ヒト免疫不全ウイルス1型（HIV-1）Gagタンパク質と宿主因子TSG101との相互作用を標的とする新規HIV薬の開発

学位の種別: 課程博士審査委員会委員 : （主査）東京大学教授 小柳津 広志, 東京大学准教授 葛山 智久, 東京大学准教授 松本 安喜, 理化学研究所ユニットリーダー 間 陽子, 東京大学准教授 栁澤 修一University of Tokyo(東京大学

Distinct MCM10 Proteasomal Degradation Profiles by Primate Lentiviruses Vpr Proteins

Viral protein R (Vpr) is an accessory protein found in various primate lentiviruses, including human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2) as well as simian immunodeficiency viruses (SIVs). Vpr modulates many processes during viral lifecycle via interaction with several of cellular targets. Previous studies showed that HIV-1 Vpr strengthened degradation of Mini-chromosome Maintenance Protein10 (MCM10) by manipulating DCAF1-Cul4-E3 ligase in proteasome-dependent pathway. However, whether Vpr from other primate lentiviruses are also associated with MCM10 degradation and the ensuing impact remain unknown. Based on phylogenetic analyses, a panel of primate lentiviruses Vpr/x covering main virus lineages was prepared. Distinct MCM10 degradation profiles were mapped and HIV-1, SIVmus and SIVrcm Vprs induced MCM10 degradation in proteasome-dependent pathway. Colocalization and interaction between MCM10 with these Vprs were also observed. Moreover, MCM10 2-7 interaction region was identified as a determinant region susceptible to degradation. However, MCM10 degradation did not alleviate DNA damage response induced by these Vpr proteins. MCM10 degradation by HIV-1 Vpr proteins was correlated with G2/M arrest, while induction of apoptosis and oligomerization formation of Vpr failed to alter MCM10 proteolysis. The current study demonstrated a distinct interplay pattern between primate lentiviruses Vpr proteins and MCM10

Vpr prevents TSG101-induced Gag accumulation at perinuclear region.

<p>(A) Schematic showing the binding motifs for TSG101 (PTAP; green) and Vpr (FRFG, ELY, and LXSLFG; red) within the Gag p6 domain. (B) HeLa cells were co-transfected with 0.8 μg of pCAGGS/Gag and 1.5 μg of pCAGGS/eCFP-TSG101 either without/with 0.5 μg of pCAGGS/HA-Vpr or 0.5 μg of pCAGGS/Vif-HA for 48 h prior to immunofluorescence staining with an anti-Gag and anti-HA antibodies, followed by an Alexa Fluor 594 goat anti-rabbit antibody and an Alexa Fluor 633 goat anti-mouse antibody. Image acquisition was performed under a confocal laser-scanning microscope. (C) Co-localization of Gag/TSG101 was analyzed by Pearson’s correlation coefficients. Data represent the means ± SD of the result of two independent experiments. *, P < 0.05 (unpaired t-test). (D-E) HeLa cells were co-transfected with 1.5 μg of pCAGGS/eCFP-TSG101 or 0.8 μg of pCAGGS/Gag-Venus (D) or 1.5 μg of pCAGGS/eCFP-TSG101 and 0.8 μg of pCAGGS/Gag-Venus either without/with 0.5 μg of pCAGGS/mRFP-Vpr (E) for 48 h before fixation and image acquisition. The precision FRET (PFRET) signal was analyzed using the sensitized emission method. The FRET (Ex.eCFP/Em.Venus) and PFRET image colors were converted by Hi/Lo function of FV10-ASW v.2.1 software (Olympus) to facilitate visualization of Gag/TSG101 co-localization. (F) FRET ratio (PFRET signal divided by the donor eCFP-TSG101 signal) of Gag/TSG101 co-localization in the absence or presence of Vpr. Data represent means ± SD of the result of one representative experiment from two independent performs. *, P < 0.05 (unpaired t-test).</p

HIV-1 Vpr Abrogates the Effect of TSG101 Overexpression to Support Virus Release

<div><p>HIV-1 budding requires interaction between Gag and cellular TSG101 to initiate viral particle assembly and release via the endosomal sorting complexes required for transport (ESCRT) pathway. However, some reports show that overexpression of TSG101 inhibits virus release by disruption of Gag targeting process. Since a HIV-1 accessory protein, Vpr binds to Gag p6 domain at the position close to the binding site for TSG101, whether Vpr implicates TSG101 overexpression effect has not been investigated. Here, we found that Vpr abrogates TSG101 overexpression effect to rescue viral production. Co-transfection of TSG101 and Gag with Vpr prevented TSG101-induced Gag accumulation in endosomes and lysosomes. In addition, Vpr rescued virus-like particle (VLP) production in a similar manner as a lysosomal inhibitor, Bafilomycin A1 indicating that Vpr inhibits TSG101-induced Gag downregulation via lysosomal pathway. Vpr and Gag interaction is required to counteract TSG101 overexpression effect since Vpr A30F mutant which is unable to interact with Gag and incorporate into virions, reduced ability to prevent Gag accumulation and to rescue VLP production. In addition, GST pull-down assays and Biacore analysis revealed that Vpr competed with TSG101 for Gag binding. These results indicate that Vpr overcomes the effects of TSG101 overexpression to support viral production by competing with TSG101 to bind Gag.</p></div

An <i>Azorhizobium caulinodans</i> ORS571 mutant with deletion of a gene encoding a TIGR02302 family protein overproduces exopolysaccharides and is defective in infection into plant host cells

<p><i>Azorhizobium caulinodans</i> is a microsymbiont of <i>Sesbania rostrata</i> Bremek. & Oberm., and is able to fix nitrogen in both the free-living and symbiotic states. In this study, we focused on the <i>ggm</i> gene (locus tag, AZC_4606) that encodes a putative membrane protein belonging to the TIGR02302 family. Although the genes encoding TIGR02302 family protein are distributed in a wide range of alpha-proteobacteria including rhizobia, the functions of this protein are still unknown. To investigate the functions of this protein in <i>A. caulinodans</i>, we made a <i>ggm</i> mutant, and analyzed its phenotypes. The <i>ggm</i> mutant produced more bubbles than the wild-type strain in L3 + N medium liquid cultures, and formed mucoid colonies on L3 + N medium agar plates, suggesting that the <i>ggm</i> mutant overproduced exopolysaccharides (EPSs). The amounts of EPSs produced by the <i>ggm</i> mutant on L3 + N plates were about 1.3-fold higher than those by the wild-type strain, and expression levels of EPS production-related genes in the <i>ggm</i> mutant grown in L3 + N liquid medium were about 2- to 4-fold higher than those of the wild-type strain. In addition, the stem nodules formed by the <i>ggm</i> mutant on the stems of <i>S. rostrata</i> showed little or no nitrogen-fixing activity. By microscopic analyses, large infection pockets and a few infected cells were observed in the stem nodules formed by <i>ggm</i> mutant, suggesting that the <i>ggm</i> mutant is defective in invasion into plant cells. Taken together, our results suggest that Ggm is involved in EPS production and that adequate levels of EPS production are required for <i>A. caulinodans</i> to invade into host cells.</p

Vpr rescues the TSG101-mediated inhibition of viral particle release.

<p>(A) HeLa cells were transfected with 0.3 μg of pNL43 Luc E^- R⁺ (Vpr⁺) or pNL43 Luc E^- R^- (Vpr^-) either without/with 0.1 μg of pCAGGS/mRFP-TSG101 and 0.1 μg of pcDNA/YFP-Vpr for 16 h prior to immunofluorescence staining with an anti-Gag AG3.0 antibody followed by a FITC-conjugated sheep anti-mouse antibody. White arrows indicate Gag/TSG101 co-localized on/near the plasma membrane observed by total internal reflection fluorescence (TIRF) microscopy. Data represents the result of one representative experiment from two independent performs. (B-C) HEK293T cells were transfected with 0.2 μg of pNL43 Luc E^- R⁺ (Vpr⁺) (B) or pNL43 Luc E^- R^- (Vpr^-) (C), together with 1 μg of pCAGGS/FLAG-TSG101 and different amounts of pCAGGS/Vpr for 48 h. Viral particles in the culture medium were collected by PEG precipitation. Whole cell lysates were prepared and samples were subjected to western blotting with anti-Gag, anti-Vpr, anti-FLAG, and anti-β-actin antibodies. Data represents the result of one representative experiment from two independent performs.</p

Vpr and Gag interaction is required to abrogate the effect of TSG101 overexpression.

<p>(A) HeLa cells were co-transfected with 0.8 μg of pCAGGS/Gag and 1.5 μg of pCAGGS/eCFP-TSG101 either without/with 0.5 μg of pCAGGS/HA-Vpr or pCAGGS/HA-Vpr A30F for 48 h prior to immunofluorescence staining with an anti-Gag and anti-HA antibodies, followed by an Alexa Fluor 594 goat anti-rabbit antibody and an Alexa Fluor 633 goat anti-mouse antibody. Co-localization of Gag/TSG101 was analyzed by Pearson’s correlation coefficients (B). Data represent the means ± SD of the result of two independent experiments. *, P < 0.05 (unpaired t-test). (C) HEK293T cells were transfected with 0.8 μg of pCAGGS/Gag, or with 0.8 μg of pCAGGS/Gag plus 1.5 μg of pCAGGS/FLAG-TSG101 either without/with different amounts of pCAGGS/Vpr A30F or 0.08 μg of pCAGGS/Vpr (positive control). After 48 h, VLPs in the cultured medium were collected by a 20% sucrose cushion. Whole cell lysates were prepared and samples were subjected to western blot analysis with anti-Gag, anti-Vpr, anti-FLAG, and anti-β-actin antibodies. The bottom panel represents intensity of VLP Gag from western blot analysis. Data represent the result of one representative experiment from two independent performs.</p

Vpr competes with TSG101 for binding to Gag.

<p>(A) GST or GST-Gag protein were immobilized on Glutathione Sepharose beads prior to overnight incubation with purified FLAG-TSG101 protein in the absence/presence of 48.3 μg/ml of PTAP short peptide (a positive control which targets the UEV domain of TSG101), 48.3 μg/ml of BSA (negative control), or 2.0 or 4.0 μg/ml of purified HA-Vpr protein. The beads were pulled-down, washed, and subjected to western blotting with anti-FLAG and anti-GST antibodies. The bottom panel represents mean ± SD intensity values of Gag-bound TSG101 from western blot analysis of four independent performs. *, P < 0.05 (unpaired t-test). (B) GST-Gag was co-incubated with purified FLAG-TSG101 and 48.3 μg/ml of PTAP short peptide or 2 μg/ml of HA-Vpr protein (competitive incubation, c.i.), or allowed to bind with PTAP short peptide or HA-Vpr protein for 3 h before co-incubation with FLAG-TSG101 protein (pre-incubation, p.i.). After overnight incubation, the beads were pulled-down, washed, and subjected to western blotting with anti-FLAG and anti-GST antibodies. The bottom panel represents intensity of Gag-bound TSG101 from western blot analysis of two independent performs. (C-D) GST-Gag protein was immobilized on Biacore sensor chip CM5 and the surface was injected with purified FLAG-TSG101 or FLAG-Vpr protein at different concentrations by Biacore T100 instrument. Kinetic sensorgrams of Gag-bound TSG101 at 0, 15.6, 31.3, 62.5, and 500.0 nM (C, left panel) and Gag-bound Vpr at 0, 15.6, 31.3, 62.5, 125.0, and 250.0 nM (C, right panel) are showed. (D) Steady stage affinity fitting curves of Gag/TSG101 (left panel) and Gag/Vpr (right panel) binding at equilibrium (4 s before injection stop). Black and red vertical lines represent equilibrium dissociation constant (KD) of Gag/TSG101 and Gag/Vpr binding affinity, representatively. Data represents the result of one representative experiment from two independent performs.</p