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Immune regulator ABIN1 suppresses HIV-1 transcription by negatively regulating the ubiquitination of Tat

View Article: PubMed Central - PubMed

ABSTRACT

Background: A20-binding inhibitor of NF-κB activation (ABIN1), an important immune regulator, was previously shown to be involved in HIV-1 replication. However, the reported studies done with overexpressed ABIN1 provided controversial results.

Results: Here we identified ABIN1 as a suppressor of HIV-1 transcription since transient knockdown of ABIN1 led to increased HIV-1 replication both in transformed Jurkat T cell line and in primary human CD4+ T lymphocytes. Depletion of ABIN1 specifically enhanced the HIV-1 transcription from the integrated genome during viral life cycle, but not the earlier steps such as reverse transcription or integration. Immunoprecipitation assays revealed that ABIN1 specifically inhibits the proto-oncogene HDM2 catalyzed K63-linked polyubiquitination of Tat at Lys71, which is critical for the transactivation activity of Tat. The ubiquitin chain binding activity of ABIN1 carried by UBAN domain turned out to be essential for the inhibitory role of ABIN1. The results of immunofluorescence localization experiments suggested that ABIN1 may obstruct Tat ubiquitination by redistributing some of HDM2 from the nucleus to the cytoplasm.

Conclusions: Our findings have revealed ABIN1 as intrinsic suppressor of HIV-1 mRNA transcription by regulating the ubiquitination of Tat.

No MeSH data available.


ABIN1 regulates the ubiquitination of Tat by modulating the distribution of HDM2. a HEK-293TABIN1-KO cells were transfected with plasmids encoding Myc-ABIN1 or Myc-QE2, together with Myc-HDM2, Flag-Tat or plasmid vectors as indicated, and subjected to IP as in Fig. 4a. b HeLa cells were transfected with expression vectors encoding or Myc-ABIN1, Myc-QE2, Myc-HDM2, Flag-HDM2 or Flag-Tat individually (upper panel) or in combination (middle and lower panel) as indicated for 24 h. Then cells were washed with PBS and harvested for Immunofluorescence analysis. Mouse anti-Myc and rabbit anti-Flag antibodies were used as primary antibodies, FITC-conjugated goat anti-rabbit IgG and Rhodamine-conjugated goat anti-mouse IgG were used to detect the two proteins. The consensus scale bar was 20 μm
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Fig7: ABIN1 regulates the ubiquitination of Tat by modulating the distribution of HDM2. a HEK-293TABIN1-KO cells were transfected with plasmids encoding Myc-ABIN1 or Myc-QE2, together with Myc-HDM2, Flag-Tat or plasmid vectors as indicated, and subjected to IP as in Fig. 4a. b HeLa cells were transfected with expression vectors encoding or Myc-ABIN1, Myc-QE2, Myc-HDM2, Flag-HDM2 or Flag-Tat individually (upper panel) or in combination (middle and lower panel) as indicated for 24 h. Then cells were washed with PBS and harvested for Immunofluorescence analysis. Mouse anti-Myc and rabbit anti-Flag antibodies were used as primary antibodies, FITC-conjugated goat anti-rabbit IgG and Rhodamine-conjugated goat anti-mouse IgG were used to detect the two proteins. The consensus scale bar was 20 μm

Mentions: How ABIN1 regulates Tat ubiquitination remains to be answered. Since ABIN1 can act neither as E3 ligases to ubiquitinate substrates nor as deubiquitin enzymes to disassembled ubiquitin chains, it may functions indirectly on Tat to regulate its ubiquitination. HDM2 emerged from previous report as the E3 ligase of Tat to promote its polyubiquitination [30]. We then tested whether ABIN1 was able to down-regulate HDM2-promoted ubiquitination of Tat. Myc-HDM2 and Flag-Tat were co-transfected into HEK-293TABIN1-KO cells with Myc-ABIN1 or Myc-ABIN1-QE2 mutant. Flag-Tat was immunoprecipitated under denaturing conditions followed by blotting with ubiquitin antibody. Results showed that wild-type ABIN1 significantly inhibited Tat ubiquitination promoted by HDM2, while ABIN1 QE2 mutant could not (Fig. 7a), supporting the hypothesis that ABIN1 inhibits HDM2-promoted Tat ubiquitination.Fig. 7


Immune regulator ABIN1 suppresses HIV-1 transcription by negatively regulating the ubiquitination of Tat
ABIN1 regulates the ubiquitination of Tat by modulating the distribution of HDM2. a HEK-293TABIN1-KO cells were transfected with plasmids encoding Myc-ABIN1 or Myc-QE2, together with Myc-HDM2, Flag-Tat or plasmid vectors as indicated, and subjected to IP as in Fig. 4a. b HeLa cells were transfected with expression vectors encoding or Myc-ABIN1, Myc-QE2, Myc-HDM2, Flag-HDM2 or Flag-Tat individually (upper panel) or in combination (middle and lower panel) as indicated for 24 h. Then cells were washed with PBS and harvested for Immunofluorescence analysis. Mouse anti-Myc and rabbit anti-Flag antibodies were used as primary antibodies, FITC-conjugated goat anti-rabbit IgG and Rhodamine-conjugated goat anti-mouse IgG were used to detect the two proteins. The consensus scale bar was 20 μm
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Related In: Results  -  Collection

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Fig7: ABIN1 regulates the ubiquitination of Tat by modulating the distribution of HDM2. a HEK-293TABIN1-KO cells were transfected with plasmids encoding Myc-ABIN1 or Myc-QE2, together with Myc-HDM2, Flag-Tat or plasmid vectors as indicated, and subjected to IP as in Fig. 4a. b HeLa cells were transfected with expression vectors encoding or Myc-ABIN1, Myc-QE2, Myc-HDM2, Flag-HDM2 or Flag-Tat individually (upper panel) or in combination (middle and lower panel) as indicated for 24 h. Then cells were washed with PBS and harvested for Immunofluorescence analysis. Mouse anti-Myc and rabbit anti-Flag antibodies were used as primary antibodies, FITC-conjugated goat anti-rabbit IgG and Rhodamine-conjugated goat anti-mouse IgG were used to detect the two proteins. The consensus scale bar was 20 μm
Mentions: How ABIN1 regulates Tat ubiquitination remains to be answered. Since ABIN1 can act neither as E3 ligases to ubiquitinate substrates nor as deubiquitin enzymes to disassembled ubiquitin chains, it may functions indirectly on Tat to regulate its ubiquitination. HDM2 emerged from previous report as the E3 ligase of Tat to promote its polyubiquitination [30]. We then tested whether ABIN1 was able to down-regulate HDM2-promoted ubiquitination of Tat. Myc-HDM2 and Flag-Tat were co-transfected into HEK-293TABIN1-KO cells with Myc-ABIN1 or Myc-ABIN1-QE2 mutant. Flag-Tat was immunoprecipitated under denaturing conditions followed by blotting with ubiquitin antibody. Results showed that wild-type ABIN1 significantly inhibited Tat ubiquitination promoted by HDM2, while ABIN1 QE2 mutant could not (Fig. 7a), supporting the hypothesis that ABIN1 inhibits HDM2-promoted Tat ubiquitination.Fig. 7

View Article: PubMed Central - PubMed

ABSTRACT

Background: A20-binding inhibitor of NF-κB activation (ABIN1), an important immune regulator, was previously shown to be involved in HIV-1 replication. However, the reported studies done with overexpressed ABIN1 provided controversial results.

Results: Here we identified ABIN1 as a suppressor of HIV-1 transcription since transient knockdown of ABIN1 led to increased HIV-1 replication both in transformed Jurkat T cell line and in primary human CD4+ T lymphocytes. Depletion of ABIN1 specifically enhanced the HIV-1 transcription from the integrated genome during viral life cycle, but not the earlier steps such as reverse transcription or integration. Immunoprecipitation assays revealed that ABIN1 specifically inhibits the proto-oncogene HDM2 catalyzed K63-linked polyubiquitination of Tat at Lys71, which is critical for the transactivation activity of Tat. The ubiquitin chain binding activity of ABIN1 carried by UBAN domain turned out to be essential for the inhibitory role of ABIN1. The results of immunofluorescence localization experiments suggested that ABIN1 may obstruct Tat ubiquitination by redistributing some of HDM2 from the nucleus to the cytoplasm.

Conclusions: Our findings have revealed ABIN1 as intrinsic suppressor of HIV-1 mRNA transcription by regulating the ubiquitination of Tat.

No MeSH data available.