HIV-1 Proteins Research Articles

HIV protein profile characteristics in patients with first-time detected infection

The HIV-infection continues to be one of the most large-scale epidemics worldwide. Many techniques have been developed to detect this disease, but the Western blot based on the identification of specific viral proteins remains the most commonly used method that allows to monitor ongoing viral processes. Despite discussions regarding the criteria for a positive test assessment and selection of a minimum number of viral proteins to reliably interpret the data, a very few studies on the protein profiles in HIV-infected patients, particularly in the Russian Federation are available. The aim of this study was to assess the prevalence of HIV viral proteins in a group of people with newly diagnosed infections analyzing 2566 blood samples from individuals with newly diagnosed HIV infection for reference testing. The samples were assessed using ELISA and IHL techniques, followed by western blotting. Subsequently, the following viral proteins were analyzed to assess HIV life cycle and the predominance of its different stages: gp160, gp120, gp41, p55, p40, p24, p17, p66, p51, and p31. For comparison, gp110/120 was chosen as the reference protein due to its lowest prevalence frequency among allenvgene products comprising 96.06%. A significantly reduced prevalence frequency was found for several protein groups: GAG — p55 (80.91%), p40 (72.14%), nucleocapsid p18/17 (67.37%); POL proteins — p68/66 (89.57%), p52/51 (81.91%), p34/31 (86.02%). Significant differences in frequency of viral proteins between age and sex groups are shown. Hypotheses explaining the obtained data are presented. By aligning anti-viral protein antibody profile with the course of the infection and patient’s condition, it will be possible to identify patterns and take necessary measures for early diagnostics with extended results, such as duration of the infection, viral load, and disease severity.

HIV Protein Nef Induces Cardiomyopathy Through Induction of Bcl2 and p21.

HIV-associated cardiovascular diseases remain a leading cause of death in people living with HIV/AIDS (PLWHA). Although antiretroviral drugs suppress the viral load, they fail to remove the virus entirely. HIV-1 Nef protein is known to play a role in viral virulence and HIV latency. Expression of Nef protein can be detected in different organs, including cardiac tissue. Despite the established role of Nef protein in HIV-1 replication, its impact on organ function inside the human body is not clear. To understand the effect of Nef at the organ level, we created a new Nef-transgenic (Nef-TG) mouse that expresses Nef protein in the heart. Our study found that Nef expression caused inhibition of cardiac function and pathological changes in the heart with increased fibrosis, leading to heart failure and early mortality. Further, we found that cellular autophagy is significantly inhibited in the cardiac tissue of Nef-TG mice. Mechanistically, we found that Nef protein causes the accumulation of Bcl2 and Beclin-1 proteins in the tissue, which may affect the cellular autophagy system. Additionally, we found Nef expression causes upregulation of the cellular senescence marker p21 and senescence-associated β-galactosidase expression. Our findings suggest that the Nef-mediated inhibition of autophagy and induction of senescence markers may promote aging in PLWHA. Our mouse model could help us to understand the effect of Nef protein on organ function during latent HIV infection.

Impact of HIV-1 tat protein on methamphetamine-induced inhibition of vesicular monoamine transporter2-mediated dopamine transport and methamphetamine conditioned place preference in HIV-1 tat transgenic mice

Perturbation of dopamine transmission has been implicated as a contributing factor in HIV-1 associated neurocognitive disorders with concurrent methamphetamine (METH) abuse. We have demonstrated that the HIV-1 protein, transactivator of transcription (Tat), decreases dopamine transport through inhibition of vesicular monoamine transporter2 (VMAT2). This study determined the effects of Tat protein on METH-inhibited VMAT2 function and METH-conditioned place preference (CPP). In vitro exposure of isolated mouse whole brain vesicles to recombinant Tat1-86 or METH displayed a concentration-dependent inhibition of the vesicular [3H]Dopamine uptake, in which a combination of Tat and METH induced a greater reduction of dopamine uptake compared to Tat or METH alone. In vivo, the maximal velocity (Vmax) of vesicular [3H]Dopamine uptake was decreased in inducible Tat transgenic (iTat-tg) mice harvested after treatment with either 21-day doxycycline (Dox) or 14-day METH (3 mg/kg, i.p., daily), whereas these mice treated with both Dox and METH displayed an additive reduction of the Vmax compared to either Tat or METH alone. Moreover, Dox-induced Tat expression increased METH-CPP in an exposure-dependent manner, with iTat-tg mice demonstrating a 2.3-fold potentiation of METH-CPP compared with Tat null control mice upon administration of Dox for 14 days. Furthermore, a 7-day administration of Dox reinstated extinguished METH-CPP. Collectively, these results suggest a synergistic effect of Tat protein and METH on inhibition of VMAT2-mediated DA transport, potentially contributing to potentiation of METH-CPP in iTat-tg mice.

HIV-1 inhibits IFITM3 expression to promote the infection of megakaryocytes.

Despite an undetectable plasma viral load as a result of antiretroviral therapy, HIV-1-infected individuals with poor immune reconstitution harbor infectious HIV-1 within their platelets. Megakaryocytes, as platelet precursors, are the likely cellular origin of these HIV-1-containing platelets. To investigate the mechanisms that allow megakaryocytes to support HIV-1 infection, we established in vitro models of viral infection using hematopoietic stem cell-derived megakaryocytes and the megakaryocytic MEG-01 cell line. We observed HIV-1 DNA provirus integration into the megakaryocyte cell genome, self-limiting virus production, and HIV-1 protein and RNA compartmentalization, which are hallmarks of HIV-1 infection in myeloid cells. In addition, following HIV-1 infection of megakaryocyte precursors, the expression of interferon-induced transmembrane protein 3 (IFITM3), an antiviral factor constitutively expressed in megakaryocytes, was inhibited in terminally differentiated HIV-1-infected megakaryocytes. IFITM3 knockdown in MEG-01 cells prior to infection led to enhanced HIV-1 infection, indicating that IFITM3 acts as an HIV-1 restriction factor in megakaryocytes. Together, these findings indicate that megakaryocyte precursors are susceptible to HIV-1 infection, leading to terminally differentiated megakaryocytes harboring virus in a process regulated by IFITM3. Megakaryocytes may thus constitute a neglected HIV-1 reservoir that warrants further study in order to develop improved antiretroviral therapies and to facilitate HIV-1 eradication.

Examining the effects of the HIV-1 protein Tat and morphine on antiretroviral accumulation and distribution within the brain.

Despite combination antiretroviral therapy effectively suppressing HIV within the periphery, neuro-acquired HIV (neuroHIV) remains a significant problem and approximately half of people living with HIV will experience HIV-associated neurocognitive disorders (HAND). Concurrent opioid use exacerbates neuroHIV by promoting neuroinflammation, neuronal injury and synaptodendritic culling, viral replication, and potentially altering antiretroviral concentrations within the brain. The present study examined the effects of HIV and morphine co-exposure on the accumulation and spatial distribution of antiretroviral drugs across multiple regions within the brain in an HIV-1 Tat transgenic mouse model by using infrared-matrix-assisted laser desorption electrospray ionization mass spectrometry imaging (IR-MALDESI MSI). Morphine exposure uniquely decreased antiretroviral concentrations in anterior cerebral (primary motor and somatosensory) cortices, corpus collosum (anterior forceps), caudoputamen, nucleus accumbens, and posterior regions including the hippocampus, corpus callosum (main body), cerebral cortex (somatosensory and auditory cortices), thalamus, and hypothalamus. Interestingly, male mice experienced greater morphine-associated decreases in antiretroviral concentrations than females. The study also assessed whether changes in antiretroviral concentrations were linked with inflammation in astroglia, assessed through the measurement of astroglial activation using glial fibrillary acidic protein (GFAP) as a marker. Alterations in antiretroviral concentrations co-registering with areas of astroglial activation exhibited sex-specific treatment differences. This study highlights the intricate interplay between HIV, opioids, and antiretroviral drugs within the CNS, elucidating distinct regional and sex variations in responsiveness. Our findings emphasize the identification of vulnerabilities within the neural landscape and underscore the necessity of carefully monitoring opioid use to maintain the efficacy of antiretroviral therapies.

Adaptation of the REINVENT neural network architecture to generate potential HIV-1 entry inhibitors

Objectives. The main purpose of this work is to adapt the architecture of the REINVENT neural network to generate potential inhibitors of the HIV-1 envelope protein gp120 using in the learning process with reinforcement of molecular docking on GPUs.Methods. To modify the initial network model, molecular docking on GPUs implemented in the learning process with reinforcement was used, and an algorithm was developed that allows converting the representations of connections generated by the SMILES network into the PDBQT format necessary for docking. To accelerate the learning of the neural network in the modified version of the REINVENT model, the AutoDock-Vina-GPU-2.1 docking program was used, and to clarify the results of its work, the procedure for revaluing the affinity of compounds to the target using the RFScore-4 evaluation function was used.Results. Using a modified version of the REINVENT model, more than 60,000 compounds were obtained, of which about 52,000 molecules have a binding energy value to the HIV-1 gp120 protein comparable to the value calculated for the HIV-1 inhibitor NBD-14204, used in calculations as a positive control. Of the 52,000 compounds selected, about 34,000 molecules satisfy the restrictions imposed on a potential drug to ensure its bioavailability when taken orally.Conclusion. The results obtained allow us to demonstrate the effectiveness of an adapted neural network by the example of designing new potential inhibitors of the gp120 HIV-1 protein capable of blocking the CD4- binding site of the gp120 virus envelope protein and preventing its penetration into host cells.

Putting a Kink in HIV-1 Particle Infectivity: Rocaglamide Inhibits HIV-1 Replication by Altering Gag-Genomic RNA Interaction.

Our examination of RNA helicases for effects on HIV-1 protein production and particle assembly identified Rocaglamide (RocA), a known modulator of eIF4A1 function, as an inhibitor of HIV-1 replication in primary CD4+ T cells and three cell systems. HIV-1 attenuation by low-nM RocA doses was associated with reduced viral particle formation without a marked decrease in Gag production. Rather, the co-localization of Gag and HIV-1 genomic RNA (gRNA) assemblies was impaired by RocA treatment in a reversible fashion. Ribonucleoprotein (RNP) immunoprecipitation studies recapitulated the loss of Gag-gRNA assemblies upon RocA treatment. Parallel biophysical studies determined that neither RocA nor eIF4A1 independently affected the ability of Gag to interact with viral RNA, but together, they distorted the structure of the HIV-1 RNP visualized by electron microscopy. Taken together, several lines of evidence indicate that RocA induces stable binding of eIF4A1 onto the viral RNA genome in a manner that interferes with the ordered assembly of Gag along Gag-gRNA assemblies required to generate infectious virions.

Probing Gag-Env dynamics at HIV-1 assembly sites using live-cell microscopy.

Human immunodeficiency virus (HIV)-1 assembly is initiated by Gag binding to the inner leaflet of the plasma membrane (PM). Gag targeting is mediated by its N-terminally myristoylated matrix (MA) domain and PM phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Upon Gag assembly, envelope (Env) glycoproteins are recruited to assembly sites; this process depends on the MA domain of Gag and the Env cytoplasmic tail. To investigate the dynamics of Env recruitment, we applied a chemical dimerizer system to manipulate HIV-1 assembly by reversible PI(4,5)P2 depletion in combination with super resolution and live-cell microscopy. This approach enabled us to control and synchronize HIV-1 assembly and track Env recruitment to individual nascent assembly sites in real time. Single virion tracking revealed that Gag and Env are accumulating at HIV-1 assembly sites with similar kinetics. PI(4,5)P2 depletion prevented Gag PM targeting and Env cluster formation, confirming Gag dependence of Env recruitment. In cells displaying pre-assembled Gag lattices, PI(4,5)P2 depletion resulted in the disintegration of the complete assembly domain, as not only Gag but also Env clusters were rapidly lost from the PM. These results argue for the existence of a Gag-induced and -maintained membrane micro-environment, which attracts Env. Gag cluster dissociation by PI(4,5)P2 depletion apparently disrupts this micro-environment, resulting in the loss of Env from the former assembly domain.IMPORTANCEHuman immunodeficiency virus (HIV)-1 assembles at the plasma membrane of infected cells, resulting in the budding of membrane-enveloped virions. HIV-1 assembly is a complex process initiated by the main structural protein of HIV-1, Gag. Interestingly, HIV-1 incorporates only a few envelope (Env) glycoproteins into budding virions, although large Env accumulations surrounding nascent Gag assemblies are detected at the plasma membrane of HIV-expressing cells. The matrix domain of Gag and the Env cytoplasmatic tail play a role in Env recruitment to HIV-1 assembly sites and its incorporation into nascent virions. However, the regulation of these processes is incompletely understood. By combining a chemical dimerizer system to manipulate HIV-1 assembly with super resolution and live-cell microscopy, our study provides new insights into the interplay between Gag, Env, and host cell membranes during viral assembly and into Env incorporation into HIV-1 virions.

HIV-TAT dysregulates microglial lipid metabolism through SREBP2/miR-124 axis: Implication of lipid droplet accumulation microglia in NeuroHIV

Chronic HIV infection can dysregulate lipid/cholesterol metabolism in the peripheral system, contributing to the higher incidences of diabetes and atherosclerosis in HIV (+) individuals. Recently, accumulating evidence indicate that HIV proteins can also dysregulate lipid/cholesterol metabolism in the brain and such dysregulation could be linked with the pathogenesis of HIV-associated neurological disorders (HAND)/NeuroHIV. To further characterize the association between lipid/cholesterol metabolism and HAND, we employed HIV-inducible transactivator of transcription (iTAT) and control mice to compare their brain lipid profiles. Our results reveal that HIV-iTAT mice possess dysregulated lipid profiles and have increased numbers of lipid droplets (LDs) accumulation microglia (LDAM) in the brains. HIV protein TAT can upregulate LDs formation through enhancing the lipid/cholesterol synthesis in vitro. Mechanistically, HIV-TAT increases the expression of sterol regulatory element-binding protein 2 (SREBP2) through microRNA-124 downregulation. Cholesterol synthesis inhibition can block HIV-TAT-mediated NLRP3 inflammasome activation and microglial activation in vitro as well as mitigate aging-related behavioral impairment and memory deficiency in HIV-iTAT mice. Taken together, our results indicate an inherent role of lipid metabolism and LDAM in the pathogenesis of NeuroHIV (immunometabolism). These findings suggest that LDAM reversal through modulating lipid/cholesterol metabolism could be a novel therapeutic target for ameliorating NeuroHIV symptoms in chronic HIV (+) individuals.

The association between HIV-1 Tat and Vif amino acid sequence variation, inflammation and Trp-Kyn metabolism: an exploratory investigation

BackgroundHIV-1 has well-established mechanisms to disrupt essential pathways in people with HIV, such as inflammation and metabolism. Moreover, diversity of the amino acid sequences in fundamental HIV-1 proteins including Tat and Vif, have been linked to dysregulating these pathways, and subsequently influencing clinical outcomes in people with HIV. However, the relationship between Tat and Vif amino acid sequence variation and specific immune markers and metabolites of the tryptophan-kynurenine (Trp-Kyn) pathway remains unclear. Therefore, this study aimed to investigate the relationship between Tat/Vif amino acid sequence diversity and Trp-Kyn metabolites (quinolinic acid (QUIN), Trp, kynurenic acid (KA), Kyn and Trp/Kyn ratio), as well as specific immune markers (sCD163, suPAR, IL-6, NGAL and hsCRP) in n = 67 South African cART-naïve people with HIV.MethodsSanger sequencing was used to determine blood-derived Tat/Vif amino acid sequence diversity. To measure Trp-Kyn metabolites, a LC–MS/MS metabolomics platform was employed using a targeted approach. To measure immune markers, Enzyme-linked immunosorbent assays and the Particle-enhanced turbidimetric assay was used.ResultsAfter adjusting for covariates, sCD163 (p = 0.042) and KA (p = 0.031) were higher in participants with Tat signatures N24 and R57, respectively, and amino acid variation at position 24 (adj R2 = 0.048, β = -0.416, p = 0.042) and 57 (adj R2 = 0.166, β = 0.535, p = 0.031) of Tat were associated with sCD163 and KA, respectively.ConclusionsThese preliminary findings suggest that amino acid variation in Tat may have an influence on underlying pathogenic HIV-1 mechanisms and therefore, this line of work merits further investigation.

Variability of VPU protein in HIV-1 sub-subtype A6 in patients with different stages of HIV infection

The aim of the study: to compare the genetic diversity of the Vpu protein in HIV-1 in the people living with HIV (PLWH) with different stages of the disease.Materials and methods. An analysis was carried out of 259 clinical samples of whole blood from HIV-infected patients with no experience of taking antiretroviral therapy, who were observed at the Center for the Prevention and Control of AIDS and Infectious Diseases, Moscow, Russia. The analysis included the following stages: extraction of proviral DNA, amplification of the region of the virus genome containing the vpu gene, sequencing of amplification products, genotyping, comparison of conservation and amino acid substitutions in the Vpu protein sequences in PLWH with different stages of the disease.Results and discussion. In 255 out of 259 (98.4%) clinical samples, a sub-subtype A6 virus variant was identified. The consensus sequence of the Vpu sub-subtype A6 protein was obtained, which contained 81 amino acids. No significant differences in the conservation of Vpu protein sequences were found between HIV-1 variants obtained from patients with different stages of the disease. Amino acid substitution P3A was significantly more common in PLWH with the second stage of HIV infection.Conclusion. The results obtained highlight the issue of the influence of non-structural proteins of HIV-1 on the course of the disease and indicate directions for possible research in the future.

Reverse vaccinology approaches to design a potent multiepitope vaccine against the HIV whole genome: immunoinformatic, bioinformatics, and molecular dynamics approaches

Substantial advances have been made in the development of promising HIV vaccines to eliminate HIV-1 infection. For the first time, one hundred of the most submitted HIV subtypes and CRFs were retrieved from the LANL database, and the consensus sequences of the eleven HIV proteins were obtained to design vaccines for human and mouse hosts. By using various servers and filters, highly qualified B-cell epitopes, as well as HTL and CD8 + epitopes that were common between mouse and human alleles and were also located in the conserved domains of HIV proteins, were considered in the vaccine constructs. With 90% coverage worldwide, the human vaccine model covers a diverse allelic population, making it widely available. Codon optimization and in silico cloning in prokaryotic and eukaryotic vectors guarantee high expression of the vaccine models in human and E. coli hosts. Molecular dynamics confirmed the stable interaction of the vaccine constructs with TLR3, TLR4, and TLR9, leading to a substantial immunogenic response to the designed vaccine. Vaccine models effectively target the humoral and cellular immune systems in humans and mice; however, experimental validation is needed to confirm these findings in silico.

Structural and Functional Dysregulation of the Brain Endothelium in HIV Infection and Substance Abuse.

Blood-brain barrier (BBB) injury and dysfunction following infection with the human immunodeficiency virus (HIV) enables viral entry into the brain, infection of resident brain cells, neuronal injury and subsequent neurodegeneration leading to HIV-associated neurocognitive disorders (HAND). Although combination antiretroviral therapy has significantly reduced the incidence and prevalence of acquired immunodeficiency syndrome and increased the life expectancy of people living with HIV, the prevalence of HAND remains high. With aging of people living with HIV associated with increased comorbidities, the prevalence of HIV-related central nervous system (CNS) complications is expected to remain high. Considering the principal role of the brain endothelium in HIV infection of the CNS and HAND, the purpose of this manuscript is to review the current literature on the pathobiology of the brain endothelium structural and functional dysregulation in HIV infection, including in the presence of HIV-1 and viral proteins (gp120, Tat, Nef, and Vpr). We summarize evidence from human and animal studies, in vitro studies, and associated mechanisms. We further summarize evidence of synergy or lack thereof between commonly abused substances (cocaine, methamphetamine, alcohol, tobacco, opioids, and cannabinoids) and HIV- or viral protein-induced BBB injury and dysfunction.

In-silico Study of Secondary Metabolites as Potential Inhibitors of NEF and P24 Protein of HIV-1

Introduction: Acquired immune deficiency syndrome (HIV/AIDS) has been a major global health concern for over 38 years. No safe and effective preventive or therapeutic vaccine has been developed although many products have been investigated. Methods: This computational study was conducted on plant-based active compounds against HIV-1 NEF and p24 protein to obtain and complexes with high binding scores were used for two-dimensional interaction studies via Ligplot to explore hydrogen bond and hydrophobic bond formation. ADMET analysis for best phytocompounds was performed using DruLito, ALOGPS, and PROTOX II. Results: According to the study conducted, phytocompounds like, Protostrychnine, Isostrychnine, Pseudo-Alpha-Colubrine, Alpha-Colubrine, Camptothecin, Benzo[f]quinoline, and (+) -Camptothecin are safe to be considered as a potential drug candidate after experimental validation against NEF and p24 proteins of HIV-1. While, Picrasidine M, Chaetochromin, 3’,3’-Binaringenin, and Sequoiaflavone displayed high binding scores of -10.8, -8.2, -9.5, -9.2 and -9.0, -8.8, -10.6, -9.0 respectively for NEF and p24 protein. All drugs belong to the toxicity class of either 4 or 5. They are inactive for hepatotoxicity and carcinogenicity but active for immunogenicity. Conclusion: For further validation of the results the phytocompounds can be extracted through solvent extraction method and tested on cell lines or animal models for their effectiveness.

Adeno-Associated Virus (AAV)-Delivered Exosomal TAT and BiTE Molecule CD4-αCD3 Facilitate the Elimination of CD4 T Cells Harboring Latent HIV-1.

Combinatorial antiretroviral therapy (cART) has transformed HIV infection from a death sentence to a controllable chronic disease, but cannot eliminate the virus. Latent HIV-1 reservoirs are the major obstacles to cure HIV-1 infection. Previously, we engineered exosomal Tat (Exo-Tat) to reactivate latent HIV-1 from the reservoir of resting CD4+ T cells. Here, we present an HIV-1 eradication platform, which uses our previously described Exo-Tat to activate latent virus from resting CD4+ T cells guided by the specific binding domain of CD4 in interleukin 16 (IL16), attached to the N-terminus of exosome surface protein lysosome-associated membrane protein 2 variant B (Lamp2B). Cells with HIV-1 surface protein gp120 expressed on the cell membranes are then targeted for immune cytolysis by a BiTE molecule CD4-αCD3, which colocalizes the gp120 surface protein of HIV-1 and the CD3 of cytotoxic T lymphocytes. Using primary blood cells obtained from antiretroviral treated individuals, we find that this combined approach led to a significant reduction in replication-competent HIV-1 in infected CD4+ T cells in a clonal in vitro cell system. Furthermore, adeno-associated virus serotype DJ (AAV-DJ) was used to deliver Exo-Tat, IL16lamp2b and CD4-αCD3 genes by constructing them in one AAV-DJ vector (the plasmid was named pEliminator). The coculture of T cells from HIV-1 patients with Huh-7 cells infected with AAV-Eliminator viruses led to the clearance of HIV-1 reservoir cells in the in vitro experiment, which could have implications for reducing the viral reservoir in vivo, indicating that Eliminator AAV viruses have the potential to be developed into therapeutic biologics to cure HIV-1 infection.

Interactome of the HIV-1 proteome and human host RNA

The human immunodeficiency virus (HIV-1) is highly dependent on a variety of host factors. Beside proteins, host RNA molecules are reported to aid HIV-1 replication and latency maintenance. Here, we implement multiple workflows of native RNA immunoprecipitation and sequencing (nRIPseq) to determine direct host RNA interaction partners of all 18 HIV-1 (poly)proteins. We identify 1,727 HIV-1 protein – human RNA interactions in the Jurkat cell line and 1,558 interactions in SupT1 cells for a subset of proteins, and discover distinct cellular pathways that seem to be used or controlled by HIV-1 on the RNA level: Tat binds mRNAs of proteins involved in the super elongation complex (AFF1-4, Cyclin-T1). Correlation of the interaction scores (based on binding abundancy) allows identifying the highest confidence interactions, for which we perform a small-scale knockdown screen that leads to the identification of three HIV-1 protein binding RNA interactors involved in HIV-1 replication (AFF2, H4C9 and RPLP0).

Selection of antibody-binding covalent aptamers

Aptamers are oligonucleotides with antibody-like binding function, selected from large combinatorial libraries. In this study, we modified a DNA aptamer library with N-hydroxysuccinimide esters, enabling covalent conjugation with cognate proteins. We selected for the ability to bind to mouse monoclonal antibodies, resulting in the isolation of two distinct covalent binding motifs. The covalent aptamers are specific for the Fc region of mouse monoclonal IgG1 and are cross-reactive with mouse IgG2a and other IgGs. Investigation into the covalent conjugation of the aptamers revealed a dependence on micromolar concentrations of Cu2+ ions which can be explained by residual catalyst remaining after modification of the aptamer library. The aptamers were successfully used as adapters in the formation of antibody-oligonucleotide conjugates (AOCs) for use in detection of HIV protein p24 and super-resolution imaging of actin. This work introduces a new method for the site-specific modification of native monoclonal antibodies and may be useful in applications requiring AOCs or other antibody conjugates.

Methodological approaches to studying phase separation and HIV-1 replication: Current and future perspectives

This article reviews tried-and-tested methodologies that have been employed in the first studies on phase separating properties of structural, RNA-binding and catalytic proteins of HIV-1. These are described here to stimulate interest for any who may want to initiate similar studies on virus-mediated liquid–liquid phase separation. Such studies serve to better understand the life cycle and pathogenesis of viruses and open the door to new therapeutics.

Tryptophan-Kynurenine Pathway Activation and Cognition in Virally Suppressed Women With HIV.

Immune and cognitive dysfunction persists even in virally suppressed women with HIV (VS-WWH). Since inflammation and HIV proteins induce the enzyme indoleamine 2,3-dioxygenase (IDO), converting tryptophan (T) to kynurenine (K) while producing downstream neurotoxic metabolites, we investigated IDO activation (KT ratio) in relation to cognition in VS-WWH and demographically similar women without HIV (WWoH). Ninety-nine VS-WWH on stable antiretroviral therapy and 102 WWoH (median age 52 vs 54 years; 73% vs 74% Black, respectively) from the New York and Chicago sites of the Women's Interagency HIV Study (WIHS) completed a neuropsychological test battery assessing motor function, processing speed, attention/working memory, verbal fluency, verbal learning and memory, and executive function and had plasma measured for tryptophan-kynurenine metabolites through liquid chromatography-tandem mass spectrometry and monocyte-derived [soluble cluster of differentiation-14 (sCD14), soluble cluster of differentiation-163 (sCD163), monocyte chemoattractant protein-1 (MCP-1)] plus general inflammatory markers [tumor necrosis factor alpha-2 receptor (TNF-R2), high-sensitivity C-reactive protein, high-sensitivity interleukin-6] through enzyme-linked immunosorbent assays between 2017 and 2020. VS-WWH had a higher KT ratio (P < 0.01) and higher sCD14 levels (P < 0.05) compared with WWoH. Higher sCD163 was associated with higher KT ratio (R = 0.29, P < 0.01) and worse fine motor function in VS-WWH; after adjusting for sCD163 and sCD14 in multivariable regressions, higher KT ratio remained significantly associated with impaired fine motor function in VS-WWH only (standardized β = -0.29, P < 0.05). IDO activation was not associated with cognition in WWoH. IDO activation (K:T) was associated with worse fine motor control in VS-WWH independent of measured systemic inflammation. Further studies investigating biological mechanisms linking IDO activation to fine motor function among VS-WWH are warranted.

Comparative analysis of retroviral Gag-host cell interactions: focus on the nuclear interactome

Retroviruses exploit host proteins to assemble and release virions from infected cells. Previously, most studies focused on interacting partners of retroviral Gag proteins that localize to the cytoplasm or plasma membrane. Given that several full-length Gag proteins have been found in the nucleus, identifying the Gag-nuclear interactome has high potential for novel findings involving previously unknown host processes. Here we systematically compared nuclear factors identified in published HIV-1 proteomic studies and performed our own mass spectrometry analysis using affinity-tagged HIV-1 and RSV Gag proteins mixed with nuclear extracts. We identified 57 nuclear proteins in common between HIV-1 and RSV Gag, and a set of nuclear proteins present in our analysis and ≥ 1 of the published HIV-1 datasets. Many proteins were associated with nuclear processes which could have functional consequences for viral replication, including transcription initiation/elongation/termination, RNA processing, splicing, and chromatin remodeling. Examples include facilitating chromatin remodeling to expose the integrated provirus, promoting expression of viral genes, repressing the transcription of antagonistic cellular genes, preventing splicing of viral RNA, altering splicing of cellular RNAs, or influencing viral or host RNA folding or RNA nuclear export. Many proteins in our pulldowns common to RSV and HIV-1 Gag are critical for transcription, including PolR2B, the second largest subunit of RNA polymerase II (RNAPII), and LEO1, a PAF1C complex member that regulates transcriptional elongation, supporting the possibility that Gag influences the host transcription profile to aid the virus. Through the interaction of RSV and HIV-1 Gag with splicing-related proteins CBLL1, HNRNPH3, TRA2B, PTBP1 and U2AF1, we speculate that Gag could enhance unspliced viral RNA production for translation and packaging. To validate one putative hit, we demonstrated an interaction of RSV Gag with Mediator complex member Med26, required for RNA polymerase II-mediated transcription. Although 57 host proteins interacted with both Gag proteins, unique host proteins belonging to each interactome dataset were identified. These results provide a strong premise for future functional studies to investigate roles for these nuclear host factors that may have shared functions in the biology of both retroviruses, as well as functions specific to RSV and HIV-1, given their distinctive hosts and molecular pathology.Graphical