Exosomes Packaging APOBEC3G Confer Human Immunodeficiency Virus Resistance to Recipient Cells▿

The human cytidine deaminase APOBEC3G (A3G) is a part of a cellular defense system against human immunodeficiency virus type 1 (HIV-1) and other retroviruses. Antiretroviral activity of A3G can be severely blunted in the presence of the HIV-1 protein Vif. However, in some cells expressing the enzymatically active low-molecular-mass form of A3G, HIV-1 replication is restricted at preintegration steps, before accumulation of Vif. Here, we show that A3G can be secreted by cells in exosomes that confer resistance to both vif-defective and wild-type HIV-1 in exosome recipient cells. Our results also suggest that A3G is the major exosomal component responsible for the anti-HIV-1 activity of exosomes. However, enzymatic activity of encapsidated A3G does not correlate with the observed limited cytidine deamination in HIV-1 DNA, suggesting that A3G-laden exosomes restrict HIV-1 through a nonenzymatic mechanism. Real-time PCR quantitation demonstrated that A3G exosomes reduce accumulation of HIV-1 reverse transcription products and steady-state levels of HIV-1 Gag and Vif proteins. Our findings suggest that A3G exosomes could be developed into a novel class of anti-HIV-1 therapeutics

BK Virus Replication in the Glomerular Vascular Unit: Implications for BK Virus Associated Nephropathy

Background: BK polyomavirus (BKV) reactivates from latency after immunosuppression in renal transplant patients, resulting in BKV-associated nephropathy (BKVAN). BKVAN has emerged as an important cause of graft dysfunction and graft loss among transplant patients. BKV infection in kidney transplant patients has increased over recent decades which correlates with the use of more potent immunosuppressive therapies. BKV infection of the Glomerular Vascular Unit (GVU) consisting of podocytes, mesangial cells, and glomerular endothelial cells could lead to glomerular inflammation and contribute to renal fibrosis. The effects of BKV on GVU infectivity have not been reported. methods: We infected GVU cells with the Dunlop strain of BKV. Viral infectivity was analyzed by microscopy, immunofluorescence, Western blot analysis, and quantitative RT-PCR (qRT-PCR). The expression of specific proinflammatory cytokines induced by BKV was analyzed by qRT-PCR. Results: BKV infection of podocytes, mesangial cells, and glomerular endothelial cells was confirmed by qRT-PCR and positive staining with antibodies to the BKV VP1 major capsid protein, or the SV40 Large T-Antigen. The increased transcriptional expression of interferon gamma-induced protein 10 (CXCL10/IP-10) and interferon beta (IFNβ) was detected in podocytes and mesangial cells at 96 h post-infection. conclusions: All cellular components of the GVU are permissive for BKV replication. Cytopathic effects induced by BKV in podocytes and glomerular endothelial cells and the expression of CXCL10 and IFNβ genes by podocytes and mesangial cells may together contribute to glomerular inflammation and cytopathology in BKVAN

Exon 4-encoded sequence is a major determinant of cytotoxicity of apolipoprotein L1

The apolipoprotein L1 (APOL1) gene (APOL1) product is toxic to kidney cells, and its G1 and G2 alleles are strongly associated with increased risk for kidney disease progression in African Americans. Variable penetrance of the G1 and G2 risk alleles highlights the significance of additional factors that trigger or modify the progression of disease. In this regard, the effect of alternative splicing in the absence or presence of G1 or G2 alleles is unknown. In this study we investigated whether alternative splicing of non-G1, non-G2 APOL1 (APOL1 G0) affects its biological activity. Among seven APOL1 exons, exons 2 and 4 are differentially expressed in major transcripts. We found that, in contrast to APOL1 splice variants B3 or C, variants A and B1 demonstrate strong toxicity in human embryonic kidney (HEK293T) cells. Subsequently, we established that exon 4 is a major determinant of toxicity of variants A and B1 and that extracellular release of these variants is dispensable for their cytotoxicity. Although only variants A and B1 induced nuclear translocation of transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy, exon 4-positive and -negative APOL1 variants stimulated perinuclear accumulation of unprocessed autophagosomes. Knockdown of endogenous TFEB did not attenuate APOL1 cytotoxicity, indicating that nuclear translocation of TFEB is dispensable for APOL1 toxicity. Our findings that a human podocyte cell line expresses exon 4-positive and -negative APOL1 transcripts suggest that these variants may play a differential role in podocyte pathology. In summary, we have identified exon 4 as a major determinant of APOL1 G0 cytotoxicity

Phospholipase D1 Couples CD4⁺ T Cell Activation to c-Myc-Dependent Deoxyribonucleotide Pool Expansion and HIV-1 Replication

<div><p>Quiescent CD4+ T cells restrict human immunodeficiency virus type 1 (HIV-1) infection at early steps of virus replication. Low levels of both deoxyribonucleotide triphosphates (dNTPs) and the biosynthetic enzymes required for their <i>de novo</i> synthesis provide one barrier to infection. CD4+ T cell activation induces metabolic reprogramming that reverses this block and facilitates HIV-1 replication. Here, we show that phospholipase D1 (PLD1) links T cell activation signals to increased HIV-1 permissivity by triggering a c-Myc-dependent transcriptional program that coordinates glucose uptake and nucleotide biosynthesis. Decreasing PLD1 activity pharmacologically or by RNA interference diminished c-Myc-dependent expression during T cell activation at the RNA and protein levels. PLD1 inhibition of HIV-1 infection was partially rescued by adding exogenous deoxyribonucleosides that bypass the need for <i>de novo</i> dNTP synthesis. Moreover, the data indicate that low dNTP levels that impact HIV-1 restriction involve decreased synthesis, and not only increased catabolism of these nucleotides. These findings uncover a unique mechanism of action for PLD1 inhibitors and support their further development as part of a therapeutic combination for HIV-1 and other viral infections dependent on host nucleotide biosynthesis.</p></div

PLD1 activity is required for normal expression of nutrient transporters in activated T cells.

<p>(A) Cell surface expression of glucose transporter Glut1 and activation marker CD25 as determined by flow cytometry on resting primary CD4+ T cells (shaded histogram) or after stimulation with anti-CD3/anti-CD28 beads for 48h in the absence (solid line) or presence of PLD1i (dashed line). (B) Western blot analysis of protein expression in whole cell lysates from primary CD4+ T cells treated as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004864#ppat.1004864.g001" target="_blank">Fig 1A</a>. (C) Q PCR analysis of expression of target genes in primary CD4+ T cells nucleofected with siRNA and then stimulated for 24 h as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004864#ppat.1004864.g001" target="_blank">Fig 1C</a>. T cells were nucleofected with non-targeting (NT) (black bars), c-Myc (grey bars), or PLD1 (open bars)-specific siRNAs. mRNA abundance in NT siRNA sample was set to1. Error bars represent standard error from the mean of triplicate samples. Data are representative of experiments from three independent donors.</p

PLD1 inhibition decreases the c-Myc-dependent dNTP biosynthetic transcriptional program.

<p>(A) Western blot analysis of protein expression in whole cell lysates prepared from resting primary human CD4+ T cells that were pretreated for 24h with DMSO vehicle, 10 or 5 μM of PLD1i, 50 or 25 μM of c-Myci, 10 μM U0126, or 100 nM of rapamycin. Cells were either left resting or stimulated with 5μg/ml PHA and 20 U/ml IL2 in the continued presence of DMSO or inhibitors for 48h before cell harvest. (B) RRM2 mRNA levels in resting primary CD4+ T cells pretreated for 24h with DMSO vehicle, 100 μM c-Myci, or 10 μM PLDi, then left resting or stimulated as in (A) as determined by quantitative real-time PCR. mRNA abundance in mock controls was set to1. Error bars represent standard error from the mean of triplicate samples. Data are representative of experiments from three independent donors. UD; undetectable levels of mRNA. (C) Resting primary human CD4+ T cells were transfected via nucleofection with siRNAs targeting PLD1 or c-Myc. Cells were allowed to recover for 24 h and then stimulated for 48 h with anti-CD3/anti-CD28 beads, harvested and western blot analysis performed as in (A). (D) In activated CD4+ T cells, PLD1 is shown to regulate c-Myc through ERK or mTOR-dependent mechanisms. This results in the increased expression of genes essential for uptake of amino acids (box 1), glucose (box 2), and synthesis of nucleotides (box 3).</p

Inhibition of PLD1 activity in activated T cells restricts HIV-1 infection in a dNTP-dependent fashion.

<p>(A) Resting primary CD4⁺ T cells were treated with 10μM PLD1i for 24 h then stimulated for 48 h with 5μg/ml PHA and 20 U/ml IL2. Cells were subsequently infected with a X4-envelope-pseudotyped HIV-1 expressing GFP. Cells were cultured in the continued presence of inhibitor. Three days post-infection, cells were analyzed for GFP expression by FACS. Where indicated, cells were treated with exogenous 50μM deoxyribonucleosides (dN) 6 h before infection. Means and SDs of triplicate samples for four independent donors are shown. Statistical significance was determined by two-tailed Student’s <i>t</i> test. *, <i>P</i> < 0.05; **, <i>P</i> < 0.01; ***, <i>P</i> < 0.001 when PLD1i-treated samples are compared with DMSO or PLD1i + dN-treated compared to PLD1i. (B) Primary CD4+ T cells were treated and infected with X4-envelope-pseudotyped GFP-reporter-expressing HIV-1(HIV-1-GFP) as in (A) and total DNA harvested 24 h after infection. Viral cDNA (ERT, LRT, or 2-LTR circles) was quantified by qPCR. Means and SDs of triplicate samples are shown. Data are representative of three independent donors. (C) Kinetics of reverse transcription and 2-LTR formation was analyzed in CD4+ T cells pre-treated with 10μM PLD1i, 50μM Myci, or DMSO vehicle for 24 h then stimulated and infected with HIV-1-GFP as in (A). Where indicated, cells were treated with 50μM deoxyribonucleosides (dN) or 10μM AZT 6 h before infection. Total DNA was harvested at 8, 16, and 24 h after infection and viral cDNA was quantified as outlined in (B). Means and SDs of triplicate samples are shown. Data are representative of two independent donors. (D) Resting primary CD4⁺ T cells were treated with 10μM PLD1i or DMSO vehicle for 24 h then stimulated and infected with HIV-1-GFP as in (Fig 5A). Where indicated, cells were treated with 50μM deoxyribonucleosides (dN) or 10μM AZT 6 h before infection. Total DNA was harvested at 24 h after infection and viral cDNA was quantified as outlined in (Fig 5B). Means and SDs of triplicate samples are shown. Data are representative of two independent donors.</p