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S100A8 and S100A9 are members of the Alarmin family; these proteins are abundantly expressed in neutrophils, form a heterodimer complex, and are secreted in plasma on pathogen infection or acute inflammatory diseases. Recently, both proteins were identified as novel biomarkers of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and were shown to play key roles in inducing an aggressive inflammatory response by mediating the release of large amounts of pro-inflammatory cytokines, called the "cytokine storm." Although co-infection with SARS-CoV-2 in people living with HIV-1 may result in an immunocompromised status, the role of the S100A8/A9 complex in HIV-1 replication in primary T cells and macrophages is still unclear. Here, we evaluated the roles of the proteins in HIV replication to elucidate their functions. We found that the complex had no impact on virus replication in both cell types; however, the subunits of S100A8 and S100A9 inhibit HIV in macrophages. These findings provide important insights into the regulation of HIV viral loads during SARS-CoV-2 co-infection.

DNA-protein interactions (DPIs) are critical to all living organisms, particularly in the regulation of gene expression, replication, packing, recombination, and DNA repair, as well as RNA transport and translation. Many laboratory techniques have been developed to study the complex interactions of proteins with DNA, such as chromatin immunoprecipitation (ChIP) assays, DNA electrophoretic mobility shift assay (EMSA), and oligonucleotide pull-down assays. Here we describe an effective approach to identify potential DNA-binding proteins: a pull-down assay using DNA-conjugated beads with a customized competition strategy, which conferred a more effective and efficient approach to determine the interaction between DNA and protein(s), therefore dramatically improving the progress to investigate novel DNA-binding proteins.

Summary We have previously identified that human Ku70, a nuclear protein, serves as a cytosolic DNA sensor. Upon transfection with DNA or infection with DNA virus, Ku70 translocates from the nucleus into the cytoplasm and then predominately induces interferon lambda1 (IFN‐λ1) rather than IFN‐alpha or IFN‐beta, through a STING‐dependent signalling pathway. However, a detailed mechanism for Ku70 cytoplasmic translocation and its correlation with IFN‐λ1 induction have not been fully elucidated. Here, we observed that cytoplasmic translocation of Ku70 only occurred in DNA‐triggered IFN‐λ1‐inducible cells. Additionally, infection by Herpes simplex virus type‐1 (HSV‐1), a DNA virus, induces cytoplasmic translocation of Ku70 and IFN‐λ1 induction in a strain‐dependent manner: the translocation and IFN‐λ1 induction were detected upon infection by HSV‐1 McKrae, but not MacIntyre, strain. A kinetic analysis indicated that cytoplasmic translocation of Ku70 was initiated right after DNA transfection and was peaked at 6 hr after DNA stimulation. Furthermore, treatment with leptomycin B, a nuclear export inhibitor, inhibited both Ku70 translocation and IFN‐λ1 induction, suggesting that Ku70 translocation is an essential and early event for its cytosolic DNA sensing. We further confirmed that enhancing the acetylation status of the cells promotes Ku70’s cytoplasmic accumulation, and therefore increases DNA‐mediated IFN‐λ1 induction. These findings provide insights into the molecular mechanism by which the versatile sensor detects pathogenic DNA in a localization‐dependent manner., Cytoplasmic‐translocated Ku70 recognizes intracellular DNA and initiates downstream STING‐TBK1‐IRFs signalling pathway to induce IFN‐λ1. The translocation of Ku70 from the nucleus to the cytoplasm is an early and required event in Ku70/DNA‐mediated IFN‐λ1 induction. Acetylation plays an essential role in modulating ku70’s cytosolic DNA sensing.

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.

Supplemental Digital Content is Available in the Text., Objectives: Interleukin-27 (IL-27) is known as an anti-HIV cytokine. We have recently demonstrated that IL-27-pretreatment promotes phytohemagglutinin-stimulated CD4(+) T cells into HIV-1-resistant cells by inhibiting an uncoating step. Purpose: To further characterize the function of the HIV resistant T cells, we investigated profiles of microRNA in the cells using microRNA sequencing (miRNA-seq) and assessed anti-HIV effect of the microRNAs. Methods: Phytohemagglutinin-stimulated CD4(+) T cells were treated with or without IL-27 for 3 days. MicroRNA profiles were analyzed using miRNA-seq. To assess anti-HIV effect, T cells or macrophages were transfected with synthesized microRNA mimics and then infected with HIVNL4.3 or HIVAD8. Anti-HIV effect was monitored by a p24 antigen enzyme-linked immunosorbent assay kit. interferon (IFN)-α, IFN-β, or IFN-λ production was quantified using each subtype-specific enzyme-linked immunosorbent assay kit. Results: A comparative analysis of microRNA profiles indicated that expression of known miRNAs was not significantly changed in IL-27-treated cells compared with untreated T cells; however, a total of 15 novel microRNAs (miRTC1 ∼ miRTC15) were identified. Anti-HIV assay using overexpression of each novel microRNA revealed that 10 nM miRTC14 (GenBank accession number: MF281439) remarkably suppressed HIV infection by (99.3 ± 0.27%, n = 9) in macrophages but not in T cells. The inhibition was associated through induction of >1000 pg/mL of IFN-αs and IFN-λ1. Conclusion: We discovered a total of 15 novel microRNAs in T cells and characterized that miRTC14, one of the novel microRNAs, was a potent IFN-inducing anti-HIV miRNA, implicating that regulation of the expression of miRTC14 may be a potent therapeutic tool for not only HIV but also other virus infection.

Inflammaging has received considerable attention because aging is characterized by low-grade, chronic and asymptomatic inflammation, concomitant with increased blood levels of senescence-associated secretory phenotype (SASP) factors, including IL-1, IL-6, IL-8, IL-18 and tumor necrosis factor-alpha (TNF-alpha). On the other hand, IL-27 is not categorized as SASP factors though it is known that IL-27 has pleiotropic roles in inflammation. Here, we evaluated the interaction between TNF-alpha and IL-27 in the context of low-grade inflammation by using in HaCaT cells. TNF-alpha induced significant upregulation of IL-6 and IL-8 through the experimental concentrations (~10 ng/ml) of TNF-alpha, while the mRNA expression levels of IL-1RA, IL-10 and IL-18BP were unchanged. After confirming the expression of IL-27 receptor in HaCaT cells, we examined the effects of IL-27 alone on the cytokine expression. IL-27 alone significantly enhanced mRNA expression levels of IL-10 and IL-18BP by 1.61-fold and 1.46-fold, respectively, and also enhanced mRNA expression levels of IL-6 by 2.32-fold. In the presence of 100 ng/ml IL-27, the expression levels of anti-inflammatory cytokines, IL-1RA, IL-10 and IL-18BP, were significantly upregulated with the treatment of a physiological concentration (1 ng/ml) TNF-alpha. Taken together, a high concentration of IL-27 exhibits anti-inflammatory effects in the presence of a low concentration of TNF-alpha when keratinocytes are the recipient of IL-27 signaling, suggesting the anti-inflammatory roles of IL-27 in inflammaging may be regulated by TNF-alpha concentration.

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

Recently, a genome-wide association study using plasma HIV RNA from antiretroviral therapy-naive patients reported that 14 naturally occurring nonsynonymous single-nucleotide polymorphisms (SNPs) in HIV derived from antiretrovirus drug-naive patients were associated with virus load (VL). Those SNPs were detected in reverse transcriptase, RNase H, integrase, envelope, and Nef. However, the impact of each mutation on viral fitness was not investigated. Here, we constructed a series of HIV variants encoding each SNP and examined their replicative abilities. An HIV variant containing a Met-to-Ile change at codon 50 in integrase [HIV(IN:M50I)] was found as an impaired virus. Despite the mutation being in integrase, the virus release was significantly suppressed (P

S100A8 and S100A9 are members of the Alarmin family; these proteins are abundantly expressed in neutrophils, form a heterodimer complex, and are secreted in plasma on pathogen infection or acute inflammatory diseases. Recently, both proteins were identified as novel biomarkers of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and were shown to play key roles in inducing an aggressive inflammatory response by mediating the release of large amounts of pro-inflammatory cytokines, called the “cytokine storm.” Although co-infection with SARS-CoV-2 in people living with HIV-1 may result in an immunocompromised status, the role of the S100A8/A9 complex in HIV-1 replication in primary T cells and macrophages is still unclear. Here, we evaluated the roles of the proteins in HIV replication to elucidate their functions. We found that the complex had no impact on virus replication in both cell types; however, the subunits of S100A8 and S100A9 inhibit HIV in macrophages. These findings provide important insights into the regulation of HIV viral loads during SARS-CoV-2 co-infection.

Macrophages play an essential role in the immune system. Recent studies have shown that long non-coding RNAs (lncRNAs) can regulate genes encoding products involved in the immune response. Interleukin (IL)-27 is a member of the IL-6/IL-12 family of cytokines with broad anti-viral effects that inhibits human immunodeficiency virus (HIV) type-1 and herpes simplex virus (HSV). However, little is known about the role of lncRNAs in macrophages affected by IL-27. Therefore, we investigated the expression profiles of mRNA and lncRNA in human monocyte-derived macrophages (MDMs) regulated by IL-27. Monocytes were differentiated in the presence of macrophage-colony stimulatory factor (M-CSF)- or human AB serum with or without IL-27, and these cells were the subject for the profile analysis using RNA-Seq. We identified 146 lncRNAs (including 88 novel ones) and 434 coding genes were differentially regulated by IL-27 in both M-CSF- and AB serum-induced macrophages. Using weighted gene co-expression network analysis, we obtained four modules. The immune system, cell cycle, and regulation of complement cascade pathways were enriched in different modules. The network of mRNAs and lncRNAs in the pathways suggest that lncRNAs might regulate immune activity in macrophages. This study provides potential insight into the roles of lncRNA in macrophages regulated by IL-27.

We previously reported that some small interfering RNA (siRNA) enhances DNA or DNA virus mediated-interferon (IFN)-λ1(a type III IFN) induction through the crosstalk between retinoic acid-inducible gene I (RIG-I) and interferon gamma-inducible protein 16 (IFI16) signalling pathway. Here we provide further evidence of a new role for siRNA. siRNA containing a 5-nucleotide (nt) motif sequence suppresses DNA-mediated not only type III IFNs, but also type I IFNs and inflammatory cytokines. We define that motif siRNA inhibits the induction when the motif is located at the 3' or 5'-terminus of siRNA. Using THP1-Lucia ISG cells with various DNA stimulants, we reveal that motif siRNA inhibits DNA or DNA virus but not RNA virus-mediated signalling. Motif siRNA specifically interrupts IFI16 but not cyclic GMP-AMP synthase (cGAS) binding to DNA and has 2.5-fold higher affinity to IFI16 than that of siRNA without the motif. We further confirm that motif siRNA potently suppresses HSV-1 virus-mediated IFNs and inflammatory cytokines, such as IFNL1, IFNB and TNFA, in human primary immature dendritic cells. Collectively, these findings may shed light on a novel function of siRNA with the unique 5-nt motif as a quencher of innate immunity and facilitate the development of potential therapeutics to regulate innate immune cascades.

Inflammaging is a subject of considerable attention, because aging is characterized by low-grade, chronic, and asymptomatic inflammation, concomitant with increased blood levels of senescence-associated secretory phenotype (SASP) factors, including IL-1, IL-6, IL-8, IL-18, and tumor necrosis factor-α (TNF-α). However, IL-27 is currently not categorized as a SASP factor, although it is known to play pleiotropic roles in inflammation. In this study, we evaluated the interaction between TNF-α and IL-27 in context of low-grade inflammation using HaCaT cells. TNF-α induced significant upregulation of the mRNA levels of IL-6 and IL-8 at the experimental concentration (~10ng/ml), while the mRNA levels of IL-1RA, IL-10, and IL-18BP were unchanged. After confirming the expression of functional IL-27 receptors in HaCaT cells, we examined the effects of IL-27 alone on cytokine expression. IL-27 alone significantly upregulated the mRNA levels of IL-10, IL-18BP and IL-6 by 1.61-fold, 1.46-fold, and 2.32-fold, respectively. In the presence of 100 ng/ml of IL-27, the mRNA levels of the anti-inflammatory cytokines IL-1RA, IL-10 and IL-18BP, were significantly upregulated upon treatment with TNF-α at the physiological concentration (1 ng/ml). Taken together, this study indicates that a high concentration of IL-27 exhibits anti-inflammatory effects in the presence of a low concentration of TNF-α, in keratinocytes, suggesting that the anti-inflammatory role of IL-27 in inflammaging may be regulated by TNF-α concentration.

Recently, a genome-wide association study using plasma HIV RNA reported that 14 naturally occurring non-synonymous single nucleotide polymorphisms (SNPs) in HIV derived from anti-retrovirus naïve patients were associated with virus load (VL). However, the impact of each mutation on viral fitness was not investigated. Here, we constructed a series of HIV variants encoding each SNP using site-directed mutagenesis and examined their replicative abilities and biological properties. An HIV variant containing Met-to-Ile change at codon 50 in integrase (HIV(IN:M50I)) was found an impaired virus. Despite the mutation being in integrase, a quantification assay demonstrated that the virus release was significantly suppressed (PImportanceA nascent HIV-1 is noninfectious. To become an infectious virus, Gag and GagPol polyproteins in the particles need to be cleaved by mature HIV protease (PR). PR is initially translated as an inactive embedded enzyme in a GagPol polyprotein. The embedded PR in homodimerized GagPol polyproteins catalyzes a proteolytic reaction to release the mature PR. This excision step by a self-cleavage is called autoprocessing. Here, during the evaluation of roles of naturally emerging non-synonymous SNPs in HIV RNA, we found that autoprocessing is inhibited by Met-to-Ile change at codon 50 in integrase in GagPol which increases the efficiency of heterodimerization with Gag. This defect was recovered by co-existing of other SNPs: Ser-to-Asn change at codon 17 in integrase or Asn-to-Ser mutation at codon 79 in RNase H, suggesting that autoprocessing is regulated by not only integrase but also RNase H in GagPol polyprotein.

Interleukin-27 (IL-27) is a pleiotropic cytokine that influences the innate and adaptive immune systems. It inhibits viral infection and regulates the expression of microRNAs (miRNAs). We recently reported that macrophages differentiated from human primary monocytes in the presence of IL-27 and human AB serum resisted human immunodeficiency virus (HIV) infection and showed significant autophagy induction. In the current study, the miRNA profiles in these cells were investigated, especially focusing on the identification of novel miRNAs regulated by IL-27-treatment. The miRNA sequencing analysis detected 38 novel miRNAs. Real-time reverse transcription polymerase chain reaction (RT-PCR) analysis confirmed that IL-27 differentially regulated the expression of 16 of the 38 miRNAs. Overexpression of the synthesized miRNA mimics by transfection revealed that miRAB40 had potent HIV-inhibiting and autophagy-inducing properties. B18R, an interferon (IFN)-neutralization protein, partially suppressed both activities, indicating that the two functions were induced via IFN-dependent and -independent pathways. Although the target mRNA(s) of miRAB40 involving in the induction of both functions was unable to identify in this study, the discovery of miRAB40, a potential HIV-inhibiting and autophagy inducing miRNA, may provide novel insights into the miRNA (small none-coding RNA)-mediated regulation of HIV inhibition and autophagy induction as an innate immune response.

OBJECTIVE: Postmortem brains of subjects diagnosed with human immunodeficiency virus-1 (HIV) associated neurocognitive disorders (HAND) exhibit loss of dendrites. However, the mechanisms by which synapses are damaged are not fully understood. DESIGN: Dendrite length and remodeling occurs via microtubules (MTs) the dynamics of which are regulated by microtubule binding proteins, including MT associated protein 2 (MAP2). The HIV protein gp120 is neurotoxic and interferes with neuronal MTs. We measured MAP2 concentrations in human cerebrospinal fluid (CSF) and MAP2 immunoreactivity in rat cortical neurons exposed to HIV and gp120. METHODS: First, we examined whether HIV affects MAP2 levels by analyzing the CSF of 27 persons living with HIV (PLH) whose neurocognitive performance had been characterized. We then used rat cortical neurons to study the mechanisms of HIV-mediated dendritic loss. RESULTS: PLH who had HAND had greater MAP2 concentrations within the CSF than cognitive normal PLH. In cortical neurons, the deleterious effect of HIV on MAP2 positive dendrites occurred through a gp120-mediated mechanism. The neurotoxic effect of HIV was blocked by a CCR5 antagonist and prevented by Helix-A, a peptide that displaces gp120 from binding to MTs, conjugated to a nanolipoprotein particle delivery platform. CONCLUSIONS: Our findings support that HIV at least partially effects its neurotoxicity via neuronal cytoskeleton modifications and provide evidence of a new therapeutic compound that could be used to prevent the HIV-associated neuropathology.

Innate immune response is insisted upon detection of foreign intracellular DNA or RNA derived from viruses and bacteria. This reaction is important to initiate an effective protective response for the host cells. This crucial step is induced by cytosolic nucleic acids sensors/binding proteins, which triggers the production of type I or type III interferons (IFNs) and proinflammatory cytokines such as Interleukin 6 (IL-6). The identification of these cytosolic DNA or RNA sensors is a key step in understanding the signaling pathways triggered by those pathogens. Here we describe an effective approach to identify potential known/novel DNA or RNA sensors: a pull-down assay using DNA/RNA-conjugated beads with a customized competition strategy, which conferred a more effective and efficient way to determine the interaction between DNA/RNA and the sensor protein(s), therefore greatly improves the progress to investigate potential novel cytosolic DNA or RNA sensors/ binding proteins •The customized method makes the traditional pull-down assays more effective and efficient to identify DNA/RNA binding protein(s).•With the competitor of your choice, the method provides specific information about the competitive binding between DNA/RNA and binding proteins., Graphical abstract Image, graphical abstract

Macrophages play an essential role in the immune system. Recent studies have shown that long non-coding RNAs (lncRNAs) can regulate genes encoding products involved in the immune response. Interleukin (IL)-27 is a member of the IL-6/IL-12 family of cytokines with broad anti-viral effects that inhibits human immunodeficiency virus (HIV) type-1 and herpes simplex virus (HSV). However, little is known about the role of lncRNAs in macrophages affected by IL-27. Therefore, we investigated the expression profiles of mRNA and lncRNA in human monocyte-derived macrophages (MDMs) regulated by IL-27. Monocytes were differentiated in the presence of macrophage-colony stimulatory factor (M-CSF)- or human AB serum with or without IL-27, and these cells were the subject for the profile analysis using RNA-Seq. We identified 146 lncRNAs (including 88 novel ones) and 434 coding genes were differentially regulated by IL-27 in both M-CSF- and AB serum-induced macrophages. Using weighted gene co-expression network analysis, we obtained four modules. The immune system, cell cycle, and regulation of complement cascade pathways were enriched in different modules. The network of mRNAs and lncRNAs in the pathways suggest that lncRNAs might regulate immune activity in macrophages. This study provides potential insight into the roles of lncRNA in macrophages regulated by IL-27.

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.

Objectives: IL-27 is known as an antiviral cytokine that inhibits HIV, hepatitis C virus, and other viruses. We have previously demonstrated that, IL-27 posttreatment after HIV-infection inhibits viral replication in primary CD4+ T cells. Design: Here, we evaluated the anti-HIV effect of IL-27 pretreatment in CD4+ T cells from healthy donors prior to HIV infection with HIVNL4.3 or vesicular stomatitis virus G glycoprotein (VSV-G)-pseudotyped HIV-luciferase virus (HIV-LUC-V). Methods: IL-27-treated CD4+ T cells were infected with HIVNL4.3 or HIV-LUC-V and assessed the anti-HIV effect. HIV infection was monitored by p24 antigen ELISA or luciferase assay. HIV fusion/entry and uncoating were determined by BlaM-Vpr assay and HIV fate of capsid and/or HIV Entry/Uncoating assay based on core-packaged RNA availability and Translation assay, respectively. HIV proviral copy number was determined by real-time PCR. Gene expression profile from IL-27-pretreated CD4+ T cells was determined using Genechip array. Posttranslational modification of global proteins from IL-27-pretreated CD4+ T cells was determined by a combination of 2-dimensional difference-in-gel-electrophoresis (2D-DIG), western-blot and protein mass spectrometry. Results: IL-27 pretreatment inhibited HIVNL4.3 and HIV-LUC-V infection in CD4+ T cells. HIV copy assay demonstrated that IL-27-treatment suppressed an early step of reverse transcription during HIV infection. A combination of 2D-DIG-electrophoresis and western blot assays demonstrated that IL-27-treatment induces a change in posttranslational modification of Y box binding protein-1 (YB-1). Overexpression of domain negative YB-1 mutants illustrated that a residue Lysine at 118 plays a key role in supporting HIV infection in CD4+ T cells. Conclusion: IL-27-pretreatment inhibits HIV-1 infection by suppressing an HIV-reverse transcription product formation/uncoating step by suppressing the acetylation of YB-1 in primary CD4+ T cells.

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.

Background: Despite successfully suppressed viremia by treatment, patients with high levels of biomarkers of coagulation/inflammation are at an increased risk of developing non-AIDS defining serious illnesses such as cardiovascular diseases. Thus, there is a relationship between persistent immune activation and coagulation/inflammation, although the mechanisms are poorly understood. Platelets play an important role in this process. Although interactions between platelets and elements of the innate immune system, such as monocytes, are well described, little is known about the interaction between platelets and the adaptive immune system. Design: We investigated the interaction of a component of the coagulation system, platelets, and the adaptive immune system T cells. Methods: Healthy controls and combination antiretroviral therapy (cART)-treated HIV-infected patients with viral loads of less than 40 copies/ml for more than 15 months were analysed for platelet–T-cell conjugate formation. Results: Platelets can form conjugates with T cells and were preferentially seen in CD4+ and CD8+ T-cell subsets with more differentiated phenotypes [memory, memory/effector and terminal effector memory (TEM)]. Compared with healthy controls, these conjugates in patients with HIV infection were more frequent, more often composed of activated platelets (CD42b+CD62P+), and were significantly associated with the D-dimer serum levels. Conclusion: These data support a model in which platelet–T-cell conjugates may play a critical role in the fast recruitment of antigen-experienced T cells to the place of injury. This mechanism can contribute in maintaining a state of coagulation/inflammation observed in these patients contributing to the pathology of the disease.

Many of the infectious diseases which have jeopardized and still are a threat to public health are caused by RNA viruses, including HIV, HCV, Influenza virus, Ebola virus and Zika virus. Because of a high rate of mutations and recombination events, rapidly evolving RNA viruses prevail within a host as a collection of closely related variants, referred to as viral quasi-species. Uncovering the genetic diversity (i.e., inferring viral haplotypes and their proportions in the population) of an RNA virus can significantly benefit the study of disease progression, antiviral drug design, vaccine design and viral pathogenesis. Recent advances in PacBio single-molecule sequencing offers sufficient throughput and contiguous long reads (>10kb) covering the full length of most genes and RNA viral genomes, providing a potential to reliably profile the viral populations. However, the relatively high error rate (2~15%) in the long-read data requires novel analysis methods to deconvolute sequences derived from complex viral mixtures. We examined samples containing complex mixtures of near-full-length HIV-1 genomes, single molecules sequenced as near-full-length (9kb) amplicons directly from PCR products, and developed a novel signature-based self-tuning spectral clustering method called SigClust to accurately determine the identity (above 99.5%) and relative abundances of viral genomes in the mixtures. Results on real HIV-1 and influenza benchmark data sets demonstrate efficacy and superior performance of SigClust.