Biology subjects

Simons, L. M.

Publications and source records attributed to Simons, L. M..

6 recordsLinked to original sources

bioRxiv2022

Rab11-FIP1C is dispensable for HIV-1 replication in primary CD4+ T cells but its role is cell-type-dependent in immortalized human T-cell lines

The HIV-1 envelope glycoprotein (Env) contains a long cytoplasmic tail harboring highly conserved motifs that direct Env trafficking and incorporation into virions and promote efficient virus spread. The cellular trafficking factor Rab11a family interacting protein 1C (FIP1C) has been implicated in the directed trafficking of Env to sites of viral assembly. In this study, we confirm that siRNA-mediated depletion of FIP1C in HeLa cells modestly reduces Env incorporation into virions. To determine whether FIP1C is required for Env incorporation and HIV-1 replication in physiologically relevant cells, CRISPR/Cas9 technology was used to knock out the expression of this protein in several human T-cell lines - Jurkat E6.1, SupT1 and H9 - and in primary human CD4+ T cells. FIP1C knock-out caused modest reductions in Env incorporation in SupT1 cells but did not inhibit virus replication in SupT1 or Jurkat E6.1 T-cell lines. In H9 cells, FIP1C knock-out caused a cell-density-dependent defect in virus replication. In primary CD4+ T cells, FIP1C knock-out had no effect on HIV-1 replication. Furthermore, HTLV-I transformed cell lines that are permissive for HIV-1 replication do not express FIP1C. Mutation of an aromatic motif in the Env cytoplasmic tail - Y795W - implicated in FIP1C-mediated Env incorporation impaired virus replication independently of FIP1C expression in SupT1, Jurkat E6.1, H9, and primary T cells. Together, these results indicate that while FIP1C may contribute to HIV-1 Env incorporation in some contexts, additional and potentially redundant host factors are likely required for Env incorporation and virus dissemination in T cells. ImportanceThe incorporation of the HIV-1 envelope (Env) glycoproteins, gp120 and gp41, into virus particles is critical for virus infectivity. Gp41 contains a long cytoplasmic tail that has been proposed to interact with host cell factors, including the trafficking factor Rab11a family interacting protein 1C (FIP1C). To investigate the role of FIP1C in relevant cell types - human T-cell lines and primary CD4+ T cells - we used CRISPR/Cas9 to knock out FIP1C expression and examined the effect on HIV-1 Env incorporation and virus replication. We observed that in two of the T-cell lines examined (Jurkat E6.1 and SupT1) and in primary CD4+ T cells, FIP1C knockout did not disrupt HIV-1 replication, whereas FIP1C knockout reduced Env expression and delayed replication in H9 cells. The results indicate that while FIP1C may contribute to Env incorporation in some cell lines, it is not an essential factor for efficient HIV-1 replication in primary CD4+ T cells.

microbiology↗

bioRxiv2022

Bioengineering multifunctional extracellular vesicles for targeted delivery of biologics to T cells

Genetically modifying T cells can enable applications ranging from cancer immunotherapy to HIV treatment, yet delivery of T cell-targeted therapeutics remains challenging. Extracellular vesicles (EVs) are nanoscale particles secreted by all cells that naturally encapsulate and transfer proteins and nucleic acids, making them an attractive and clinically-relevant platform for engineering biocompatible delivery vehicles. We report a suite of technologies for genetically engineering cells to produce multifunctional EV vehicles--without employing chemical modifications that complicate biomanufacturing. We display high affinity targeting domains on the EV surface to achieve specific, efficient binding to T cells, identify a protein tag to confer active cargo loading into EVs, and display fusogenic glycoproteins to increase EV uptake and fusion with recipient cells. We demonstrate integration of these technologies by delivering Cas9-sgRNA complexes to edit primary human T cells. These approaches could enable targeting vesicles to a range of cells for the efficient delivery of cargo.

bioengineering↗

bioRxiv2022

Blockade of TGF-β signaling reactivates HIV-1/SIV reservoirs and immune responses in vivo

Elevated levels of TGF-{beta}, a potent immunosuppressive factor, are present in HIV-1 infected individuals even after years of antiretroviral therapy (ART). TGF-{beta} plays a critical role in maintaining immune cells in a resting state by inhibiting cell activation and proliferation. Resting HIV-1 target cells represent one of the main cellular reservoirs after long term ART and the low inducibility of the latent provirus constitutes one of the major obstacles to "kick and kill" cure strategies. We hypothesized that releasing cells from TGF-{beta}-driven signaling would promote latency reversal. To test our hypothesis, we compared ex vivo models of HIV-1 latency reactivation with and without TGF-{beta} and a TGF-{beta} type 1 receptor (TGFBR1) inhibitor, galunisertib. We also tested the effect of galunisertib in SIV infected, ART treated macaques by monitoring SIV envelope (env) protein expression via PET/CT using the Cu64-anti gp120 Fab (7D3) probe, along with plasma and tissue viral loads (VL). Exogenous TGF-1{beta} reduced HIV-1 reactivation in U1 and ACH2 latency models. Galunisertib increased HIV-1 latency reversal both in ex vivo models and in PBMC from HIV-1 infected, cART treated aviremic donors. In vivo, oral galunisertib promoted increased SIV env protein total standardized uptake values (SUVtot) in PET/CT images of tissues (gut and lymph nodes) of 5 out of 7 aviremic, long-term ART-treated, SIV-infected, macaques. This increase correlated with an increase in SIV RNA in gut tissue. Two out of 7 animals also exhibited increases in plasma viral load. Higher anti-SIV T cell responses and anti-SIV env antibody titers were detected after galunisertib treatment in most animals. In summary, our data suggest that blocking TGF-{beta} signaling simultaneously increases retroviral reactivation events and enhances anti-SIV immune responses.

immunology↗

bioRxiv2022

An immunoPET probe to SARS-CoV-2 reveals early infection of the male genital tract in rhesus macaques

The systemic nature of SARS-CoV-2 infection is highly recognized, but poorly characterized. A non-invasive and unbiased method is needed to clarify whole body spatiotemporal dynamics of SARS-CoV-2 infection after transmission. We recently developed a probe based on the anti-SARS-CoV-2 spike antibody CR3022 to study SARS-CoV-2 pathogenesis in vivo. Herein, we describe its use in immunoPET to investigate SARS-CoV-2 infection of three rhesus macaques. Using PET/CT imaging of macaques at different times post-SARS-CoV-2 inoculation, we track the 64Cu-labelled CR3022-F(ab)2 probe targeting the spike protein of SARS-CoV-2 to study the dynamics of infection within the respiratory tract and uncover novel sites of infection. Using this method, we uncovered differences in lung pathology between infection with the WA1 isolate and the delta variant, which were readily corroborated through computed tomography scans. The 64Cu-CR3022-probe also demonstrated dynamic changes occurring between 1- and 2-weeks post-infection. Remarkably, a robust signal was seen in the male genital tract (MGT) of all three animals studied. Infection of the MGT was validated by immunofluorescence imaging of infected cells in the testicular and penile tissue and severe pathology was observed in the testes of one animal at 2-weeks post-infection. The results presented here underscore the utility of using immunoPET to study the dynamics of SARS-CoV-2 infection to understand its pathogenicity and discover new anatomical sites of viral replication. We provide direct evidence for SARS-CoV-2 infection of the MGT in rhesus macaques revealing the possible pathologic outcomes of viral replication at these sites. Graphic AbstractPET/CT detected SARS-CoV-2 infection of 4 different tissues in the male genital tract illuminates the cause of COVID-19 clinical sequalae of male sexual health and fertility O_FIG O_LINKSMALLFIG WIDTH=189 HEIGHT=200 SRC="FIGDIR/small/481974v2_ufig1.gif" ALT="Figure 1"> View larger version (74K): org.highwire.dtl.DTLVardef@188c53aorg.highwire.dtl.DTLVardef@4c639forg.highwire.dtl.DTLVardef@120799forg.highwire.dtl.DTLVardef@110edb1_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Diagram shows schematic illustration of the male genital tract of the rhesus macaque. Virus icon shows sites of SARS-CoV-2 PET signal. Text highlighting the clinical sequalae associated with each sight of infection is shown in text adjacent to each infection site. C_FIG

immunology↗

bioRxiv2022

Recognition of HIV-1 capsid licenses innate immune response to viral infection

Cyclic GMP-AMP synthase (cGAS) is a primary sensor of aberrant DNA that governs an innate immune signaling cascade, leading to the induction of the type-I interferon response. We have previously identified polyglutamine binding protein 1, PQBP1, as an adaptor molecule required for cGAS-mediated innate immune response of lentiviruses, including the human immunodeficiency virus 1 (HIV-1), but dispensable for the recognition of DNA viruses. HIV-1- encoded DNA is synthesized as a single copy from its RNA genome, and is subsequently integrated into the host chromatin. HIV-1 then produces progeny through amplification and packaging of its RNA genome, thus, in contrast to DNA viruses, HIV-1 DNA is both transient and of low abundance. However, the molecular basis for the detection and verification of this low abundance HIV-1 DNA pathogen-associated molecular pattern (PAMP) is not understood. Here, we elucidate a two-factor authentication strategy that is employed by the innate immune surveillance machinery to selectively respond to the low concentration of PAMP, while discerning these species from extranuclear DNA molecules. We find that, upon HIV-1 infection, PQBP1 decorates intact viral capsid, which serves as a primary verification step for the viral nucleic acid cargo. As the reverse transcription and capsid disassembly initiate, cGAS protein is then recruited to the capsid in a PQBP1-dependent manner, enabling cGAS molecules to be co-positioned at the site of PAMP generation. Thus, these data indicate that PQBP1 recognition of the HIV-1 capsid sanctions a robust cGAS-dependent response to a limited abundance and short-lived DNA PAMP. Critically, this illuminates a molecular strategy wherein the modular recruitment of co-factors to germline encoded pattern recognition receptors (PRRs) serves to enhance repertoire of pathogens that can be sensed by the innate immune surveillance machinery.

molecular biology↗

bioRxiv2020

TRIM5α restriction of HIV-1-N74D viruses in lymphocytes is caused by a loss of cyclophilin A protection

The core of HIV-1 viruses bearing the capsid change N74D (HIV-1-N74D) do not bind the human protein cleavage and polyadenylation specificity factor subunit 6 (CPSF6). In addition, HIV-1-N74D viruses have altered patterns of integration site preference in human cell lines. In primary human CD4+ T cells, HIV-1-N74D viruses exhibit infectivity defects when compared to wild type. The reason for this loss of infectivity in primary cells is unknown. We first investigated whether loss of CPSF6 binding accounts for the loss of infectivity. Depletion of CPSF6 in human CD4+ T cells did not affect the early stages of wild-type HIV-1 replication, suggesting that defective infectivity in the case of HIV-1-N74D is not due to the loss of CPSF6 binding. Based on our previous result that cyclophilin A (Cyp A) protected HIV-1 from human tripartite motif-containing protein 5 (TRIM5hu) restriction in CD4+ T cells, we tested whether TRIM5hu was involved in the decreased infectivity observed for HIV-1-N74D. Depletion of TRIM5hu in CD4+ T cells rescued the infectivity of HIV-1-N74D, suggesting that HIV-1-N74D cores interacted with TRIM5hu. Accordingly, TRIM5hu binding to HIV-1-N74D cores was increased compared with that of wild-type cores, and consistently, HIV-1-N74D cores lost their ability to bind Cyp A. In conclusion, we showed that the decreased infectivity of HIV-1-N74D in CD4+ T cells is due to a loss of Cyp A protection from TRIM5hu restriction activity.

microbiology↗