27 research outputs found
Progress Toward a Human CD4/CCR5 Transgenic Rat Model for De Novo Infection by Human Immunodeficiency Virus Type 1
The development of a permissive small animal model for the study of human immunodeficiency virus type (HIV)-1 pathogenesis and the testing of antiviral strategies has been hampered by the inability of HIV-1 to infect primary rodent cells productively. In this study, we explored transgenic rats expressing the HIV-1 receptor complex as a susceptible host. Rats transgenic for human CD4 (hCD4) and the human chemokine receptor CCR5 (hCCR5) were generated that express the transgenes in CD4+ T lymphocytes, macrophages, and microglia. In ex vivo cultures, CD4+ T lymphocytes, macrophages, and microglia from hCD4/hCCR5 transgenic rats were highly susceptible to infection by HIV-1 R5 viruses leading to expression of abundant levels of early HIV-1 gene products comparable to those found in human reference cultures. Primary rat macrophages and microglia, but not lymphocytes, from double-transgenic rats could be productively infected by various recombinant and primary R5 strains of HIV-1. Moreover, after systemic challenge with HIV-1, lymphatic organs from hCD4/hCCR5 transgenic rats contained episomal 2–long terminal repeat (LTR) circles, integrated provirus, and early viral gene products, demonstrating susceptibility to HIV-1 in vivo. Transgenic rats also displayed a low-level plasma viremia early in infection. Thus, transgenic rats expressing the appropriate human receptor complex are promising candidates for a small animal model of HIV-1 infection
PARP2 Is the Predominant Poly(ADP-Ribose) Polymerase in Arabidopsis DNA Damage and Immune Responses
<div><p>Poly (ADP-ribose) polymerases (PARPs) catalyze the transfer of multiple poly(ADP-ribose) units onto target proteins. Poly(ADP-ribosyl)ation plays a crucial role in a variety of cellular processes including, most prominently, auto-activation of PARP at sites of DNA breaks to activate DNA repair processes. In humans, PARP1 (the founding and most characterized member of the PARP family) accounts for more than 90% of overall cellular PARP activity in response to DNA damage. We have found that, in contrast with animals, in <i>Arabidopsis thaliana</i> PARP2 (At4g02390), rather than PARP1 (At2g31320), makes the greatest contribution to PARP activity and organismal viability in response to genotoxic stresses caused by bleomycin, mitomycin C or gamma-radiation. Plant PARP2 proteins carry SAP DNA binding motifs rather than the zinc finger domains common in plant and animal PARP1 proteins. PARP2 also makes stronger contributions than PARP1 to plant immune responses including restriction of pathogenic <i>Pseudomonas syringae</i> pv. <i>tomato</i> growth and reduction of infection-associated DNA double-strand break abundance. For poly(ADP-ribose) glycohydrolase (PARG) enzymes, we find that Arabidopsis PARG1 and not PARG2 is the major contributor to poly(ADP-ribose) removal from acceptor proteins. The activity or abundance of PARP2 is influenced by PARP1 and PARG1. PARP2 and PARP1 physically interact with each other, and with PARG1 and PARG2, suggesting relatively direct regulatory interactions among these mediators of the balance of poly(ADP-ribosyl)ation. As with plant PARP2, plant PARG proteins are also structurally distinct from their animal counterparts. Hence core aspects of plant poly(ADP-ribosyl)ation are mediated by substantially different enzymes than in animals, suggesting the likelihood of substantial differences in regulation.</p></div
Arabidopsis <i>parp</i> mutants are compromised in basal resistance.
<p>(A) Bacterial population sizes of <i>Pst</i> within leaves. <i>Pst</i> DC3000 strains with or without <i>avrRpt2</i> were syringe infiltrated into leaf mesophyll at 1×10<sup>5</sup> cfu/ml and bacterial populations were measured 3 d post-inoculation. Mean ± standard error of mean for one experiment shown. Experiments were performed three times with similar results; * indicates significant difference from Col-0 across the three experiments (ANOVA, Tukey pairwise comparisons, <i>P</i> < 0.05). (B) Flg22-induced callose deposition. Seedlings exposed to 1 μM flg22 for 24 h were fixed and stained with aniline blue to highlight callose deposition. Left panel: representative images of the four genotypes; right panel: data summary for all tested leaves (n = 24 per genotype). * indicates significant difference from Col-0 across the three experiments (ANOVA, Tukey pairwise comparisons, <i>P</i> < 0.05). (C) Seedling growth inhibition due to chronic flg22-induced defense activation. Ratio is weight of individual seedlings grown for 14 d in liquid MS media + 1 μM flg22, divided by mean of seedlings of same genotype grown without flg22 within same experiment (mean ± standard error of mean). * indicates significant difference from Col-0 across the three experiments (ANOVA, Tukey pairwise comparisons, <i>P</i> < 0.05). (D) Flg22-triggered oxidative burst. Reactive oxygen species from leaf discs of the indicated genotype were measured for 30 min after treatment with 1 μM flg22. RLU: relative luminescence units. (E) <i>Pst</i>-induced γ-H2AX accumulation. Arabidopsis plants of the indicated genotype were vacuum-infiltrated with the indicated <i>Pst</i> strain at 1×10<sup>7</sup> cfu/ml. The level of γ-H2AX in leaf samples from the indicated time points after inoculation was assessed by immunoblot using anti-γ-H2AX antibody. Equivalent loading of lanes was verified using Ponceau S stain. Upper and lower blots are from separate experiments. Similar results obtained in two separate experiments.</p
PARP2 plays a dominant role in response to ionizing irradiation.
<p>(A) Two-week old Arabidopsis plants grown on MS plates were irradiated with 150 Gy of γ-radiation and then flash-frozen 20, 40 or 60 min after removal from the radiation source. Total proteins were then extracted, separated by SDS-PAGE and analyzed by immunoblotting with an anti-PAR antibody. 0 min sample not exposed to γ-radiation source. All samples shown in (A) were processed in parallel within the same experiment. (B) The level of γ-H2AX was assessed at 20, 40 and 60 min after irradiation as in (A), using an anti-γ-H2AX antibody. Samples all processed in parallel from same experiment. Shorter and longer time exposures of same immunoblot are shown. Equivalent loading of lanes was verified using Ponceau S stain. Experiments were performed three times with similar results.</p
Subcellular localization of Arabidopsis PARP1/2 and PARG1/2.
<p>Paired confocal fluorescence microscopy images show same sample; green wavelengths (GFP) on left and red wavelengths (chlorophyll) on right. (A) Arabidopsis PARP1 and PARP2 localized in the nucleus. <i>35S</i>:<i>AtPARP1-GFP</i> and <i>35S</i>:<i>AtPARP2-GFP</i> transiently expressed in <i>N</i>. <i>benthamiana</i> epidermal cells within leaves were imaged. (B) PARP1 and PARP2 localized in the nucleus in Arabidopsis. Subcellular localization was carried out in stable transgenic lines carrying <i>35S</i>:<i>AtPARP1-GFP</i> and <i>35S</i>:<i>AtPARP2-GFP</i> in the wild-type Col-0 background. (C) Arabidopsis PARG1 and PARG2 localized in the cytoplasm and nucleus. <i>35S</i>:<i>AtPARG1-GFP</i> and <i>35S</i>:<i>AtPARG2-GFP</i> were transiently expressed in <i>N</i>. <i>benthamiana</i> and the images were taken 2 d after inoculation.</p
PARG1 is more active than PARG2 in removal of poly(ADP-ribosyl)ation after bleomycin treatment.
<p>Two-week-old <i>parg1-2</i>, <i>parg2-1</i> and Col-0 Arabidopsis plants were treated with 2.5 μg/ml bleomycin and samples were collected at indicated times. Total proteins were extracted, separated by SDS-PAGE and analyzed by immunoblotting with anti-PAR (A) or anti-PARP2 (B) antibody. Equivalent loading of lanes was verified using Ponceau S stain. Similar results obtained in two separate experiments.</p
Interactions between PARPs and PARGs.
<p>(A) PARP1 associates <i>in vivo</i> with PARP2. (B) PARG1 associates <i>in vivo</i> with PARG2. (C) PARP1 associates <i>in vivo</i> with PARG1 and PARG2. (D) PARP2 associates <i>in vivo</i> with PARG1 and PARG2. The indicated proteins were transiently expressed in <i>N</i>. <i>benthamiana</i>. Input lanes were loaded with total protein extracts, IP lanes were loaded with immunoprecipitation products. Immunoprecipitations were performed with anti-GFP antibodies and immunoblots were analyzed with anti-GFP or anti-myc antibodies, as noted. These experiments were repeated twice with similar results.</p
A transcriptomics approach uncovers novel roles for poly(ADP-ribosyl)ation in the basal defense response in Arabidopsis thaliana.
Pharmacological inhibition of poly(ADP-ribose) polymerase (PARP) or loss of Arabidopsis thaliana PARG1 (poly(ADP-ribose) glycohydrolase) disrupt a subset of plant defenses. In the present study we examined the impact of altered poly(ADP-ribosyl)ation on early gene expression induced by the microbe-associate molecular patterns (MAMPs) flagellin (flg22) and EF-Tu (elf18). Stringent statistical analyses and filtering identified 178 genes having MAMP-induced mRNA abundance patterns that were altered by either PARP inhibitor 3-aminobenzamide (3AB) or PARG1 knockout. From the identified set of 178 genes, over fifty Arabidopsis T-DNA insertion lines were chosen and screened for altered basal defense responses. Subtle alterations in callose deposition and/or seedling growth in response to those MAMPs were observed in knockouts of At3g55630 (FPGS3, a cytosolic folylpolyglutamate synthetase), At5g15660 (containing an F-box domain), At1g47370 (a TIR-X (Toll-Interleukin Receptor domain)), and At5g64060 (a predicted pectin methylesterase inhibitor). Over-represented GO terms for the gene expression study included "innate immune response" for elf18/parg1, highlighting a subset of elf18-activated defense-associated genes whose expression is altered in parg1 plants. The study also allowed a tightly controlled comparison of early mRNA abundance responses to flg22 and elf18 in wild-type Arabidopsis, which revealed many differences. The PARP inhibitor 3-methoxybenzamide (3MB) was also used in the gene expression profiling, but pleiotropic impacts of this inhibitor were observed. This transcriptomics study revealed targets for further dissection of MAMP-induced plant immune responses, impacts of PARP inhibitors, and the molecular mechanisms by which poly(ADP-ribosyl)ation regulates plant responses to MAMPs
Experimental design.
<p><b>A.</b> Three biological replicate pools of 48 ten day-old wild-type (Col-0) seedlings were pre-treated for two hours with either 3AB, 3-MB, or vehicle (DMSO), and then treated for one hour with either flg22 flagellin peptide or sterile H<sub>2</sub>O. <b>B.</b> Three biological replicate pools of 48 ten day-old wild-type (Col-0) or <i>parg1-2</i> knockout seedlings were treated for one hour with either elf18 EF-TU peptide or sterile H<sub>2</sub>O.</p
Gene ontology over-representation in genes differentially regulated by either flg22 or elf18.
<p>Gene ontology over-representation in genes differentially regulated by either flg22 or elf18.</p