Biology subjects

Pillai, S. K.

Publications and source records attributed to Pillai, S. K..

10 recordsLinked to original sources

bioRxiv2025

A Cluster of Acidic Residues in the Cytoplasmic Domain of SARS-CoV-2 Spike is Required for Virion-Incorporation and Infectivity

Abstract/SummaryLike all coronaviruses, the infectivity of SARS-CoV-2 virus particles (virions) requires incorporation of the Spike glycoprotein. Yet, the mechanisms that support the virion-incorporation of Spike are incompletely defined. We noted an unusual feature of human sarbecovirus Spike proteins: their cytoplasmic domains (CDs) contain a stretch of acidic amino acids (DEDDSE). This sequence resembles a cluster of acidic residues, or acidic cluster (AC) motif, found in the cytoplasmic domain of the cellular endoprotease Furin. In Furin, the acidic cluster acts as a protein sorting signal, supporting its intracellular localization at the trans-Golgi network (TGN). We tested the contribution of the acidic cluster motif in the Spike CD to protein interactions and to the infectivity of SARS-CoV-2. We used virus-like particles (VLPs) as a model for viral "infection" (transduction). The SARS-CoV2 VLPs were produced by co-expressing Spike (S), Membrane (M), Envelope (E) and Nucleocapsid (N) proteins and deliver an RNA encoding luciferase to target cells expressing the ACE2 receptor. Remarkably, when all five acidic residues of the DEDDSE sequence were replaced with alanines, the VLPs were rendered non-infectious. The N-terminal DE residues provided most of the activity of the acidic cluster. These virologi-cally-impaired Spike mutants were able to reach the cell surface and induce the formation of syncytia, indicating that they are fusogenic and capable of anterograde traffic through the biosynthetic pathway to the plasma membrane. Despite this, they failed to efficiently incorporate into virions. We observed acidic cluster motif-dependent interactions of the Spike CD with several cellular proteins that could potentially support its role in virion-incorporation, including the ERM proteins Ezrin, Radixin, and Moesin; the retromer subunit Vps35, and the medium subunits of the clathrin adaptor complexes AP1 and AP2. While the key cofactor and mechanism of action remains to be defined, this region of acidic residues in the Spike CD appears to be a novel determinant of SARS-CoV-2 infectivity.

molecular biology↗

bioRxiv2024

High-resolution Inference of Multiplexed Anti-HIV Gene Editing using Single-Cell Targeted DNA Sequencing

Gene therapy-based HIV cure strategies typically aim to excise the HIV provirus directly, or target host dependency factors (HDFs) that support viral persistence. Cure approaches will likely require simultaneous co-targeting of multiple sites within the HIV genome to prevent evolution of resistance, and/or co-targeting of multiple HDFs to fully render host cells refractory to HIV infection. Bulk cell-based methods do not enable inference of co-editing within individual viral or target cell genomes, and do not discriminate between monoallelic and biallelic gene disruption. Here, we describe a targeted single-cell DNA sequencing (scDNA-seq) platform characterizing the near full-length HIV genome and 50 established HDF genes, designed to evaluate anti-HIV gene therapy strategies. We implemented the platform to investigate the capacity of multiplexed CRISPR-Cas9 ribonucleoprotein complexes (Cas9-RNPs) to simultaneously 1) inactivate the HIV provirus, and 2) knockout the CCR5 and CXCR4 HDF (entry co-receptor) genes in microglia and primary monocyte-derived macrophages (MDMs). Our scDNA-seq pipeline revealed that antiviral gene editing is rarely observed at multiple loci (or both alleles of a locus) within an individual cell, and editing probabilities across sites are linked. Our results demonstrate that single-cell sequencing is critical to evaluate the true efficacy and therapeutic potential of HIV gene therapy.

genomics↗

bioRxiv2022

The Hypoxia-regulated Ectonucleotidase CD73 is a Host Determinant of HIV Latency

Deciphering the mechanisms underlying viral persistence is critical to achieving a cure for HIV infection. We implemented a systems approach to discover molecular signatures of HIV latently-infected CD4+ T cells, identifying the immunosuppressive, adenosine-producing ectonucleotidase CD73 as a key surface marker of latent cells. Hypoxic conditioning, reflecting the lymphoid tissue microenvironment, increased the frequency of CD73+ CD4+ T cells and promoted HIV latency. Transcriptomic profiles of CD73+ CD4+ T cells favored viral quiescence, immune evasion, and cell survival. CD73+ CD4+ T cells were capable of harboring a functional HIV reservoir and reinitiating productive infection ex vivo. CD73 or adenosine receptor blockade facilitated latent HIV reactivation in vitro, mechanistically linking adenosine signaling to viral quiescence. Finally, tissue imaging of lymph nodes from HIV-infected individuals on antiretroviral therapy revealed spatial association between CD73 expression and HIV persistence in vivo. Our findings warrant development of HIV cure strategies targeting the hypoxia-CD73-adenosine axis.

microbiology↗

bioRxiv2022

Human Galectin-9 Potently Enhances SARS-CoV-2 Replication and Inflammation in Airway Epithelial Cells

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has caused a global economic and health crisis. Recently, plasma levels of galectin-9 (Gal-9), a {beta}-galactoside-binding lectin involved in immune regulation and viral immunopathogenesis, were reported to be elevated in the setting of severe COVID-19 disease. However, the impact of Gal-9 on SARS-CoV-2 infection and immunopathology remained to be elucidated. Here, we demonstrate that Gal-9 treatment potently enhances SARS-CoV-2 replication in human airway epithelial cells (AECs), including primary AECs in air-liquid interface (ALI) culture. Gal-9-glycan interactions promote SARS-CoV-2 attachment and entry into AECs in an ACE2-dependent manner, enhancing the binding affinity of the viral spike protein to ACE2. Transcriptomic analysis revealed that Gal-9 and SARS-CoV-2 infection synergistically induce the expression of key pro-inflammatory programs in AECs including the IL-6, IL-8, IL-17, EIF2, and TNF signaling pathways. Our findings suggest that manipulation of Gal-9 should be explored as a therapeutic strategy for SARS-CoV-2 infection. ImportanceCOVID-19 continues to have a major global health and economic impact. Identifying host molecular determinants that modulate SARS-CoV-2 infectivity and pathology is a key step in discovering novel therapeutic approaches for COVID-19. Several recent studies have revealed that plasma concentrations of the human {beta}-galactoside-binding protein galectin-9 (Gal-9) are highly elevated in COVID-19 patients. In this study, we investigated the impact of Gal-9 on SARS-CoV-2 pathogenesis ex vivo in airway epithelial cells (AECs), the critical initial targets of SARS-CoV-2 infection. Our findings reveal that Gal-9 potently enhances SARS-CoV-2 replication in AECs, interacting with glycans to enhance the binding between viral particles and entry receptors on the target cell surface. Moreover, we determined that Gal-9 accelerates and exacerbates several virus-induced pro-inflammatory programs in AECs that are established signature characteristics of COVID-19 disease and SARS-CoV-2-induced acute respiratory distress syndrome (ARDS). Our findings suggest that Gal-9 is a promising pharmacological target for COVID-19 therapies.

microbiology↗

bioRxiv2020

Durable Control of HIV-1 Using a Staphylococcus aureus Cas9-Expressing Lentivirus Co-Targeting Viral Latency and Host Susceptibility

CRISPR/Cas9 gene editing has the potential to revolutionize the clinical management of HIV-1 infection, and may eliminate the need for antiretroviral therapy (ART). Current gene therapies attempt to either excise HIV-1 provirus or target HIV-1 entry receptors to prevent infection of new cells. Using a viral dynamic model, we determined that combining these two interventions, in the presence or absence of ART, significantly lowers the gene editing efficacy thresholds required to achieve an HIV-1 cure. To implement this dual-targeting approach, we engineered a single lentiviral vector that simultaneously targets multiple highly-conserved regions of the provirus and the host CXCR4 coreceptor, and developed a novel coculture system enabling real-time monitoring of latent infection, viral reactivation, and infection of new target cells. Simultaneous dual-targeting depleted HIV-1-infected cells with significantly greater potency than vectors targeting either virus or host independently, highlighting its potential as an HIV-1 cure strategy.

microbiology↗

bioRxiv2020

FOXO1 promotes HIV Latency by suppressing ER stress in T cells

Quiescence is a hallmark of CD4+ T cells latently infected with HIV-1. While reversing this quiescence is an effective approach to reactivate latent HIV from T cells in culture, it can cause deleterious cytokine dysregulation in patients. Here we report that FOXO1, a key regulator of T-cell quiescence, promotes latency and suppresses productive HIV infection. In resting T cells, FOXO1 inhibition induces ER stress and activates two associated transcription factors: activating transcription factor 4 (ATF4) and nuclear factor of activated T cells (NFAT). Both factors associate with HIV chromatin and are necessary for HIV reactivation. Indeed, inhibition of PKR-like endoplasmic reticulum kinase (PERK), a known link between ER stress and ATF4, and calcineurin, a calcium-dependent regulator of NFAT, synergistically suppress HIV reactivation induced by FOXO1 inhibition. Thus, our studies uncover a novel link between FOXO1, ER stress, and HIV infection that could be therapeutically exploited to selectively reverse T-cell quiescence and reduce the size of the latent viral reservoir.

immunology↗

bioRxiv2020

Efficient Generation of Isogenic Primary Human Myeloid Cells using CRISPR-Cas9 Ribonucleoproteins

Genome engineering of primary human cells with CRISPR-Cas9 has revolutionized experimental and therapeutic approaches to cell biology, but human myeloid-lineage cells have remained largely genetically intractable. We present a method for delivery of CRISPR-Cas9 ribonucleoprotein (RNP) complexes by nucleofection directly into CD14+ human monocytes purified from peripheral blood, leading to high rates of precise gene knockout. These cells can be efficiently differentiated into monocyte-derived macrophages or dendritic cells. This process yields genetically-edited cells that retain critical markers of both myeloid differentiation and phagocytic function. Genetic ablation of the restriction factor SAMHD1 increased HIV-1 infection more than fifty-fold, demonstrating the power of this system for genotype-phenotype interrogation. This fast, flexible and scalable platform can be used for genetic studies of human myeloid cells in immune signaling, inflammation, cancer immunology, host-pathogen interactions, and beyond, and could facilitate development of novel myeloid cellular therapies.

genetics↗

bioRxiv2020

μ-Lat: A High-Throughput Humanized Mouse Model for Preclinical Evaluation of Human Immunodeficiency Virus Eradication Strategies

A critical barrier to the development of a human immunodeficiency virus (HIV) cure is the lack of a scalable animal model that enables robust evaluation of eradication approaches prior to testing in humans. We established a humanized mouse model of latent HIV infection by transplanting "J-Lat" cells, Jurkat cells harboring a latent HIV provirus encoding an enhanced green fluorescent protein (GFP) reporter, into irradiated adult NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. J-Lat cells exhibited successful engraftment in several tissues including spleen, bone barrow, peripheral blood, and lung, in line with the diverse natural tissue tropism of HIV. Administration of tumor necrosis factor (TNF)-, an established HIV latency reversal agent, significantly induced GFP expression in engrafted cells across tissues, reflecting viral reactivation. These data suggest that our murine latency ("-Lat") model enables efficient determination of how effectively viral eradication agents, including latency reversal agents, penetrate and function in diverse anatomical sites harboring HIV in vivo.

microbiology↗

bioRxiv2020

TCF-1 regulates the stem-like memory potential of HIV-specific CD8+ T cells in elite controllers

Although many HIV cure strategies seek to expand HIV-specific CD8+ T cells to control the virus, all are likely to fail if cellular exhaustion is not prevented. A loss in stem-like memory properties (i.e., the ability to proliferate and generate secondary effector cells) is a key feature of exhaustion; little is known, however, about how these properties are regulated in human virus-specific CD8+ T cells. We found that virus-specific CD8+ T cells from humans and non-human primates naturally controlling HIV/SIV infection express more of the transcription factor, TCF-1, than non-controllers. HIV-specific CD8+ T cell TCF-1 expression correlated with memory marker expression and proliferative capacity and declined with antigenic stimulation. CRISPR-Cas9 editing of TCF-1 in human primary T cells demonstrated a direct role in regulating expansion capacity. Collectively, these data suggest that TCF-1 controls the stem-like memory properties of HIV-specific CD8+ T cells and provides a rationale for enhancing this pathway in T cell-based therapeutic strategies for HIV. One Sentence SummaryTCF-1 is highly expressed in HIV-specific CD8+ T cells from elite controllers and directly regulates human CD8+ T cell expansion capacity in response to T cell receptor stimulation.

immunology↗

bioRxiv2018

Chromatin Functional States Correlate with HIV Latency Reversal in Infected Primary CD4+ T Cells

Human immunodeficiency virus (HIV) infection cannot be cured due to a small reservoir of latently infected CD4+ T cells in treated patients. The \"shock and kill\" approach proposes to eliminate the reservoir by inducing its activation and the direct or indirect killing of infected cells. Current latency reversing agents (LRAs) do not reduce the viral reservoir in treated patients.\n\nWe use a novel dual-fluorescent HIV reporter to identify and purify latent cells, and to determine the fraction of latent cells that undergo viral reactivation after infection of primary CD4+ T cells. Unexpectedly, LRAs reactivate less than 5% of latent proviruses. Analysis of HIV integration sites from induced and non-induced latent populations reveals distinct provirus integration sites between these two populations in terms of chromatin functional states.\n\nThese findings challenge \"shock and kill\", and suggest the need of more potent LRAs in combination with immunomodulatory approaches to eradicate HIV reservoir.

microbiology↗