Limits...
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.

Show MeSH

Related in: MedlinePlus

Expression of TRIM22 in HOS-CD4/CXCR4 cells blocks late steps in HIV replication.A) Induction of TRIM22 RNA levels by IFNβ treatment of HOS-CD4/CXCR4 cells. Cells were pre-treated with 1000 units/ml of IFNβ, RNA isolated and subjected to Northern blot analysis. The TRIM22 3′UTR was labeled and used as a probe. B) Stable expression of HA-tagged TRIM22 in HOS-CD4/CXCR4 cells verified by Western blotting. C) Inhibition of HIV replication in cells stably expressing TRIM22. Cells were infected with different concentrations of replication-competent HIV-1 R9. After 72 hours, virus released into the supernatant was pelleted and assayed for p24CA levels by Western blot. The numbers below the Western blots correspond to the amounts of virus added to each well, quantified according to the amount of reverse transcriptase activity. D) TRIM22 does not inhibit early HIV replication steps, subsequent to entry. The TRIM22 expressing cells were infected with different amounts of HIV-1/VSVG pseudotyped virus and analyzed by FACS for GFP expression. Data obtained from a second independently generated cell line expressing TRIM22 (#2) are also shown. Data shown are representative of two independent infections for each performed in quadruplicate (+/−StdDev).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2279259&req=5

ppat-1000007-g001: Expression of TRIM22 in HOS-CD4/CXCR4 cells blocks late steps in HIV replication.A) Induction of TRIM22 RNA levels by IFNβ treatment of HOS-CD4/CXCR4 cells. Cells were pre-treated with 1000 units/ml of IFNβ, RNA isolated and subjected to Northern blot analysis. The TRIM22 3′UTR was labeled and used as a probe. B) Stable expression of HA-tagged TRIM22 in HOS-CD4/CXCR4 cells verified by Western blotting. C) Inhibition of HIV replication in cells stably expressing TRIM22. Cells were infected with different concentrations of replication-competent HIV-1 R9. After 72 hours, virus released into the supernatant was pelleted and assayed for p24CA levels by Western blot. The numbers below the Western blots correspond to the amounts of virus added to each well, quantified according to the amount of reverse transcriptase activity. D) TRIM22 does not inhibit early HIV replication steps, subsequent to entry. The TRIM22 expressing cells were infected with different amounts of HIV-1/VSVG pseudotyped virus and analyzed by FACS for GFP expression. Data obtained from a second independently generated cell line expressing TRIM22 (#2) are also shown. Data shown are representative of two independent infections for each performed in quadruplicate (+/−StdDev).

Mentions: We focused our study on TRIM22, which was induced at least 86-fold after IFNβ-treatment. Other TRIM genes that were notably up-regulated included TRIM19 variants (2.8 to 18-fold), TRIM21 (2.6-fold), TRIM34 variant 1 (2.4-fold) and TRIM5delta (2.4-fold), consistent with previous studies [26],[36],[37],[38],[39]. Gene chip data on TRIM22 induction was confirmed using Northern blot analysis (Figure 1A). These results support previous studies of other cell types, although we observed a higher level of IFNβ-induced TRIM22 expression than previously reported [4],[27].


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

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

Expression of TRIM22 in HOS-CD4/CXCR4 cells blocks late steps in HIV replication.A) Induction of TRIM22 RNA levels by IFNβ treatment of HOS-CD4/CXCR4 cells. Cells were pre-treated with 1000 units/ml of IFNβ, RNA isolated and subjected to Northern blot analysis. The TRIM22 3′UTR was labeled and used as a probe. B) Stable expression of HA-tagged TRIM22 in HOS-CD4/CXCR4 cells verified by Western blotting. C) Inhibition of HIV replication in cells stably expressing TRIM22. Cells were infected with different concentrations of replication-competent HIV-1 R9. After 72 hours, virus released into the supernatant was pelleted and assayed for p24CA levels by Western blot. The numbers below the Western blots correspond to the amounts of virus added to each well, quantified according to the amount of reverse transcriptase activity. D) TRIM22 does not inhibit early HIV replication steps, subsequent to entry. The TRIM22 expressing cells were infected with different amounts of HIV-1/VSVG pseudotyped virus and analyzed by FACS for GFP expression. Data obtained from a second independently generated cell line expressing TRIM22 (#2) are also shown. Data shown are representative of two independent infections for each performed in quadruplicate (+/−StdDev).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2279259&req=5

ppat-1000007-g001: Expression of TRIM22 in HOS-CD4/CXCR4 cells blocks late steps in HIV replication.A) Induction of TRIM22 RNA levels by IFNβ treatment of HOS-CD4/CXCR4 cells. Cells were pre-treated with 1000 units/ml of IFNβ, RNA isolated and subjected to Northern blot analysis. The TRIM22 3′UTR was labeled and used as a probe. B) Stable expression of HA-tagged TRIM22 in HOS-CD4/CXCR4 cells verified by Western blotting. C) Inhibition of HIV replication in cells stably expressing TRIM22. Cells were infected with different concentrations of replication-competent HIV-1 R9. After 72 hours, virus released into the supernatant was pelleted and assayed for p24CA levels by Western blot. The numbers below the Western blots correspond to the amounts of virus added to each well, quantified according to the amount of reverse transcriptase activity. D) TRIM22 does not inhibit early HIV replication steps, subsequent to entry. The TRIM22 expressing cells were infected with different amounts of HIV-1/VSVG pseudotyped virus and analyzed by FACS for GFP expression. Data obtained from a second independently generated cell line expressing TRIM22 (#2) are also shown. Data shown are representative of two independent infections for each performed in quadruplicate (+/−StdDev).
Mentions: We focused our study on TRIM22, which was induced at least 86-fold after IFNβ-treatment. Other TRIM genes that were notably up-regulated included TRIM19 variants (2.8 to 18-fold), TRIM21 (2.6-fold), TRIM34 variant 1 (2.4-fold) and TRIM5delta (2.4-fold), consistent with previous studies [26],[36],[37],[38],[39]. Gene chip data on TRIM22 induction was confirmed using Northern blot analysis (Figure 1A). These results support previous studies of other cell types, although we observed a higher level of IFNβ-induced TRIM22 expression than previously reported [4],[27].

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
Related in: MedlinePlus