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Interleukin (IL)-27, a member of the IL-12 family of cytokines, induces human immunodeficiency virus (HIV)-resistant monocyte-derived macrophages and T cells. This resistance is mediated via the downregulation of spectrin beta, non-erythrocytic 1 (SPTBN1), induction of autophagy, or suppression of the acetylation of Y-box binding protein-1 (YB-1); however, the role of IL-27 administration during the induction of immature monocyte-derived dendritic cells (iDC) is poorly investigated. In the current study, we investigated the function of IL-27-induced iDC (27DC) on HIV infection. 27DC inhibited HIV infection by 95 ± 3% without significant changes in the expression of CD4, CCR5, and SPTBN1 expression, autophagy induction and acetylation of YB-1 compared to iDC. An HIV proviral DNA copy number assay displayed that 27DC suppressed reverse transcriptase (RT) reaction without influencing the virus entry. A DNA microarray analysis was performed to identify the differentially expressed genes between 27DC and iDC. Compared to iDC, 51 genes were differentially expressed in 27DC, with more than 3-fold changes in four independent donors. Cross-reference analysis with the reported 2,214 HIV regulatory host genes identified nine genes as potential interests: Ankyrin repeat domain 22, Guanylate binding protein (GBP)-1, -2, -4, -5, Stabilin 1, Serpin family G member 1 (SERPING1), Interferon alpha inducible protein 6, and Interferon-induced protein with tetratricopeptide repeats 3. A knock-down study using si-RNA failed to determine a key factor associated with the anti-HIV activity due to the induction of robust amounts of off-target effects. Overexpression of each protein in cells had no impact on HIV infection. Thus, we could not define the mechanism of the anti-HIV effect in 27DC. However, our findings indicated that IL-27 differentiates monocytes into HIV-resistant DC, and the inhibitory mechanism differs from IL-27-induced HIV-resistant macrophages and T cells. [ABSTRACT FROM AUTHOR]

Accounting for genetic variation is an essential consideration during human immunodeficiency virus type 1 (HIV-1) investigation. Nanopore sequencing preserves proviral integrity by passing long genomic fragments through ionic channels, allowing reads that span the entire genome of different viral quasispecies (vQS). However, this sequencing method has suffered from high error rates, limiting its utility. This was the inspiration behind HIV-Quasipore: an HIV-1-specific Nanopore basecaller suite designed to overcome these error rates through training with gold-standard data. It comprises three deep learning-based R9.4.1 basecallers: fast, high accuracy (HAC), super accuracy (SUP), and two R10.3 deep learning-based basecallers: HAC and SUP. This was accomplished by sequencing the HIV-1 J-Lat 10.6 cell line using Nanopore and highquality Sanger techniques. Training significantly reduced basecaller error rates across all models (Student's one-sided t-test; p = 0.0) where median error rates were 0.0189, 0.0018, 0.0008, for R9.4.1 HIV-Quasipore-fast, HAC, SUP, and 0.0007, 0.0011 for R10.3 HIV-Quasipore-HAC, and SUP, respectively. This improved quality reduces the resolution needed to accurately detect a vQS from 22.4 to 2.6% of total positional coverage for R9.4.1 HIV-Quasipore-fast, 6.9 to 0.5% for R9.4.1 HIV-Quasipore-HAC, 4.5 to 0.3% for R9.4.1 HIV-Quasipore-SUP, 8.0 to 0.3% for R10.3 HIV-Quasipore-HAC, and 5.4 to 0.3% for R10.3 HIV-Quasipore-SUP. This was consistently observed across the entire J-Lat 10.6 genome and maintained across longer reads. Reads with greater than 8,000 nucleotides display a median nucleotide identity of 0.9819, 0.9982, and 0.9991, for R9.4.1 HIV-Quasipore-fast, HAC, SUP, and 0.9993, 0.9988 for R10.3 HIV-Quasipore-HAC, and SUP, respectively. To evaluate the robustness of this tool against unseen data, HIV-Quasipore and their corresponding pretrained basecallers were used to sequence the J-Lat 9.2 cell line and a clinical isolate acquired from the Drexel Medicine CARES cohort. When sample reads were compared against their corresponding consensus sequence, all HIV-Quasipore basecallers displayed higher median alignment accuracies than their pretrained counterparts for both the J-Lat 9.2 cell line and clinical isolate. Using Nanopore sequencing can allow investigators to explore topics, such as vQS profile detection, HIV-1 integration site analysis, whole genome amplification, gene coevolution, and CRISPR-induced indel detection, among others. HIV-Quasipore basecallers can be acquired here: https://github.com/DamLabResources/HIV-Quasipore-basecallers. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Despite antiretroviral therapy human immunodeficiency virus type-1 (HIV-1) infection results in neuroinflammation of the central nervous system that can cause HIV-associated neurocognitive disorders (HAND). The molecular mechanisms involved in the development of HAND are unclear, however, they are likely due to both direct and indirect consequences of HIV-1 infection and inflammation of the central nervous system. Additionally, opioid abuse in infected individuals has the potential to exacerbate HIV-comorbidities, such as HAND. Although restricted for productive HIV replication, astrocytes (comprising 40–70% of all brain cells) likely play a significant role in neuropathogenesis in infected individuals due to the production and response of viral proteins. The HIV-1 protein Tat is critical for viral transcription, causes neuroinflammation, and can be secreted from infected cells to affect uninfected bystander cells. The Wnt/β-catenin signaling cascade plays an integral role in restricting HIV-1 infection in part by negatively regulating HIV-1 Tat function. Conversely, Tat can overcome this negative regulation and inhibit β-catenin signaling by sequestering the critical transcription factor TCF-4 from binding to β-catenin. Here, we aimed to explore how opiate exposure affects Tat-mediated suppression of β-catenin in astrocytes and the downstream modulation of neuroinflammatory genes. We observed that morphine can potentiate Tat suppression of β-catenin activity in human astrocytes. In contrast, Tat mutants deficient in secretion, and lacking neurotoxic effects, do not affect β-catenin activity in the presence or absence of morphine. Finally, morphine treatment of astrocytes was sufficient to reduce the expression of genes involved in neuroinflammation. Examining the molecular mechanisms of how HIV-1 infection and opiate exposure exacerbate neuroinflammation may help us inform or predict disease progression prior to HAND development. [ABSTRACT FROM AUTHOR]

The human immunodeficiency virus type 1 (HIV‐1) transactivator of transcription (Tat) protein functions both intracellularly and extracellularly. Intracellularly, the main function is to enhance transcription of the viral promoter. However, this process only requires a small amount of intracellular Tat. The majority of Tat is secreted through an unconventional mechanism by binding to phosphatidylinositol‐4,5‐bisphosphate (PtdIns(4,5)P2), a phospholipid in the inner leaflet of the plasma membrane that is required for secretion. This interaction is mediated by the basic domain of Tat (residues 48‐57) and a conserved tryptophan (residue 11). After binding to PtdIns(4,5)P2, Tat secretion diverges into multiple pathways, which we categorized as oligomerization‐mediated pore formation, spontaneous translocation and incorporation into exosomes. Extracellular Tat has been shown to be neurotoxic and toxic to other cells of the central nervous system (CNS) and periphery, able to recruit immune cells to the CNS and cerebrospinal fluid, and alter the gene expression and morphology of uninfected cells. The effects of extracellular Tat have been examined in HIV‐1‐associated neurocognitive disorders (HAND); however, only a small number of studies have focused on the mechanisms underlying Tat secretion. In this review, the molecular mechanisms of Tat secretion will be examined in a variety of biologically relevant cell types. [ABSTRACT FROM AUTHOR]

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 gene editing system has been shown to be effective at inhibiting human immunodeficiency virus type 1 (HIV-1). Studies have not consistently used a trackable dual reporter system to determine what cells received the Cas9/gRNA to determine the overall knockdown of HIV. Some studies have used stably transduced cells under drug selection to accomplish this goal. Here a two-color system was used that allows tracking of viral protein expression and which cells received the CRISPR/Cas9 system. These experiments ensured that each gRNA used was a perfect match to the intended target to remove this variable. The data showed that gRNAs targeting the transactivation response element (TAR) region or other highly conserved regions of the HIV-1 genome were effective at stopping viral gene expression, with multiple assays demonstrating greater than 95 percent reduction. Conversely, gRNAs targeting conserved sites of the 5' portion of the U3 region were largely ineffective, demonstrating that the location of edits in the long terminal repeat (LTR) matter with respect to function. In addition, it was observed that a gRNA targeting Tat was effective in a T-cell model of HIV-1 latency. Taken together, these studies demonstrated gRNAs designed to highly conserved functional regions have near 100% efficacy in vitro in cells known to have received the Cas9/gRNA pair., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Allen, Chung, Worrell, Nwaozo, Madrid, Mele, Dampier, Nonnemacher and Wigdahl.)

The full length of HIV/R7/E - /GFP integrated in the J-Lat 10.6 cell line was sequenced in this study. The single copy of the integrated virus, including the breakpoints from the human chromosome to the provirus, was amplified by two separate PCRs. A 10,200-bp genome sequence was acquired, analyzed, and deposited in GenBank., (Copyright © 2020 Chung et al.)

The human immunodeficiency virus type 1 (HIV-1) transactivator of transcription (Tat) protein functions both intracellularly and extracellularly. Intracellularly, the main function is to enhance transcription of the viral promoter. However, this process only requires a small amount of intracellular Tat. The majority of Tat is secreted through an unconventional mechanism by binding to phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P 2 ), a phospholipid in the inner leaflet of the plasma membrane that is required for secretion. This interaction is mediated by the basic domain of Tat (residues 48-57) and a conserved tryptophan (residue 11). After binding to PtdIns(4,5)P 2 , Tat secretion diverges into multiple pathways, which we categorized as oligomerization-mediated pore formation, spontaneous translocation and incorporation into exosomes. Extracellular Tat has been shown to be neurotoxic and toxic to other cells of the central nervous system (CNS) and periphery, able to recruit immune cells to the CNS and cerebrospinal fluid, and alter the gene expression and morphology of uninfected cells. The effects of extracellular Tat have been examined in HIV-1-associated neurocognitive disorders (HAND); however, only a small number of studies have focused on the mechanisms underlying Tat secretion. In this review, the molecular mechanisms of Tat secretion will be examined in a variety of biologically relevant cell types., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)