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Characterization of novel inhibitors of HIV-1 replication that function via alteration of viral RNA processing and rev function.

Wong RW, Balachandran A, Haaland M, Stoilov P, Cochrane A - Nucleic Acids Res. (2013)

Bottom Line: Although only 8-Azaguanine dramatically decreased accumulation of HIV-1 unspliced and singly spliced RNAs and altered splice site usage, both compounds blocked Gag and Env expression without affecting production of Tat (p16) and Rev regulatory proteins.This conclusion is supported by the determination that both drugs block the nuclear export of genomic HIV-1 RNA to the cytoplasm.Testing confirmed that these compounds suppress HIV-1 expression in T cells at doses below those previously used in humans for tumour chemotherapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto M5S 1A8, Canada, Department of Molecular Genetics, University of Toronto, Toronto M5S 1A8, Canada and Department of Biochemistry, West Virginia University, Morgantown, WV 26506, USA.

ABSTRACT
Expression of the complete HIV-1 genome depends on the appropriate processing of viral RNA. Altering the balance of viral RNA processing impairs replication of the virus. In this report, we characterize two small molecule modulators of HIV-1 RNA processing, 8-azaguanine and 2-(2-(5-nitro-2-thienyl)vinyl)quinoline (5350150), which function by distinct mechanisms to suppress viral gene expression. Although only 8-Azaguanine dramatically decreased accumulation of HIV-1 unspliced and singly spliced RNAs and altered splice site usage, both compounds blocked Gag and Env expression without affecting production of Tat (p16) and Rev regulatory proteins. Subsequent analyses suggest that these compounds affect Rev-mediated RNA transport by different mechanisms. Both compounds induced cytoplasmic accumulation of Rev, suggesting that they function, in part, by impairing Rev function. This conclusion is supported by the determination that both drugs block the nuclear export of genomic HIV-1 RNA to the cytoplasm. Testing confirmed that these compounds suppress HIV-1 expression in T cells at doses below those previously used in humans for tumour chemotherapy. Together, our observations demonstrate that small molecules can be used to inhibit HIV-1 replication by altering another avenue of viral RNA processing, offering the potential for the development of novel therapeutics for controlling this disease.

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Related in: MedlinePlus

Both 8-Aza and 5350150 alter the subcellular distribution of HIV-1 Rev but not nuclear proteins of the host cell. HeLa Rev cells stably expressing HIV-1 Rev were (A) control treated (DMSO) or incubated with 4 µg/ml of Act. D with (+) or without LB for 2 h or (B) treated with compounds overnight with 45 µM 8-Aza or 2 µM 5350150 as described in Figure 1. After fixation and permeabilization, localization of Rev was detected by immunofluorescence using a rabbit anti-Rev antibody followed by either a FITC- or Cy5-conjugated anti-rabbit IgG antibody. In parallel, cells were stained with antibodies for (C) hnRNP A1 or (D) SRp20 as detailed in ‘Materials and Methods’ section. Shown are representative images (gray scale) from three independent trials. Cells were stained with DAPI to allow imaging of the nuclei. Magnification 400×. (E) To determine whether 8-Aza or 5350150 were altering Rev localization by affecting Rev import, HeLa Rev cells were treated with DMSO, 2 h with Act. D (as a control that induces Rev cytoplasmic accumulation) or overnight with either 45 µM 8-Aza or 2 µM 5350150, and then left untreated or treated with LB (+LB) for 2 h before fixation. Rev distribution was assessed in individual cells by immunofluorescent staining using a rabbit anti-Rev antibody as detailed in ‘Materials and Methods’ section. Individual cells were scored as having predominately nuclear (N > C, white), whole cell (N = C, grey) or predominately cytoplasmic (N < C, black) patterns of Rev distribution. Shown are data averaged from three independent experiments, error bars are SEM and significant changes from control indicated by asterisks as described in ‘Materials and Methods’ section. Representative images of the distribution patterns observed are provided in Supplementary Figure S3C.
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gkt727-F5: Both 8-Aza and 5350150 alter the subcellular distribution of HIV-1 Rev but not nuclear proteins of the host cell. HeLa Rev cells stably expressing HIV-1 Rev were (A) control treated (DMSO) or incubated with 4 µg/ml of Act. D with (+) or without LB for 2 h or (B) treated with compounds overnight with 45 µM 8-Aza or 2 µM 5350150 as described in Figure 1. After fixation and permeabilization, localization of Rev was detected by immunofluorescence using a rabbit anti-Rev antibody followed by either a FITC- or Cy5-conjugated anti-rabbit IgG antibody. In parallel, cells were stained with antibodies for (C) hnRNP A1 or (D) SRp20 as detailed in ‘Materials and Methods’ section. Shown are representative images (gray scale) from three independent trials. Cells were stained with DAPI to allow imaging of the nuclei. Magnification 400×. (E) To determine whether 8-Aza or 5350150 were altering Rev localization by affecting Rev import, HeLa Rev cells were treated with DMSO, 2 h with Act. D (as a control that induces Rev cytoplasmic accumulation) or overnight with either 45 µM 8-Aza or 2 µM 5350150, and then left untreated or treated with LB (+LB) for 2 h before fixation. Rev distribution was assessed in individual cells by immunofluorescent staining using a rabbit anti-Rev antibody as detailed in ‘Materials and Methods’ section. Individual cells were scored as having predominately nuclear (N > C, white), whole cell (N = C, grey) or predominately cytoplasmic (N < C, black) patterns of Rev distribution. Shown are data averaged from three independent experiments, error bars are SEM and significant changes from control indicated by asterisks as described in ‘Materials and Methods’ section. Representative images of the distribution patterns observed are provided in Supplementary Figure S3C.

Mentions: Although the alteration in HIV-1 US, SS, and MS RNAs induced by 8-Aza could account for the reduced expression of the corresponding proteins (Gag and Env), a similar case cannot be made for 5350150. The loss of both Gag and Env expression on addition of 5350150 suggested that it might impair Rev function. The ability of Rev to shuttle between the nucleus and cytoplasm is critical for its ability to export US and SS viral RNAs to the cytoplasm and their subsequent translation (28,29). Consequently, alterations in either Rev nuclear import or export could reduce its function and be reflected in changes in its subcellular distribution or localization of Rev-dependent viral RNAs. As a test of this hypothesis, the effect of 8-Aza or 5350150 on Rev subcellular distribution was examined by immunofluorescence microscopy. To monitor changes in Rev localization in response to drugs, we used a HeLa Rev cell line that constitutively expresses Rev alone at higher levels than the HeLa rtTA-HIV-ΔMls cell line to better assess changes in Rev activity. As previously reported and shown in Figure 5A, Rev is predominately localized to the nucleus and, in particular, the nucleolus in the absence of any treatment (DMSO) (30). However, on addition of actinomycin D (Act. D), Rev accumulates in the cytoplasm (31). This response can be reversed by leptomycin B (LB, an inhibitor of the Rev export mediator, Crm1) (32). Act. D acts to invert the relative rates of Rev import into and export out of the nucleus, resulting in export being the dominant pathway. LB, which inhibits Rev export by inactivating Crm1, induces nuclear accumulation of Rev by allowing only import to occur. To assess whether either 8-Aza or 5350150 also affects Rev transport, cells were treated with either compound overnight. As shown in Figure 5B, addition of either 8-Aza or 5350150 induced Rev accumulation in the cytoplasm (similar to the effect on Act. D treatment). Subsequent time course experiments determined that 8-Aza induced a shift in Rev subcellular distribution within 4 h, whereas the same effect required ∼16 h of treatment with 5350150 (Supplementary Figure S3). To determine whether the effects of 8-Aza or 5350150 on Rev subcellular distribution reflected a general perturbation in nuclear protein distribution, we also examined for changes in SC35 as well as known shuttling factors, hnRNP A1 and SRp20 (33,34). As shown in Figure 5C and D and Supplementary Figure S4, neither 8-Aza nor 5350150 induced any detectable alteration in distribution of other nuclear or nuclear shuttling proteins examined, suggesting that the responses seen for Rev are selective.Figure 5.


Characterization of novel inhibitors of HIV-1 replication that function via alteration of viral RNA processing and rev function.

Wong RW, Balachandran A, Haaland M, Stoilov P, Cochrane A - Nucleic Acids Res. (2013)

Both 8-Aza and 5350150 alter the subcellular distribution of HIV-1 Rev but not nuclear proteins of the host cell. HeLa Rev cells stably expressing HIV-1 Rev were (A) control treated (DMSO) or incubated with 4 µg/ml of Act. D with (+) or without LB for 2 h or (B) treated with compounds overnight with 45 µM 8-Aza or 2 µM 5350150 as described in Figure 1. After fixation and permeabilization, localization of Rev was detected by immunofluorescence using a rabbit anti-Rev antibody followed by either a FITC- or Cy5-conjugated anti-rabbit IgG antibody. In parallel, cells were stained with antibodies for (C) hnRNP A1 or (D) SRp20 as detailed in ‘Materials and Methods’ section. Shown are representative images (gray scale) from three independent trials. Cells were stained with DAPI to allow imaging of the nuclei. Magnification 400×. (E) To determine whether 8-Aza or 5350150 were altering Rev localization by affecting Rev import, HeLa Rev cells were treated with DMSO, 2 h with Act. D (as a control that induces Rev cytoplasmic accumulation) or overnight with either 45 µM 8-Aza or 2 µM 5350150, and then left untreated or treated with LB (+LB) for 2 h before fixation. Rev distribution was assessed in individual cells by immunofluorescent staining using a rabbit anti-Rev antibody as detailed in ‘Materials and Methods’ section. Individual cells were scored as having predominately nuclear (N > C, white), whole cell (N = C, grey) or predominately cytoplasmic (N < C, black) patterns of Rev distribution. Shown are data averaged from three independent experiments, error bars are SEM and significant changes from control indicated by asterisks as described in ‘Materials and Methods’ section. Representative images of the distribution patterns observed are provided in Supplementary Figure S3C.
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gkt727-F5: Both 8-Aza and 5350150 alter the subcellular distribution of HIV-1 Rev but not nuclear proteins of the host cell. HeLa Rev cells stably expressing HIV-1 Rev were (A) control treated (DMSO) or incubated with 4 µg/ml of Act. D with (+) or without LB for 2 h or (B) treated with compounds overnight with 45 µM 8-Aza or 2 µM 5350150 as described in Figure 1. After fixation and permeabilization, localization of Rev was detected by immunofluorescence using a rabbit anti-Rev antibody followed by either a FITC- or Cy5-conjugated anti-rabbit IgG antibody. In parallel, cells were stained with antibodies for (C) hnRNP A1 or (D) SRp20 as detailed in ‘Materials and Methods’ section. Shown are representative images (gray scale) from three independent trials. Cells were stained with DAPI to allow imaging of the nuclei. Magnification 400×. (E) To determine whether 8-Aza or 5350150 were altering Rev localization by affecting Rev import, HeLa Rev cells were treated with DMSO, 2 h with Act. D (as a control that induces Rev cytoplasmic accumulation) or overnight with either 45 µM 8-Aza or 2 µM 5350150, and then left untreated or treated with LB (+LB) for 2 h before fixation. Rev distribution was assessed in individual cells by immunofluorescent staining using a rabbit anti-Rev antibody as detailed in ‘Materials and Methods’ section. Individual cells were scored as having predominately nuclear (N > C, white), whole cell (N = C, grey) or predominately cytoplasmic (N < C, black) patterns of Rev distribution. Shown are data averaged from three independent experiments, error bars are SEM and significant changes from control indicated by asterisks as described in ‘Materials and Methods’ section. Representative images of the distribution patterns observed are provided in Supplementary Figure S3C.
Mentions: Although the alteration in HIV-1 US, SS, and MS RNAs induced by 8-Aza could account for the reduced expression of the corresponding proteins (Gag and Env), a similar case cannot be made for 5350150. The loss of both Gag and Env expression on addition of 5350150 suggested that it might impair Rev function. The ability of Rev to shuttle between the nucleus and cytoplasm is critical for its ability to export US and SS viral RNAs to the cytoplasm and their subsequent translation (28,29). Consequently, alterations in either Rev nuclear import or export could reduce its function and be reflected in changes in its subcellular distribution or localization of Rev-dependent viral RNAs. As a test of this hypothesis, the effect of 8-Aza or 5350150 on Rev subcellular distribution was examined by immunofluorescence microscopy. To monitor changes in Rev localization in response to drugs, we used a HeLa Rev cell line that constitutively expresses Rev alone at higher levels than the HeLa rtTA-HIV-ΔMls cell line to better assess changes in Rev activity. As previously reported and shown in Figure 5A, Rev is predominately localized to the nucleus and, in particular, the nucleolus in the absence of any treatment (DMSO) (30). However, on addition of actinomycin D (Act. D), Rev accumulates in the cytoplasm (31). This response can be reversed by leptomycin B (LB, an inhibitor of the Rev export mediator, Crm1) (32). Act. D acts to invert the relative rates of Rev import into and export out of the nucleus, resulting in export being the dominant pathway. LB, which inhibits Rev export by inactivating Crm1, induces nuclear accumulation of Rev by allowing only import to occur. To assess whether either 8-Aza or 5350150 also affects Rev transport, cells were treated with either compound overnight. As shown in Figure 5B, addition of either 8-Aza or 5350150 induced Rev accumulation in the cytoplasm (similar to the effect on Act. D treatment). Subsequent time course experiments determined that 8-Aza induced a shift in Rev subcellular distribution within 4 h, whereas the same effect required ∼16 h of treatment with 5350150 (Supplementary Figure S3). To determine whether the effects of 8-Aza or 5350150 on Rev subcellular distribution reflected a general perturbation in nuclear protein distribution, we also examined for changes in SC35 as well as known shuttling factors, hnRNP A1 and SRp20 (33,34). As shown in Figure 5C and D and Supplementary Figure S4, neither 8-Aza nor 5350150 induced any detectable alteration in distribution of other nuclear or nuclear shuttling proteins examined, suggesting that the responses seen for Rev are selective.Figure 5.

Bottom Line: Although only 8-Azaguanine dramatically decreased accumulation of HIV-1 unspliced and singly spliced RNAs and altered splice site usage, both compounds blocked Gag and Env expression without affecting production of Tat (p16) and Rev regulatory proteins.This conclusion is supported by the determination that both drugs block the nuclear export of genomic HIV-1 RNA to the cytoplasm.Testing confirmed that these compounds suppress HIV-1 expression in T cells at doses below those previously used in humans for tumour chemotherapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto M5S 1A8, Canada, Department of Molecular Genetics, University of Toronto, Toronto M5S 1A8, Canada and Department of Biochemistry, West Virginia University, Morgantown, WV 26506, USA.

ABSTRACT
Expression of the complete HIV-1 genome depends on the appropriate processing of viral RNA. Altering the balance of viral RNA processing impairs replication of the virus. In this report, we characterize two small molecule modulators of HIV-1 RNA processing, 8-azaguanine and 2-(2-(5-nitro-2-thienyl)vinyl)quinoline (5350150), which function by distinct mechanisms to suppress viral gene expression. Although only 8-Azaguanine dramatically decreased accumulation of HIV-1 unspliced and singly spliced RNAs and altered splice site usage, both compounds blocked Gag and Env expression without affecting production of Tat (p16) and Rev regulatory proteins. Subsequent analyses suggest that these compounds affect Rev-mediated RNA transport by different mechanisms. Both compounds induced cytoplasmic accumulation of Rev, suggesting that they function, in part, by impairing Rev function. This conclusion is supported by the determination that both drugs block the nuclear export of genomic HIV-1 RNA to the cytoplasm. Testing confirmed that these compounds suppress HIV-1 expression in T cells at doses below those previously used in humans for tumour chemotherapy. Together, our observations demonstrate that small molecules can be used to inhibit HIV-1 replication by altering another avenue of viral RNA processing, offering the potential for the development of novel therapeutics for controlling this disease.

Show MeSH
Related in: MedlinePlus