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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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TRIM22 alters the sub-cellular localization of Gag protein.A) Analysis of Gag localization by fluorescence microscopy. HOS-CD4/CXCR4 cells were co-transfected with pTRIM22 and pGag/GFP for 3 hours, washed and incubated with 100 µM cycloheximide for 3 hours. IFNβ pre-treatment was done for 16 hours prior to transfection with pGag/GFP. Cells were washed and incubated in fresh media for a further 48 hours. Gag-GFP localization was assessed using fluorescence microscopy. High magnification images of the white squares are shown to the right. B) Quantifications of images as in A from three independent experiments. GFP-positive cells were classified as showing either punctate fluorescence at the cell periphery or diffuse cytoplasmic fluorescence. The number of cells counted was: pcDNA, n = 332; TRIM22, n = 386; −IFNβ = 497; +IFNβ = 398. C) HOS-CD4/CXCR4 cells were co-transfected with pGag in the absence or presence of pTRIM22 and pulse-chase analysis of intracellular Gag protein was done. D) Detection of 3H-myristic acid-labeled Gag. Cells were co-transfected with pGag in the absence or presence of pTRIM22 for 24 hours and then labeled with 3H-myristic acid overnight. Gag was immunoprecipitated and resolved on a SDS-PAGE gel before exposure to film (“Labeled Gag”) or Western blot analysis using anti-p24CA (“Total Gag”).
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ppat-1000007-g005: TRIM22 alters the sub-cellular localization of Gag protein.A) Analysis of Gag localization by fluorescence microscopy. HOS-CD4/CXCR4 cells were co-transfected with pTRIM22 and pGag/GFP for 3 hours, washed and incubated with 100 µM cycloheximide for 3 hours. IFNβ pre-treatment was done for 16 hours prior to transfection with pGag/GFP. Cells were washed and incubated in fresh media for a further 48 hours. Gag-GFP localization was assessed using fluorescence microscopy. High magnification images of the white squares are shown to the right. B) Quantifications of images as in A from three independent experiments. GFP-positive cells were classified as showing either punctate fluorescence at the cell periphery or diffuse cytoplasmic fluorescence. The number of cells counted was: pcDNA, n = 332; TRIM22, n = 386; −IFNβ = 497; +IFNβ = 398. C) HOS-CD4/CXCR4 cells were co-transfected with pGag in the absence or presence of pTRIM22 and pulse-chase analysis of intracellular Gag protein was done. D) Detection of 3H-myristic acid-labeled Gag. Cells were co-transfected with pGag in the absence or presence of pTRIM22 for 24 hours and then labeled with 3H-myristic acid overnight. Gag was immunoprecipitated and resolved on a SDS-PAGE gel before exposure to film (“Labeled Gag”) or Western blot analysis using anti-p24CA (“Total Gag”).

Mentions: We next investigated whether TRIM22-mediated inhibition was associated with altered Gag trafficking by monitoring the subcellular localization of HIV Gag fused to GFP (pGag-GFP) [41],[42],[43],[44]. We transfected pGag-GFP in the presence or absence of pTRIM22, then 3 hours later treated the cells with cycloheximide for 3 hours. Forty-eight hours after release of the cycloheximide block, the localization of the Gag-GFP protein was visualized by fluorescence microscopy by taking optical slices through the center of cells (Figure 5A). In the control cells, 65% of GFP-positive cells had punctate fluorescence at or near the plasma membrane and 35% of the cells showed diffuse cytoplasmic fluorescence. In contrast, in cells expressing TRIM22, 12% of the cells had punctate fluorescence at or near the plasma membrane and 88% of the cells yielded diffuse cytoplasmic localization without visible puncta (Figure 5B). The observed difference in proportions was highly significant (P<0.0001, Chi-square test).


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

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

TRIM22 alters the sub-cellular localization of Gag protein.A) Analysis of Gag localization by fluorescence microscopy. HOS-CD4/CXCR4 cells were co-transfected with pTRIM22 and pGag/GFP for 3 hours, washed and incubated with 100 µM cycloheximide for 3 hours. IFNβ pre-treatment was done for 16 hours prior to transfection with pGag/GFP. Cells were washed and incubated in fresh media for a further 48 hours. Gag-GFP localization was assessed using fluorescence microscopy. High magnification images of the white squares are shown to the right. B) Quantifications of images as in A from three independent experiments. GFP-positive cells were classified as showing either punctate fluorescence at the cell periphery or diffuse cytoplasmic fluorescence. The number of cells counted was: pcDNA, n = 332; TRIM22, n = 386; −IFNβ = 497; +IFNβ = 398. C) HOS-CD4/CXCR4 cells were co-transfected with pGag in the absence or presence of pTRIM22 and pulse-chase analysis of intracellular Gag protein was done. D) Detection of 3H-myristic acid-labeled Gag. Cells were co-transfected with pGag in the absence or presence of pTRIM22 for 24 hours and then labeled with 3H-myristic acid overnight. Gag was immunoprecipitated and resolved on a SDS-PAGE gel before exposure to film (“Labeled Gag”) or Western blot analysis using anti-p24CA (“Total Gag”).
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Related In: Results  -  Collection

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ppat-1000007-g005: TRIM22 alters the sub-cellular localization of Gag protein.A) Analysis of Gag localization by fluorescence microscopy. HOS-CD4/CXCR4 cells were co-transfected with pTRIM22 and pGag/GFP for 3 hours, washed and incubated with 100 µM cycloheximide for 3 hours. IFNβ pre-treatment was done for 16 hours prior to transfection with pGag/GFP. Cells were washed and incubated in fresh media for a further 48 hours. Gag-GFP localization was assessed using fluorescence microscopy. High magnification images of the white squares are shown to the right. B) Quantifications of images as in A from three independent experiments. GFP-positive cells were classified as showing either punctate fluorescence at the cell periphery or diffuse cytoplasmic fluorescence. The number of cells counted was: pcDNA, n = 332; TRIM22, n = 386; −IFNβ = 497; +IFNβ = 398. C) HOS-CD4/CXCR4 cells were co-transfected with pGag in the absence or presence of pTRIM22 and pulse-chase analysis of intracellular Gag protein was done. D) Detection of 3H-myristic acid-labeled Gag. Cells were co-transfected with pGag in the absence or presence of pTRIM22 for 24 hours and then labeled with 3H-myristic acid overnight. Gag was immunoprecipitated and resolved on a SDS-PAGE gel before exposure to film (“Labeled Gag”) or Western blot analysis using anti-p24CA (“Total Gag”).
Mentions: We next investigated whether TRIM22-mediated inhibition was associated with altered Gag trafficking by monitoring the subcellular localization of HIV Gag fused to GFP (pGag-GFP) [41],[42],[43],[44]. We transfected pGag-GFP in the presence or absence of pTRIM22, then 3 hours later treated the cells with cycloheximide for 3 hours. Forty-eight hours after release of the cycloheximide block, the localization of the Gag-GFP protein was visualized by fluorescence microscopy by taking optical slices through the center of cells (Figure 5A). In the control cells, 65% of GFP-positive cells had punctate fluorescence at or near the plasma membrane and 35% of the cells showed diffuse cytoplasmic fluorescence. In contrast, in cells expressing TRIM22, 12% of the cells had punctate fluorescence at or near the plasma membrane and 88% of the cells yielded diffuse cytoplasmic localization without visible puncta (Figure 5B). The observed difference in proportions was highly significant (P<0.0001, Chi-square test).

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