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The interferon response inhibits HIV particle production by induction of TRIM22.

Barr SD, Smiley JR, Bushman FD - PLoS Pathog. (2008)

Bottom Line: To assess the role of TRIM22 expressed under natural inducing conditions, we compared the effects of interferon in cells depleted for TRIM22 using RNAi and found that HIV particle release was significantly increased in the knockdown, implying that TRIM22 acts as a natural antiviral effector.TRIM22 did not block the release of MLV or EIAV Gag particles.Inhibition was associated with diffuse cytoplasmic staining of HIV Gag rather than accumulation at the plasma membrane, suggesting TRIM22 disrupts proper trafficking.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology and Immunology, University of Alberta, Alberta Institute for Viral Immunology, Edmonton, Alberta, Canada. stephen.barr@ualberta.ca

ABSTRACT
Treatment of human cells with Type 1 interferons restricts HIV replication. Here we report that the tripartite motif protein TRIM22 is a key mediator. We used transcriptional profiling to identify cellular genes that were induced by interferon treatment and identified TRIM22 as one of the most strongly up-regulated genes. We confirmed, as in previous studies, that TRIM22 over-expression inhibited HIV replication. To assess the role of TRIM22 expressed under natural inducing conditions, we compared the effects of interferon in cells depleted for TRIM22 using RNAi and found that HIV particle release was significantly increased in the knockdown, implying that TRIM22 acts as a natural antiviral effector. Further studies showed that TRIM22 inhibited budding of virus-like particles containing Gag only, indicating that Gag was the target of TRIM22. TRIM22 did not block the release of MLV or EIAV Gag particles. Inhibition was associated with diffuse cytoplasmic staining of HIV Gag rather than accumulation at the plasma membrane, suggesting TRIM22 disrupts proper trafficking. Mutational analyses of TRIM22 showed that the catalytic amino acids Cys15 and Cys18 of the RING domain are required for TRIM22 antiviral activity. These data disclose a pathway by which Type 1 interferons obstruct HIV replication.

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Knockdown of TRIM22 by RNA interference abrogates interferon-induced inhibition of HIV replication.A) HOS-CD4/CXCR4 cells were transfected with the empty vector control plasmid pLKO.1 or pLKO.1/TRIM22 shRNA#1 followed by treatment with 0, 50, 500 or 1000 units/ml of IFNβ (represented by triangles at the top of the Northern blots). Total RNA was extracted and subjected to Northern blot analysis to detect TRIM22 RNA transcripts. TRIM34 (variant 3) RNA transcript levels were also detected for a comparison (middle row). Input RNA was normalized using Northern blots probed with an actin fragment (bottom row). B) Quantitation of the Northern blots in A, indicating the fold induction of TRIM22 RNA in response to the IFNβ treatment. C) Cells were transfected with the empty vector pLKO.1 or pLKO.1/TRIM22shRNA#1 and treated with 1000 units/ml of IFNβ. Cells were then transfected with pR9 and Western blot analysis of HIV capsid antigen in cells and supernatants was analyzed using an anti-p24 antibody. D) Cells were transfected with the control plasmids pLKO.1, pLKO.1/eGFPshRNA or pLKO.1/scrambledshRNA or TRIM22 shRNA plasmids pLKO.1/TRIM22shRNA#1 and pLKO.1/TRIM22shRNA#2 and treated with 1000 units/ml of IFNβ. Following transfection with pR9, HIV capsid antigen released into the supernatant was detected by Western blot and quantified densitometrically. Data represents the averages (+/−StdDev) of at least 3 experiments performed in triplicate. E) A comparison of the average fold inhibition in particle release of the pooled control shRNAs and the pooled TRIM22 shRNAs (+/−StdDev). *, P = 0.0002 (Mann-Whitney).
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ppat-1000007-g003: Knockdown of TRIM22 by RNA interference abrogates interferon-induced inhibition of HIV replication.A) HOS-CD4/CXCR4 cells were transfected with the empty vector control plasmid pLKO.1 or pLKO.1/TRIM22 shRNA#1 followed by treatment with 0, 50, 500 or 1000 units/ml of IFNβ (represented by triangles at the top of the Northern blots). Total RNA was extracted and subjected to Northern blot analysis to detect TRIM22 RNA transcripts. TRIM34 (variant 3) RNA transcript levels were also detected for a comparison (middle row). Input RNA was normalized using Northern blots probed with an actin fragment (bottom row). B) Quantitation of the Northern blots in A, indicating the fold induction of TRIM22 RNA in response to the IFNβ treatment. C) Cells were transfected with the empty vector pLKO.1 or pLKO.1/TRIM22shRNA#1 and treated with 1000 units/ml of IFNβ. Cells were then transfected with pR9 and Western blot analysis of HIV capsid antigen in cells and supernatants was analyzed using an anti-p24 antibody. D) Cells were transfected with the control plasmids pLKO.1, pLKO.1/eGFPshRNA or pLKO.1/scrambledshRNA or TRIM22 shRNA plasmids pLKO.1/TRIM22shRNA#1 and pLKO.1/TRIM22shRNA#2 and treated with 1000 units/ml of IFNβ. Following transfection with pR9, HIV capsid antigen released into the supernatant was detected by Western blot and quantified densitometrically. Data represents the averages (+/−StdDev) of at least 3 experiments performed in triplicate. E) A comparison of the average fold inhibition in particle release of the pooled control shRNAs and the pooled TRIM22 shRNAs (+/−StdDev). *, P = 0.0002 (Mann-Whitney).

Mentions: The above data and results in [27] indicated that ectopic expression of TRIM22 can interfere with HIV replication, but from over-expression data alone it is uncertain whether expression at physiological levels would result in antiviral activity. For this reason, we tested whether depleting TRIM22 using short hairpin ribonucleic acid (shRNA) in the context of an interferon response allowed increased production of extracellular HIV particles. HOS-CD4/CXCR4 cells were transiently transfected for 24 hours with either pLKO.1/TRIM22shRNA#1 or the empty vector control (pLKO.1). In control cells, TRIM22 RNA accumulation increased in the presence of increasing concentrations of IFNβ. In the cells expressing the TRIM22 shRNA, TRIM22 RNA levels were significantly reduced (Figure 3A and B). As a control, expression of the related TRIM34 gene was tested and found to be unaffected by the TRIM22 shRNA (Figure 3A).


The interferon response inhibits HIV particle production by induction of TRIM22.

Barr SD, Smiley JR, Bushman FD - PLoS Pathog. (2008)

Knockdown of TRIM22 by RNA interference abrogates interferon-induced inhibition of HIV replication.A) HOS-CD4/CXCR4 cells were transfected with the empty vector control plasmid pLKO.1 or pLKO.1/TRIM22 shRNA#1 followed by treatment with 0, 50, 500 or 1000 units/ml of IFNβ (represented by triangles at the top of the Northern blots). Total RNA was extracted and subjected to Northern blot analysis to detect TRIM22 RNA transcripts. TRIM34 (variant 3) RNA transcript levels were also detected for a comparison (middle row). Input RNA was normalized using Northern blots probed with an actin fragment (bottom row). B) Quantitation of the Northern blots in A, indicating the fold induction of TRIM22 RNA in response to the IFNβ treatment. C) Cells were transfected with the empty vector pLKO.1 or pLKO.1/TRIM22shRNA#1 and treated with 1000 units/ml of IFNβ. Cells were then transfected with pR9 and Western blot analysis of HIV capsid antigen in cells and supernatants was analyzed using an anti-p24 antibody. D) Cells were transfected with the control plasmids pLKO.1, pLKO.1/eGFPshRNA or pLKO.1/scrambledshRNA or TRIM22 shRNA plasmids pLKO.1/TRIM22shRNA#1 and pLKO.1/TRIM22shRNA#2 and treated with 1000 units/ml of IFNβ. Following transfection with pR9, HIV capsid antigen released into the supernatant was detected by Western blot and quantified densitometrically. Data represents the averages (+/−StdDev) of at least 3 experiments performed in triplicate. E) A comparison of the average fold inhibition in particle release of the pooled control shRNAs and the pooled TRIM22 shRNAs (+/−StdDev). *, P = 0.0002 (Mann-Whitney).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2279259&req=5

ppat-1000007-g003: Knockdown of TRIM22 by RNA interference abrogates interferon-induced inhibition of HIV replication.A) HOS-CD4/CXCR4 cells were transfected with the empty vector control plasmid pLKO.1 or pLKO.1/TRIM22 shRNA#1 followed by treatment with 0, 50, 500 or 1000 units/ml of IFNβ (represented by triangles at the top of the Northern blots). Total RNA was extracted and subjected to Northern blot analysis to detect TRIM22 RNA transcripts. TRIM34 (variant 3) RNA transcript levels were also detected for a comparison (middle row). Input RNA was normalized using Northern blots probed with an actin fragment (bottom row). B) Quantitation of the Northern blots in A, indicating the fold induction of TRIM22 RNA in response to the IFNβ treatment. C) Cells were transfected with the empty vector pLKO.1 or pLKO.1/TRIM22shRNA#1 and treated with 1000 units/ml of IFNβ. Cells were then transfected with pR9 and Western blot analysis of HIV capsid antigen in cells and supernatants was analyzed using an anti-p24 antibody. D) Cells were transfected with the control plasmids pLKO.1, pLKO.1/eGFPshRNA or pLKO.1/scrambledshRNA or TRIM22 shRNA plasmids pLKO.1/TRIM22shRNA#1 and pLKO.1/TRIM22shRNA#2 and treated with 1000 units/ml of IFNβ. Following transfection with pR9, HIV capsid antigen released into the supernatant was detected by Western blot and quantified densitometrically. Data represents the averages (+/−StdDev) of at least 3 experiments performed in triplicate. E) A comparison of the average fold inhibition in particle release of the pooled control shRNAs and the pooled TRIM22 shRNAs (+/−StdDev). *, P = 0.0002 (Mann-Whitney).
Mentions: The above data and results in [27] indicated that ectopic expression of TRIM22 can interfere with HIV replication, but from over-expression data alone it is uncertain whether expression at physiological levels would result in antiviral activity. For this reason, we tested whether depleting TRIM22 using short hairpin ribonucleic acid (shRNA) in the context of an interferon response allowed increased production of extracellular HIV particles. HOS-CD4/CXCR4 cells were transiently transfected for 24 hours with either pLKO.1/TRIM22shRNA#1 or the empty vector control (pLKO.1). In control cells, TRIM22 RNA accumulation increased in the presence of increasing concentrations of IFNβ. In the cells expressing the TRIM22 shRNA, TRIM22 RNA levels were significantly reduced (Figure 3A and B). As a control, expression of the related TRIM34 gene was tested and found to be unaffected by the TRIM22 shRNA (Figure 3A).

Bottom Line: To assess the role of TRIM22 expressed under natural inducing conditions, we compared the effects of interferon in cells depleted for TRIM22 using RNAi and found that HIV particle release was significantly increased in the knockdown, implying that TRIM22 acts as a natural antiviral effector.TRIM22 did not block the release of MLV or EIAV Gag particles.Inhibition was associated with diffuse cytoplasmic staining of HIV Gag rather than accumulation at the plasma membrane, suggesting TRIM22 disrupts proper trafficking.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology and Immunology, University of Alberta, Alberta Institute for Viral Immunology, Edmonton, Alberta, Canada. stephen.barr@ualberta.ca

ABSTRACT
Treatment of human cells with Type 1 interferons restricts HIV replication. Here we report that the tripartite motif protein TRIM22 is a key mediator. We used transcriptional profiling to identify cellular genes that were induced by interferon treatment and identified TRIM22 as one of the most strongly up-regulated genes. We confirmed, as in previous studies, that TRIM22 over-expression inhibited HIV replication. To assess the role of TRIM22 expressed under natural inducing conditions, we compared the effects of interferon in cells depleted for TRIM22 using RNAi and found that HIV particle release was significantly increased in the knockdown, implying that TRIM22 acts as a natural antiviral effector. Further studies showed that TRIM22 inhibited budding of virus-like particles containing Gag only, indicating that Gag was the target of TRIM22. TRIM22 did not block the release of MLV or EIAV Gag particles. Inhibition was associated with diffuse cytoplasmic staining of HIV Gag rather than accumulation at the plasma membrane, suggesting TRIM22 disrupts proper trafficking. Mutational analyses of TRIM22 showed that the catalytic amino acids Cys15 and Cys18 of the RING domain are required for TRIM22 antiviral activity. These data disclose a pathway by which Type 1 interferons obstruct HIV replication.

Show MeSH