mediated cell activation induced gene expression profiles that were unique to CD4(+) T cells from SIV-naive sooty mangabeys and rhesus macaques. More importantly, distinct and reproducible gene expression patterns were detected in CD4(+) T cells as a result of in vivo SIV infection in animals from each of the two species. In addition, SIV infection in both species showed significant differential effects on TCR activation-induced expression with a reproducible alteration of 10 genes highlighted by discordant effects on expression of Cyclin D3, Cyclin B, and RAD17. Therefore SIV infection in rhesus macaques and sooty mangabeys exhibits distinct and reproducible effects on cell cycle regulation in CD4(+) T cells during T cell activation that may be the basis for disease susceptibility vs. resistance in these two species, respectively.

Complete mitochondrial genome of the sooty mangabey, Cercocebus atys atys (Mammalia: Primates: Cercopithecidae).

The sooty mangabey (Cercocebus atys atys) is an Old World monkey belonging to family Cercopithecidae. It has recently been widely used as an important animal model for researches related to human immunodeficiency virus (HIV) and AIDS. In this study, the mitochondrial genome sequence of this species is determined and described for the first time. It is 16,536 bp in length and consists of 13 protein-coding genes, 22 tRNA genes, 2 ribosomal RNA genes and 1 putative control region (CR). The genome organization, nucleotide composition and codon usage are similar to those reported from other monkey mitochondrial genomes. The descending order of the base composition on heavy strand is 32.5% A, 29.5% C, 25.8% T, and 12.2% G, with a relatively lower level of G and a slightly A+T bias of 58.3%. All genes are distributed on the H-strand except for the ND6 subunit gene and seven tRNA genes, which are encoded on the L-strand. This mitochondrial genome data are potentially important for the study of molecular evolution, conservation genetics and disease infection in C. a. atys.

Coexpression Network Analysis of Benign and Malignant Phenotypes of SIV-Infected Sooty Mangabey and Rhesus Macaque.

To explore the differences between the extreme SIV infection phenotypes, nonprogression (BEN: benign) to AIDS in sooty mangabeys (SMs) and progression to AIDS (MAL: malignant) in rhesus macaques (RMs), we performed an integrated dual positive-negative connectivity (DPNC) analysis of gene coexpression networks (GCN) based on publicly available big data sets in the GEO database of NCBI. The microarray-based gene expression data sets were generated, respectively, from the peripheral blood of SMs and RMs at several time points of SIV infection. Significant differences of GCN changes in DPNC values were observed in SIV-infected SMs and RMs. There are three groups of enriched genes or pathways (EGPs) that are associated with three SIV infection phenotypes (BEN+, MAL+ and mixed BEN+/MAL+). The MAL+ phenotype in SIV-infected RMs is specifically associated with eight EGPs, including the protein ubiquitin proteasome system, p53, granzyme A, gramzyme B, polo-like kinase, Glucocorticoid receptor, oxidative phosyphorylation and mitochondrial signaling. Mitochondrial (endosymbiotic) dysfunction is solely present in RMs. Specific BEN+ pattern changes in four EGPs are identified in SIV-infected SMs, including the pathways contributing to interferon signaling, BRCA1/DNA damage response, PKR/INF induction and LGALS8. There are three enriched pathways (PRR-activated IRF signaling, RIG1-like receptor and PRR pathway) contributing to the mixed (BEN+/MAL+) phenotypes of SIV infections in RMs and SMs, suggesting that these pathways play a dual role in the host defense against viral infections. Further analysis of Hub genes in these GCNs revealed that the genes LGALS8 and IL-17RA, which positively regulate the barrier function of the gut mucosa and the immune homeostasis with the gut microbiota (exosymbiosis), were significantly differentially expressed in RMs and SMs. Our data suggest that there exists an exo- (dysbiosis of the gut microbiota) and endo- (mitochondrial dysfunction) symbiotic imbalance (EESI) in HIV/SIV infections. Dissecting the mechanisms of the exo-endo symbiotic balance (EESB) that maintains immune homeostasis and the EESI problems in HIV/SIV infections may lead to a better understanding of the pathogenesis of AIDS and the development of novel interventions for the rational control of this disease.

Mind the gap: upgrading genomes with Pacific Biosciences RS long-read sequencing technology.

Many genomes have been sequenced to high-quality draft status using Sanger capillary electrophoresis and/or newer short-read sequence data and whole genome assembly techniques. However, even the best draft genomes contain gaps and other imperfections due to limitations in the input data and the techniques used to build draft assemblies. Sequencing biases, repetitive genomic features, genomic polymorphism, and other complicating factors all come together to make some regions difficult or impossible to assemble. Traditionally, draft genomes were upgraded to "phase 3 finished" status using time-consuming and expensive Sanger-based manual finishing processes. For more facile assembly and automated finishing of draft genomes, we present here an automated approach to finishing using long-reads from the Pacific Biosciences RS (PacBio) platform. Our algorithm and associated software tool, PBJelly, (publicly available at https://sourceforge.net/projects/pb-jelly/) automates the finishing process using long sequence reads in a reference-guided assembly process. PBJelly also provides "lift-over" co-ordinate tables to easily port existing annotations to the upgraded assembly. Using PBJelly and long PacBio reads, we upgraded the draft genome sequences of a simulated Drosophila melanogaster, the version 2 draft Drosophila pseudoobscura, an assembly of the Assemblathon 2.0 budgerigar dataset, and a preliminary assembly of the Sooty mangabey. With 24× mapped coverage of PacBio long-reads, we addressed 99% of gaps and were able to close 69% and improve 12% of all gaps in D. pseudoobscura. With 4× mapped coverage of PacBio long-reads we saw reads address 63% of gaps in our budgerigar assembly, of which 32% were closed and 63% improved. With 6.8× mapped coverage of mangabey PacBio long-reads we addressed 97% of gaps and closed 66% of addressed gaps and improved 19%. The accuracy of gap closure was validated by comparison to Sanger sequencing on gaps from the original D. pseudoobscura draft assembly and shown to be dependent on initial reference quality.

Comparative transcriptomics of extreme phenotypes of human HIV-1 infection and SIV infection in sooty mangabey and rhesus macaque.

High levels of HIV-1 replication during the chronic phase of infection usually correlate with rapid progression to severe immunodeficiency. However, a minority of highly viremic individuals remains asymptomatic and maintains high CD4⁺ T cell counts. This tolerant profile is poorly understood and reminiscent of the widely studied nonprogressive disease model of SIV infection in natural hosts. Here, we identify transcriptome differences between rapid progressors (RPs) and viremic nonprogressors (VNPs) and highlight several genes relevant for the understanding of HIV-1-induced immunosuppression. RPs were characterized by a specific transcriptome profile of CD4⁺ and CD8⁺ T cells similar to that observed in pathogenic SIV-infected rhesus macaques. In contrast, VNPs exhibited lower expression of interferon-stimulated genes and shared a common gene regulation profile with nonpathogenic SIV-infected sooty mangabeys. A short list of genes associated with VNP, including CASP1, CD38, LAG3, TNFSF13B, SOCS1, and EEF1D, showed significant correlation with time to disease progression when evaluated in an independent set of CD4⁺ T cell expression data. This work characterizes 2 minimally studied clinical patterns of progression to AIDS, whose analysis may inform our understanding of HIV pathogenesis.

Global genomic analysis reveals rapid control of a robust innate response in SIV-infected sooty mangabeys.

Natural SIV infection of sooty mangabeys (SMs) is nonprogressive despite chronic virus replication. Strikingly, it is characterized by low levels of immune activation, while pathogenic SIV infection of rhesus macaques (RMs) is associated with chronic immune activation. To elucidate the mechanisms underlying this intriguing phenotype, we used high-density oligonucleotide microarrays to longitudinally assess host gene expression in SIV-infected SMs and RMs. We found that acute SIV infection of SMs was consistently associated with a robust innate immune response, including widespread upregulation of IFN-stimulated genes (ISGs) in blood and lymph nodes. While SMs exhibited a rapid resolution of ISG expression and immune activation, both responses were observed chronically in RMs. Systems biology analysis indicated that expression of the lymphocyte inhibitory receptor LAG3, a marker of T cell exhaustion, correlated with immune activation in SIV-infected RMs but not SMs. Our findings suggest that active immune regulatory mechanisms, rather than intrinsically attenuated innate immune responses, underlie the low levels of immune activation characteristic of SMs chronically infected with SIV.

CD4+ T cells from simian immunodeficiency virus disease-resistant sooty mangabeys produce more IL-2 than cells from disease-susceptible species: involvement of p300 and CREB at the proximal IL-2 promoter in IL-2 up-regulation.

IL-2 is an important cytokine required for the physiological function of CD4(+) T cells. Immunological unresponsiveness-anergy- of CD4(+) T cells is characterized by the inability of these cells to synthesize IL-2. Both progressive HIV infection leading to AIDS in humans and SIV infection in rhesus macaques (RM) are associated with dysregulation of IL-2 synthesis. In certain nonhuman primate species, such as sooty mangabeys (SM), SIV infection does not lead to AIDS. We have shown that this is associated with the resistance of the CD4(+) T cells from SM to undergo anergy in vitro. In this study, we show that CD4(+) T cells from SM spontaneously synthesize 2- to 3-fold higher levels of IL-2 than corresponding cells from RM. Proximal IL-2 promoter constructs derived from SM show significantly higher activity than the RM-derived constructs in primary CD4(+) T cells, which is associated with an element at approximately nt -200. Activity of both constructs was up-regulated by p300 and down-regulated by CREB to a similar degree. Chromatin immunoprecipitation analysis showed significantly higher binding of p300 and lower binding of CREB to the SM promoter in vivo. Two single nucleotide substitutions present in the SM sequence around position -200 and -180 seem to increase the affinity of these sites for the binding of transcription factors, one of which was identified as Oct-1. These unique characteristics of the proximal IL-2 promoter in SM therefore can represent one of the mechanisms contributing to the resistance of these cells to undergo anergy.

Sooty mangabey (Cercocebus torquatus atys) IGHG and IGHA genes.

Antibodies are adaptor molecules of the immune system that link antigen recognition with the effector mechanisms responsible for antigen clearance. Several nonhuman primate species are widely used in biomedical research, especially for vaccine development and for AIDS-related studies. However, nonhuman primate antibody molecules have been characterized only partially and only in a few species. Here, we describe sooty mangabey (Cercocebus torquatus atys) IGHG and IGHA genes, which encode the heavy-chain constant region of IgG and IgA molecules, respectively. The four mangabey IGHG genes are highly homologous to the rhesus macaque and baboon IGHG genes (percent identity varies between 94.0 and 98.8, depending on the subclass), with most amino acid differences located in the hinge regions. Results obtained by real-time reverse transcription polymerase chain reaction show that the four IGHG genes are expressed at least at the mRNA level. The mangabey IGHA gene is highly homologous to the corresponding gene from rhesus macaques (percent identity ranges from 88.6 to 96.7, depending on the allele considered), the only other nonhominoid primate species for which the complete sequence of the IGHA gene is currently available. In the mangabey analyzed, two IGHA alleles are present, confirming that high levels of IGHA gene heterozygosity are present in monkey species. These results show that nonhuman primate gamma and alpha heavy chains differ from each other mostly at the level of the hinge region and that alpha sequence heterogeneity in nonhuman primate species is also present in other gamma regions. In addition, these results provide sequence information that can be used for residue frequency analysis of antibody heavy-chain constant region sequences.

Genealogical and demographic influences on infant abuse and neglect in group-living sooty mangabeys (Cercocebus atys).

This study investigated the occurrence of infant abuse and neglect in a large population of group-living mangabeys over a period of almost 3 decades. The prevalence of infant abuse and neglect did not differ significantly among the 9 families comprising the population, but within some families there was evidence of genealogical effects on infant abuse. Maternal inexperience and infant age were risk factors for neglect but not for abuse. Whereas neglecting mothers neglected only 1 of their offspring, usually their first-born infant, abusive mothers abused several of their offspring, and risk of severe abuse increased with later births. Infant sex was not a risk factor for neglect or abuse. These and other results concur with the findings of a previous investigation of infant abuse and neglect in a different primate species in indicating that neglect and abuse are different phenomena and in emphasizing genealogical influences on infant abuse in primates. The investigation of biological, experiential, and social determinants of the spontaneous occurrence of infant abuse and neglect in relatively undisturbed primate populations could significantly enhance our understanding of the etiology of child abuse and neglect in humans.

Phylogenetic analysis of the Friedreich ataxia GAA trinucleotide repeat.

Friedreich ataxia is an autosomal recessive neurodegenerative disorder associated with a GAA repeat expansion in the first intron of the gene (FRDA) encoding a novel, highly conserved, 210 amino acid protein known as frataxin. Normal variation in repeat size was determined by analysis of more than 600 DNA samples from seven human populations. This analysis showed that the most frequent allele had nine GAA repeats, and no alleles with fewer than five GAA repeats were found. The European and Syrian populations had the highest percentage of alleles with 10 or more GAA repeats, while the Papua New Guinea population did not have any alleles carrying more than 10 GAA repeats. The distributions of repeat sizes in the European, Syrian, and African American populations were significantly different from those in the Asian and Papua New Guinea populations (p < 0.001). The GAA repeat size was also determined in five nonhuman primates. Samples from 10 chimpanzees, 3 orangutans, 1 gorilla, 1 rhesus macaque, 1 mangabey, and 1 tamarin were analyzed. Among those primates belonging to the Pongidae family, the chimpanzees were found to carry three or four GAA repeats, the orangutans had four or five GAA repeats, and the gorilla carried three GAA repeats. In primates belonging to the Cercopithecidae family, three GAA repeats were found in the mangabey and two in the rhesus macaque. However, an AluY subfamily member inserted in the poly(A) tract preceding the GAA repeat region in the rhesus macaque, making the amplified sequence approximately 300 bp longer. The GAA repeat was also found in the tamarin, suggesting that it arose at least 40 million years ago and remained relatively small throughout the majority of primate evolution, with a punctuated expansion in the human genome.

Quantification of thymic function by measuring T cell receptor excision circles within peripheral blood and lymphoid tissues in monkeys.

The thymus is the primary organ responsible for the production of mature TCR alpha / beta T cells. Quantification of a DNA excision circle that is produced during TCR rearrangement, termed a signal joint TCR rearrangement excision circle (sjTREC) can be used as a measure of thymic function. Here sjTREC measurement has been applied to two monkey species used as animal models of human disease, rhesus macaques (Asian origin) and sooty mangabeys (African origin). Initial PCR analysis determined that the TCR deltaRec-PsiJalpha rearrangement leading to sjTREC formation occurs in both species. Primers to a DNA sequence conserved in macaques, mangabeys and humans were used in a quantitative competitive PCR assay to quantify sjTREC. We found that as in humans, sjTREC in these two monkey species decline with age. sjTREC are first generated in thymocytes during the early stages of TCR rearrangement. Lymph node CD4(+) and CD8(+) T cells contain more sjTREC than peripheral blood T cell populations, suggesting that recent thymic emigrants home to the lymphoid tissues. The sjTREC level is significantly higher within the peripheral blood CD4(+) and CD8(+) T cells of mangabeys compared to macaques. Removal of the thymus in four macaques led to a profound decrease in peripheral blood sjTREC level by 1 year post-thymectomy, indicating the lack of a significant extra-thymic source of peripheral naive T cells in macaques. Our results indicate that production, trafficking, and proliferation of recent thymic emigrants in these two monkey species represents a useful animal model system for understanding human immunological disorders.