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Interactions with DCAF1 and DDB1 in the CRL4 E3 ubiquitin ligase are required for Vpr-mediated G2 arrest

Yoshiyuki Hakata12*, Masaaki Miyazawa2 and Nathaniel R Landau1

Author Affiliations

1 Department of Microbiology, New York University School of Medicine, 522 First Avenue, New York, NY 10016, USA

2 Department of Immunology, Kinki University Faculty of Medicine, 377-2 Ohno-Higashi, Osaka-Sayama, Osaka 589-8511, Japan

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Virology Journal 2014, 11:108  doi:10.1186/1743-422X-11-108

Published: 9 June 2014

Additional files

Additional file 1: Figure S1:

Association of VprQ65R mutant with DCAF1 and DDB1. 293T cells were cotransfected with HA-Vpr or HA-VpQ65R and Flag-DCAF1 expression vectors. Vpr was immunoprecipitated with anti-HA antibody and the immunoprecipitates were subjected to immunoblot analysis with anti-Flag MAb, anti-DDB1 antibody, and anti-HA antibody.

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Additional file 2: Figure S2:

Cell cycle analysis of VprR90K and VprR90D mutants. (A) 293T cells were transfected with HIV-1 Vpr or Vpr mutant, Flag-DCAF1, and EGFP expression vectors. After staining with propidium iodide, the cells were analyzed by flow cytometry. The G2:G1 ratio was calculated after gating for the GFP+ cells. The results are representative of three independent experiments. (B) A portion of the cells used in (A) was subjected to an immunoblot analysis to confirm Vpr and Vpr mutants expression. The βtubulin was a loading control.

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Additional file 3: Figure S3:

Colocalization of VprR90K with DDB1. HeLa cells were transfected with HA-Vpr or HA-VprR90K and Flag-DCAF1 expression vectors. The transfected cells were permeabilized, fixed, and then incubated with anti-DDB1 and anti-HA antibodies followed by Alexa Fluor 594-anti-rabbit IgG and Alexa Fluor 488-anti-rat IgG. The percentage of Vpr foci colocalized with DDB1 among total Vpr foci was calculated. More than 380 Vpr foci were evaluated for each sample and three independent experiments were done. The data are the mean values with standard deviations. P values were calculated by the Student’s t-test with P < 0.05 considered significant.

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Additional file 4: Figure S4:

SIVagm HA-Vpr shows a defect in UNG2 degradation. 293T cells were transfected with increasing amounts (0.05 μg, 0.1 μg, and 0.2 μg) of HA-Vpr or SIVagm HA-Vpr expression vector together with a constant amount of HA-UNG2 and Flag-DCAF1 expression vectors. The cells were lysed, and then Vpr and UNG2 were detected by immunoblot analysis with anti-HA antibody. The βtubulin was a loading control.

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Additional file 5: Figure S5:

Phosphorylation of H2AX by Vpr. HeLa cells (1 x 105) were transfected with 0.1 μg of HA-Vpr or SIVagm HA-Vpr expression vector. Twenty-four hours after transfection, the cells were lysed in sample buffer. Vpr and γH2AX in the cell lysate were detected by immunoblot analysis. The βtubulin was a loading control.

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