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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.


Related in: MedlinePlus

ABIN1 knockdown increases HIV-1 replication. a Jurkat T cells were transfected with siRNAs targeting ABIN1 (siABIN1-1 or siABIN1-2) or scramble siRNA (siNC) as described in “Methods”. 36 h post transfection (hpt), cells were counted and challenged with HIV-1 strain NL4-3. At 1, 3, 5 days post infection, the cells and supernatants were harvested to measure viral production by p24 ELISA of the supernatants, against a standard curve. Cells were lysed and subjected to Western Blots to determine the knockdown efficiency of ABIN1. The lower panel showed the ABIN1 knockdown efficiency at day 5 as the representative. b The experiments were conducted similarly as in (a), except that human primary CD4+ T lymphocytes were used. c, d Determination of ABIN1 mRNA level after viral infection. Jurkat cells were challenged with HIV(NL4-3) as in a, b, at 0, 24, 72 hpi, cells were harvested for RNA extraction. The mRNA levels of ABIN1 and HIV-1 gag was measured by real time PCR, normalized to cellular GAPDH. Data are shown as mean ± SD of triplicate samples and are representative of at least three independent experiments. p values were calculated based on unpaired t test and significant changes relative to siNC transfected cells or samples collected at 0 hpi. *p < 0.05; **p < 0.01; ***p < 0.001
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Fig1: ABIN1 knockdown increases HIV-1 replication. a Jurkat T cells were transfected with siRNAs targeting ABIN1 (siABIN1-1 or siABIN1-2) or scramble siRNA (siNC) as described in “Methods”. 36 h post transfection (hpt), cells were counted and challenged with HIV-1 strain NL4-3. At 1, 3, 5 days post infection, the cells and supernatants were harvested to measure viral production by p24 ELISA of the supernatants, against a standard curve. Cells were lysed and subjected to Western Blots to determine the knockdown efficiency of ABIN1. The lower panel showed the ABIN1 knockdown efficiency at day 5 as the representative. b The experiments were conducted similarly as in (a), except that human primary CD4+ T lymphocytes were used. c, d Determination of ABIN1 mRNA level after viral infection. Jurkat cells were challenged with HIV(NL4-3) as in a, b, at 0, 24, 72 hpi, cells were harvested for RNA extraction. The mRNA levels of ABIN1 and HIV-1 gag was measured by real time PCR, normalized to cellular GAPDH. Data are shown as mean ± SD of triplicate samples and are representative of at least three independent experiments. p values were calculated based on unpaired t test and significant changes relative to siNC transfected cells or samples collected at 0 hpi. *p < 0.05; **p < 0.01; ***p < 0.001

Mentions: The effect of ABIN1 on HIV-1 replication has been previously studied, but the results seem to be controversial and only cell lines such as HEK-293T or HeLa T4 cells transfected with overexpressed ABIN1 were employed during the research. To examine whether ABIN1 is able to inhibit HIV-1 replication under physiological condition, two non-overlapping siRNAs which target ABIN1 open reading frame were introduced into different cells challenged with HIV-1 (NL4-3). The results showed that transient knockdown of ABIN1 led to increased HIV-1 replication not only in Jurkat T cell line (Fig. 1a) but also in primary human CD4+ T lymphocytes, the primary target cells for HIV-1 from healthy donors (Fig. 1b).Fig. 1


Immune regulator ABIN1 suppresses HIV-1 transcription by negatively regulating the ubiquitination of Tat
ABIN1 knockdown increases HIV-1 replication. a Jurkat T cells were transfected with siRNAs targeting ABIN1 (siABIN1-1 or siABIN1-2) or scramble siRNA (siNC) as described in “Methods”. 36 h post transfection (hpt), cells were counted and challenged with HIV-1 strain NL4-3. At 1, 3, 5 days post infection, the cells and supernatants were harvested to measure viral production by p24 ELISA of the supernatants, against a standard curve. Cells were lysed and subjected to Western Blots to determine the knockdown efficiency of ABIN1. The lower panel showed the ABIN1 knockdown efficiency at day 5 as the representative. b The experiments were conducted similarly as in (a), except that human primary CD4+ T lymphocytes were used. c, d Determination of ABIN1 mRNA level after viral infection. Jurkat cells were challenged with HIV(NL4-3) as in a, b, at 0, 24, 72 hpi, cells were harvested for RNA extraction. The mRNA levels of ABIN1 and HIV-1 gag was measured by real time PCR, normalized to cellular GAPDH. Data are shown as mean ± SD of triplicate samples and are representative of at least three independent experiments. p values were calculated based on unpaired t test and significant changes relative to siNC transfected cells or samples collected at 0 hpi. *p < 0.05; **p < 0.01; ***p < 0.001
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Fig1: ABIN1 knockdown increases HIV-1 replication. a Jurkat T cells were transfected with siRNAs targeting ABIN1 (siABIN1-1 or siABIN1-2) or scramble siRNA (siNC) as described in “Methods”. 36 h post transfection (hpt), cells were counted and challenged with HIV-1 strain NL4-3. At 1, 3, 5 days post infection, the cells and supernatants were harvested to measure viral production by p24 ELISA of the supernatants, against a standard curve. Cells were lysed and subjected to Western Blots to determine the knockdown efficiency of ABIN1. The lower panel showed the ABIN1 knockdown efficiency at day 5 as the representative. b The experiments were conducted similarly as in (a), except that human primary CD4+ T lymphocytes were used. c, d Determination of ABIN1 mRNA level after viral infection. Jurkat cells were challenged with HIV(NL4-3) as in a, b, at 0, 24, 72 hpi, cells were harvested for RNA extraction. The mRNA levels of ABIN1 and HIV-1 gag was measured by real time PCR, normalized to cellular GAPDH. Data are shown as mean ± SD of triplicate samples and are representative of at least three independent experiments. p values were calculated based on unpaired t test and significant changes relative to siNC transfected cells or samples collected at 0 hpi. *p < 0.05; **p < 0.01; ***p < 0.001
Mentions: The effect of ABIN1 on HIV-1 replication has been previously studied, but the results seem to be controversial and only cell lines such as HEK-293T or HeLa T4 cells transfected with overexpressed ABIN1 were employed during the research. To examine whether ABIN1 is able to inhibit HIV-1 replication under physiological condition, two non-overlapping siRNAs which target ABIN1 open reading frame were introduced into different cells challenged with HIV-1 (NL4-3). The results showed that transient knockdown of ABIN1 led to increased HIV-1 replication not only in Jurkat T cell line (Fig. 1a) but also in primary human CD4+ T lymphocytes, the primary target cells for HIV-1 from healthy donors (Fig. 1b).Fig. 1

View Article: PubMed Central - PubMed

ABSTRACT

Background: A20-binding inhibitor of NF-&kappa;B 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.


Related in: MedlinePlus