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Objectives Elevated levels of interleukin-6 (IL-6), D-dimer, and C-reactive protein (hsCRP) are associated with increased incidence of comorbid disease and mortality among people living with HIV (PLWH). Prior studies suggest a genetic basis for these biomarker elevations in the general population. The study objectives are to identify the genetic basis for these biomarkers among PLWH. Methods Baseline levels of hsCRP, D-dimer and IL-6, and single nucleotide polymorphisms (SNPs) were determined for 7,768 participants in three HIV treatment trials. Single variant analysis was performed for each biomarker on samples from each of three ethnic groups (African [AFR], Admixed American [AMR], European [EUR]) within each trial including covariates relevant to biomarker levels. For each ethnic group, the results were pooled across trials, then further pooled across ethnicities. Results The transethnic analysis identified three, two and one known loci associated with hsCRP, D-dimer and IL-6 levels, respectively, and two novel loci, FGB and GCNT1, associated with D-dimer levels. Lead SNPs exhibited similar effects across ethnicities. Additionally, three novel, ethnic-specific loci were identified: CATSPERG associated with D-dimer in AFR and PROX1-AS1 and TRAPPC9 associated with IL-6 in AFR and AMR, respectively. Conclusions Eleven loci associated with three biomarker levels were identified in PLWH from the three studies including six loci known in the general population and five novel loci associated with D-dimer and IL-6 levels. These findings support the hypothesis that host genetics may partially contribute to chronic inflammation in PLWH and help to identify potential targets for intervention of serious non-AIDS complications.

Significance In HIV-infected patients on combination antiretroviral therapy (cART), greater than 95% of proviruses in the peripheral blood are “defective.” Historically, these defective proviruses have been thought to be dead-end products with no real pathophysiological significance, as they do not encode replication-competent viruses. Contrary to this view, we have identified cells in tissue culture and from cART-treated patients that harbor defective proviruses and produce viral proteins. Features found in these translationally competent yet defective proviruses suggest that HIV-1 infection results in modification of the CD4+ T cell genome analogous to human endogenous retroviruses. We propose that these defective HIV-1 proviruses are biologically significant, despite being “replication incompetent,” have the potential to elicit immune activation, and may serve as a barrier to HIV-1 cure., HIV-1 proviruses persist in the CD4+ T cells of HIV-infected individuals despite years of combination antiretroviral therapy (cART) with suppression of HIV-1 RNA levels

Herpes simplex virus 1 (HSV-1) strain McKrae is highly virulent and relatively neuroinvasive in animal models compared with other wild-type HSV-1 strains. To identify the genetic determinants that lead to the unique phenotypes of the McKrae strain, we sequenced its genome with PacBio single-molecule real-time (SMRT) technology and resolved the complete sequence.

Herpes simplex virus type 1 (HSV-1) strain MacIntyre has a severe defect in the anterograde spread after replication in the nucleus. To better understand and identify the genetic determinants that lead to the unique phenotypes of the MacIntyre strain, we sequenced its genome with PacBio single-molecule real-time sequencing technology and resolved the complete sequence.

BACKGROUND: HIV-infected patients with poor virologic control and multidrug-resistant virus have limited therapeutic options. The current study was undertaken to evaluate the safety, immunologic effects, and antiviral activity of peripheral lymphocytes transferred from an elite controller, whose immune system is able to control viral replication without antiretroviral medications, to an HLA-B*2705–matched progressor. METHODS: Approximately 22 billion cells were collected from an elite controller by lymphapheresis and infused within 6 hours into a recipient with a preinfusion CD4(+) T cell count of 10 cells/μL (1%) and HIV plasma viral load of 114,993 copies/mL. RESULTS: Donor cells were cleared from the recipient’s peripheral blood by day 8. A transient decrease in viral load to 58,421 (day 3) was followed by a rebound to 702,972 (day 6) before returning to baseline values by day 8. The decreased viral load was temporally associated with peak levels of donor T cells, including CD8(+) T cells that had high levels of expression of Ki67, perforin, and granzyme B. Notably, recipient CD8(+) T cells also showed increased expression of these markers, especially in HIV-specific tetramer–positive cells. CONCLUSION: These results suggest that the adoptive transfer of lymphocytes from an HIV-infected elite controller to an HIV-infected patient with progressive disease may be able to perturb the immune system of the recipient in both positive and negative ways. TRIAL REGISTRATION: ClinicalTrials.gov NCT00559416. FUNDING: Intramural Research Programs of the US NIH Clinical Center and the National Institute of Allergy and Infectious Diseases (NIAID); the National Cancer Institute.

The impact of variation in host genetics on replication of human immunodeficiency virus type 1 (HIV-1) in demographically diverse populations remains uncertain. In the current study, we performed a genome-wide screen for associations of single-nucleotide polymorphisms (SNPs) to viral load (VL) in antiretroviral therapy–naive participants (n = 2440) with varying demographics from the Strategic Timing of AntiRetroviral Treatment (START) trial. Associations were assessed using genotypic data generated by a customized SNP array, imputed HLA alleles, and multiple linear regression. Genome-wide significant associations between SNPs and VL were observed in the major histocompatibility complex class I region (MHC I), with effect sizes ranging between 0.14 and 0.39 log10 VL (copies/mL). Supporting the SNP findings, we identified several HLA alleles significantly associated with VL, extending prior observations that the (MHC I) is a major host determinant of HIV-1 control with shared genetic variants across diverse populations and underscoring the limitations of genome-wide association studies as being merely a screening tool.

After initiating antiretroviral therapy (ART), a rapid decline in plasma viremia is followed by reservoir stabilization. Viral outgrowth assay suggests the reservoir continues to decline slowly, but variation over time and among individuals complicates our understanding of selective pressures during ART. We used full-length sequencing to study more than 800 HIV proviruses of two subjects on ART at four time points over nine years to investigate the selection pressures influencing the dynamics of the reservoir. We found that intact as well as defective proviruses capable of significant protein expression decrease over time. Moreover, proviruses lacking genetic elements to promote viral protein expression, yet containing strong splice donor sequences increase relative to other defectives over time, especially among clones. Our work suggests that HIV expression occurs to a significant extent during ART and results in HIV clearance, but this is obscured by clones generated by donor splice site-enhanced clonal expansion.

We have previously demonstrated that Interleukin-27 differentially regulates the expression of seven novel microRNAs. Here we elucidate the functional significance of these novel microRNAs. Of the seven microRNAs, over expression of miRNA-6852 (miR-SX4) mimic induces cell cycle arrest at G2/M phase and induces necrosis in HEK293 and panel of cervical cancer cells (Human Papilloma Virus (HPV) infected cell lines; HeLa, CaSki and SiHa cells). To define the mechanism of the miR-SX4-mediated G2/M arrest, a microarray gene chip array and western blot analysis were performed. FoxM1, a transcription factor is identified as a key protein down-regulated by miR-SX4, even though the miR-SX4 does not target 3’UTR of FoxM1. Knock down of FoxM1 using si-RNA demonstrate that FoxM1 silenced cell induces G2/M cell cycle arrest and necrosis. Our data demonstrated for the first time that miR-SX4 could be a potent anti-cancer microRNA.

We previously identified Ku70, a subunit of a DNA repair protein complex, as a cytosolic DNA sensor that induces the production of interferon-λ1 (IFN-λ1) by human primary cells and cell lines. IFN-λ1 is a type III IFN and has similar antiviral activity to that of the type I IFNs (IFN-α and IFN-β). We observed that human embryonic kidney (HEK) 293T cells, which are deficient in the innate immune adaptor protein STING (stimulator of IFN genes), did not produce IFN-λ1 in response to DNA unless they were reconstituted with STING. Conversely, parental HEK 293 cells produced IFN-λ1 after they were exposed to exogenous DNA; however, when STING was knocked out in the HEK 293 cells through the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 genome editing system, they lost this response. Through confocal microscopy, we demonstrated that endogenous Ku70 was located in the nucleus and then translocated to the cytoplasm upon DNA exposure to form a complex with STING. Additionally, the DNA binding domain of Ku70 was essential for formation of the Ku70-STING complex. Knocking down STING in primary human macrophages inhibited their ability to produce IFN-λ1 in response to transfection with DNA or infection with the DNA virus HSV-2 (herpes simplex virus–2). Together, these data suggest that STING mediates the Ku70-mediated IFN-λ1 innate immune response to exogenous DNA or DNA virus infection.

MicroRNAs (miRNAs) regulate gene expression and thereby influence cell fate and function. Recent studies suggest that an abundant class of miRNAs play important roles in immune cells, such as T cells, natural killer (NK) cells, B cells, and dendritic cells (DCs). Interleukin (IL)-27 is a member of the IL-12 family of cytokines with broad anti-viral effects. It is a potent inhibitor of HIV-1 infection in CD4+ T cells and macrophages, as well as monocyte-derived immature dendritic cells (iDCs). This pilot study compared miRNA profiles between iDCs and IL-27-treated iDCs (27DCs) using deep sequencing methods and identified 46 known miRNAs that were significantly differentially expressed in 27DCs: 36 were upregulated and 10 downregulated by IL-27. Many of the potential target genes of these miRNAs are involved in IL-27 associated pathways, such as JAK/STAT, MAPKs, and PI3K and several were also previously reported to be involved in the regulation of human DC function. This study found that these miRNAs also potentially target several viral genomes and therefore may have antiviral effects. Four of these differential miRNAs (miR-99a-5p, miR-222-3p, miR-138-5p, and miR-125b-5p) were validated using quantitative reverse transcription PCR (RT-qPCR). Twenty-two novel miRNAs were discovered from deep sequencing and confirmed using RT-qPCR. This study furthers the understanding of the role of IL-27 in immunity and lays a foundation for future characterization of the role of specific miRNAs in DCs.

β-glucans, which can activate innate immune responses, are a major component in the cell wall of the cyst form of Pneumocystis. In the current study, we examined whether β-1,3-glucans are masked by surface proteins in Pneumocystis and what role β-glucans play in Pneumocystis-associated inflammation. For 3 species, including Pneumocystis jirovecii, which causes Pneumocystis pneumonia in humans, Pneumocystis carinii, and Pneumocystis murina, β-1,3-glucans were masked in most organisms, as demonstrated by increased exposure following trypsin treatment. Using quantitative polymerase chain reaction and microarray techniques, we demonstrated in a mouse model of Pneumocystis pneumonia that treatment with caspofungin, an inhibitor of β-1,3-glucan synthesis, for 21 days decreased expression of a broad panel of inflammatory markers, including interferon γ, tumor necrosis factor α, interleukin 1β, interleukin 6, and multiple chemokines/chemokine ligands. Thus, β-glucans in Pneumocystis cysts are largely masked, which likely decreases innate immune activation; this mechanism presumably was developed for interactions with immunocompetent hosts, in whom organism loads are substantially lower. In immunosuppressed hosts with a high organism burden, organism death and release of glucans appears to be an important contributor to deleterious host inflammatory responses.

Pneumocystis jirovecii is a major cause of life-threatening pneumonia in immunosuppressed patients including transplant recipients and those with HIV/AIDS, yet surprisingly little is known about the biology of this fungal pathogen. Here we report near complete genome assemblies for three Pneumocystis species that infect humans, rats and mice. Pneumocystis genomes are highly compact relative to other fungi, with substantial reductions of ribosomal RNA genes, transporters, transcription factors and many metabolic pathways, but contain expansions of surface proteins, especially a unique and complex surface glycoprotein superfamily, as well as proteases and RNA processing proteins. Unexpectedly, the key fungal cell wall components chitin and outer chain N-mannans are absent, based on genome content and experimental validation. Our findings suggest that Pneumocystis has developed unique mechanisms of adaptation to life exclusively in mammalian hosts, including dependence on the lungs for gas and nutrients and highly efficient strategies to escape both host innate and acquired immune defenses., Pneumocystis jirovecii is a fungus that can cause life-threatening pneumonia in immunocompromised patients. Here, the authors sequence the genomes of P. jirovecii and two other Pneumocystis species, and show the unexpected absence of chitin (a near universal fungal cell wall component).

Background HIV-1 set-point viral load (SPVL) is predictive of disease progression and shows variability across HIV-1-positive (HIV+) persons. Various factors may influence SPVL including viral features, environmental exposure and host genetics. To identify single nucleotide polymorphisms (SNPs) associated with SPVL, we performed a genome-wide association study (GWAS) on a subset of participants from the Strategic Timing of AntiRetroviral Treatment (START) study covering a demographically diverse population. Methods. Consenting participants were antiretroviral therapy (ART)-naïve and SPVL was taken as log10(HIV RNA) at study entry. Genotypic data were generated on a custom content Affymetrix Axiom SNP array covering 770,558 probes. The Ensembl Gene database, assembly GRCh37.p13, was used for annotation. Principal component analysis (PCA) was used to identify population structures, and analysis of variance (ANOVA) was performed to detect associations between SNPs and SPVL. SNPs with zero variance or minor allele frequency (MAF) ≤0.05 were removed. Results. Among the 2,544 participants, PCA showed distinct population structures with strong separation between black (n = 578) and nonblack (n = 1,966) participants, Figure 1. ANOVA was performed independently on both subsets. Two SNPs located in the Major Histocompatibility Complex (MHC) class I region of chromosome six reached genome-wide significance (P < 5 × 10–8) in the non-black population: rs4418214 (P = 1.74 × 10-10), and rs57989216 (P = 3.96 × 10–8), Figure 2. Two additional SNPs, rs9264942 (P = 5.99 × 10–8) and rs7356880 (P = 9.69 × 10–8), in the same region approached significance. The minor alleles of all four SNPs were associated with lower SPVL, Figure 3. While no SNPs reached genome-wide significance in the black group, we observed similar trends toward lower SPVL for both rs4418214 and rs57989216. Conclusion. In this study we confirm the association of a previously reported SNP (rs4418214) and identify a novel candidate SNP (rs57989216) associated with lower SPVL in a population of nonblack, ART-naïve HIV+ persons. Current findings suggest that the effects of these SNPs are consistent across race groups, but further studies are required to confirm this. Our results support previous findings that variation in the MHC class I region is a major host determinant of HIV-1 control. Disclosures D. D. Murray, Centre of Excellence for Health, Immunity and Infectious diseases (CHIP), Department of Infectious Diseases, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark: Employee, Salary. J. M. Molina, Gilead: Scientific Advisor, Consulting fee. Merck: Scientific Advisor, Consulting fee. ViiV: Scientific Advisor, Consulting fee. Teva: Scientific Advisor, Consulting fee.

Functional analysis of large gene lists, derived in most cases from emerging high-throughput genomic, proteomic and bioinformatics scanning approaches, is still a challenging and daunting task. The gene-annotation enrichment analysis is a promising high-throughput strategy that increases the likelihood for investigators to identify biological processes most pertinent to their study. Approximately 68 bioinformatics enrichment tools that are currently available in the community are collected in this survey. Tools are uniquely categorized into three major classes, according to their underlying enrichment algorithms. The comprehensive collections, unique tool classifications and associated questions/issues will provide a more comprehensive and up-to-date view regarding the advantages, pitfalls and recent trends in a simpler tool-class level rather than by a tool-by-tool approach. Thus, the survey will help tool designers/developers and experienced end users understand the underlying algorithms and pertinent details of particular tool categories/tools, enabling them to make the best choices for their particular research interests.

Development of HIV-1 drug resistance mutations (HDRMs) is one of the major reasons for the clinical failure of antiretroviral therapy. Treatment success rates can be improved by applying personalized anti-HIV regimens based on a patient’s HDRM profile. However, the sensitivity and specificity of the HDRM profile is limited by the methods used for detection. Sanger-based sequencing technology has traditionally been used for determining HDRM profiles at the single nucleotide variant (SNV) level, but with a sensitivity of only ≥ 20% in the HIV population of a patient. Next Generation Sequencing (NGS) technologies offer greater detection sensitivity (~ 1%) and larger scope (hundreds of samples per run). However, NGS technologies produce reads that are too short to enable the detection of the physical linkages of individual SNVs across the haplotype of each HIV strain present. In this article, we demonstrate that the single-molecule long reads generated using the Third Generation Sequencer (TGS), PacBio RS II, along with the appropriate bioinformatics analysis method, can resolve the HDRM profile at a more advanced quasispecies level. The case studies on patients’ HIV samples showed that the quasispecies view produced using the PacBio method offered greater detection sensitivity and was more comprehensive for understanding HDRM situations, which is complement to both Sanger and NGS technologies. In conclusion, the PacBio method, providing a promising new quasispecies level of HDRM profiling, may effect an important change in the field of HIV drug resistance research.

UNLABELLED Our current biological knowledge is spread over many independent bioinformatics databases where many different types of gene and protein identifiers are used. The heterogeneous and redundant nature of these identifiers limits data analysis across different bioinformatics resources. It is an even more serious bottleneck of data analysis for larger datasets, such as gene lists derived from microarray and proteomic experiments. The DAVID Gene ID Conversion Tool (DICT), a web-based application, is able to convert user's input gene or gene product identifiers from one type to another in a more comprehensive and high-throughput manner with a uniquely enhanced ID-ID mapping database. AVAILABILITY http://david.abcc.ncifcrf.gov/conversion.jsp.

Background Due to the complex and distributed nature of biological research, our current biological knowledge is spread over many redundant annotation databases maintained by many independent groups. Analysts usually need to visit many of these bioinformatics databases in order to integrate comprehensive annotation information for their genes, which becomes one of the bottlenecks, particularly for the analytic task associated with a large gene list. Thus, a highly centralized and ready-to-use gene-annotation knowledgebase is in demand for high throughput gene functional analysis. Description The DAVID Knowledgebase is built around the DAVID Gene Concept, a single-linkage method to agglomerate tens of millions of gene/protein identifiers from a variety of public genomic resources into DAVID gene clusters. The grouping of such identifiers improves the cross-reference capability, particularly across NCBI and UniProt systems, enabling more than 40 publicly available functional annotation sources to be comprehensively integrated and centralized by the DAVID gene clusters. The simple, pair-wise, text format files which make up the DAVID Knowledgebase are freely downloadable for various data analysis uses. In addition, a well organized web interface allows users to query different types of heterogeneous annotations in a high-throughput manner. Conclusion The DAVID Knowledgebase is designed to facilitate high throughput gene functional analysis. For a given gene list, it not only provides the quick accessibility to a wide range of heterogeneous annotation data in a centralized location, but also enriches the level of biological information for an individual gene. Moreover, the entire DAVID Knowledgebase is freely downloadable or searchable at http://david.abcc.ncifcrf.gov/knowledgebase/.

Background Gene isoforms are commonly found in both prokaryotes and eukaryotes. Since each isoform may perform a specific function in response to changing environmental conditions, studying the dynamics of gene isoforms is important in understanding biological processes and disease conditions. However, genome-wide identification of gene isoforms is technically challenging due to the high degree of sequence identity among isoforms. Traditional targeted sequencing approach, involving Sanger sequencing of plasmid-cloned PCR products, has low throughput and is very tedious and time-consuming. Next-generation sequencing technologies such as Illumina and 454 achieve high throughput but their short read lengths are a critical barrier to accurate assembly of highly similar gene isoforms, and may result in ambiguities and false joining during sequence assembly. More recently, the third generation sequencer represented by the PacBio platform offers sufficient throughput and long reads covering the full length of typical genes, thus providing a potential to reliably profile gene isoforms. However, the PacBio long reads are error-prone and cannot be effectively analyzed by traditional assembly programs. Results We present a clustering-based analysis pipeline integrated with PacBio sequencing data for profiling highly similar gene isoforms. This approach was first evaluated in comparison to de novo assembly of 454 reads using a benchmark admixture containing 10 known, cloned msg genes encoding the major surface glycoprotein of Pneumocystis jirovecii. All 10 msg isoforms were successfully reconstructed with the expected length (~1.5 kb) and correct sequence by the new approach, while 454 reads could not be correctly assembled using various assembly programs. When using an additional benchmark admixture containing 22 known P. jirovecii msg isoforms, this approach accurately reconstructed all but 4 these isoforms in their full-length (~3 kb); these 4 isoforms were present in low concentrations in the admixture. Finally, when applied to the original clinical sample from which the 22 known msg isoforms were cloned, this approach successfully identified not only all known isoforms accurately (~3 kb each) but also 48 novel isoforms. Conclusions PacBio sequencing integrated with the clustering-based analysis pipeline achieves high-throughput and high-resolution discrimination of highly similar sequences, and can serve as a new approach for genome-wide characterization of gene isoforms and other highly repetitive sequences. Electronic supplementary material The online version of this article (doi:10.1186/s13040-016-0090-8) contains supplementary material, which is available to authorized users.

The DAVID gene functional classification tool uses a novel fuzzy clustering algorithm to condense a list of genes or associated biological terms into organized classes of related genes or biology, called biological modules., The DAVID Gene Functional Classification Tool uses a novel agglomeration algorithm to condense a list of genes or associated biological terms into organized classes of related genes or biology, called biological modules. This organization is accomplished by mining the complex biological co-occurrences found in multiple sources of functional annotation. It is a powerful method to group functionally related genes and terms into a manageable number of biological modules for efficient interpretation of gene lists in a network context.