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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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Effect of 8-Aza and 5350150 on HIV-1 and host RNA processing. The effects of 8-Aza or 5350150 on HIV-1 mRNA levels were assessed by qRT-PCR (as described in ‘Materials and Methods’ section) of HeLa rtTA-HIV-ΔMls cells treated with 45 µM 8-Aza, 2 µM 5350150 or DMSO (control) for 24 h with doxycycline before harvest. (A) The position of the primers (arrow points) used in qRT-PCR. (B) Abundance of HIV-1 unspliced (US, gray), singly spliced (SS, white), and multiply spliced (MS, black) mRNAs are shown relative to DMSO (+) controls. The housekeeping gene β-actin served as an internal loading control for the normalization of these data. Shown are data averaged from ≥ 5 experiments, error bars are SEM, and significant changes from control indicated by asterisks as described in ‘Materials and Methods’ section. (C-E) Changes in HIV-1 splice site use were assayed by RT-PCR of the 2 kb, MS class of HIV-1 mRNA (as outlined in ‘Materials and Methods’ section). (C) The position of the primers (arrow points) used to amplify MS mRNA species. (D) Representative RT-PCR gel of the levels of each MS mRNA species (arrows) from HeLa rtTA-HIV-ΔMls cells treated with doxycycline and 45 µM 8-Aza, 2 µM 5350150 or DMSO as performed in Figure 1. See Supplementary Figure S1 for a description of the PCR products generated. (E) Graph summarizing the effects of 8-Aza (white), 5350150 (black), and control treatments (gray) on the level of each MS mRNA species (x-axis) relative to the total HIV-1 MS mRNA (y-axis), displayed as percentage (%) of the total HIV-1 MS RNA. Data were averaged from ≥ 6 experiments, error bars are SEM, and significant differences from control indicated by asterisks as described in ‘Materials and Methods’ section. (F and G) RNAs from treatments outlined earlier in the text were subsequently used to analyse for changes in host RNA splicing. Primer pairs spanning host RNA alternative splicing event were used to amplify from cDNA and amplicons quantitated following resolution by capillary electrophoresis. Shown is a comparison of the level of alternative exon inclusion following treatment with (F) 5350150 or (G) 8-Aza relative to DMSO-treated cells. Values are the average of three independent trials, and events significantly different from control (DMSO) at P < 0.05 are indicated by open diamonds. The dotted line (grey) reflects the anticipated results if the drugs had no effect on host RNA splicing. The solid line (black) is linear regression of the data with strength of the correlation indicated.
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gkt727-F2: Effect of 8-Aza and 5350150 on HIV-1 and host RNA processing. The effects of 8-Aza or 5350150 on HIV-1 mRNA levels were assessed by qRT-PCR (as described in ‘Materials and Methods’ section) of HeLa rtTA-HIV-ΔMls cells treated with 45 µM 8-Aza, 2 µM 5350150 or DMSO (control) for 24 h with doxycycline before harvest. (A) The position of the primers (arrow points) used in qRT-PCR. (B) Abundance of HIV-1 unspliced (US, gray), singly spliced (SS, white), and multiply spliced (MS, black) mRNAs are shown relative to DMSO (+) controls. The housekeeping gene β-actin served as an internal loading control for the normalization of these data. Shown are data averaged from ≥ 5 experiments, error bars are SEM, and significant changes from control indicated by asterisks as described in ‘Materials and Methods’ section. (C-E) Changes in HIV-1 splice site use were assayed by RT-PCR of the 2 kb, MS class of HIV-1 mRNA (as outlined in ‘Materials and Methods’ section). (C) The position of the primers (arrow points) used to amplify MS mRNA species. (D) Representative RT-PCR gel of the levels of each MS mRNA species (arrows) from HeLa rtTA-HIV-ΔMls cells treated with doxycycline and 45 µM 8-Aza, 2 µM 5350150 or DMSO as performed in Figure 1. See Supplementary Figure S1 for a description of the PCR products generated. (E) Graph summarizing the effects of 8-Aza (white), 5350150 (black), and control treatments (gray) on the level of each MS mRNA species (x-axis) relative to the total HIV-1 MS mRNA (y-axis), displayed as percentage (%) of the total HIV-1 MS RNA. Data were averaged from ≥ 6 experiments, error bars are SEM, and significant differences from control indicated by asterisks as described in ‘Materials and Methods’ section. (F and G) RNAs from treatments outlined earlier in the text were subsequently used to analyse for changes in host RNA splicing. Primer pairs spanning host RNA alternative splicing event were used to amplify from cDNA and amplicons quantitated following resolution by capillary electrophoresis. Shown is a comparison of the level of alternative exon inclusion following treatment with (F) 5350150 or (G) 8-Aza relative to DMSO-treated cells. Values are the average of three independent trials, and events significantly different from control (DMSO) at P < 0.05 are indicated by open diamonds. The dotted line (grey) reflects the anticipated results if the drugs had no effect on host RNA splicing. The solid line (black) is linear regression of the data with strength of the correlation indicated.

Mentions: Based on the effect of these compounds on HIV-1 gene expression and recently reported modification of RNA splicing (Percifield et al., unpublished data), we examined whether either compound altered HIV-1 RNA accumulation or splice site selection. As indicated in Figure 2A and B, qRT-PCR analyses determined that 8-Aza treatment significantly altered HIV-1 RNA accumulation (similar to chlorhexidine) (24). Treatment with this compound reduced both US and SS viral RNA abundance to ∼20% of control (+Dox) levels, whereas MS RNA accumulation was increased by ∼1.5-fold. In contrast, 5350150 treatment led to only modest changes, US RNA being reduced to ∼60% of control, no effect on SS RNA accumulation, and a ∼1.5-fold increase in MS RNA abundance. In addition, there were no significant effects on the overall RNA concentration or copies of the housekeeping gene, actin (data not shown). In light of these alterations, we also investigated whether either compound elicited any changes in the frequency of splice site use. To this end, RT-PCR was performed to amplify viral MS RNAs, and the products were analysed by separation on denaturing gels. As seen in Figure 2C–E, treatment with 5350150 had little effect on the frequency of splice site usage within MS viral RNAs. In contrast, 8-Aza induced a marked alteration in splice site usage, strongly promoting the generation of Nef1 product [generated by the joining of the first 5′splice site of HIV (SD1) with the last 3′splice site (SA7)] with a comparable reduction in Nef2 RNA. In addition, there was a modest reduction in the level of Rev1/2 and increase of Tat1 and Tat2 RNAs.Figure 2.


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)

Effect of 8-Aza and 5350150 on HIV-1 and host RNA processing. The effects of 8-Aza or 5350150 on HIV-1 mRNA levels were assessed by qRT-PCR (as described in ‘Materials and Methods’ section) of HeLa rtTA-HIV-ΔMls cells treated with 45 µM 8-Aza, 2 µM 5350150 or DMSO (control) for 24 h with doxycycline before harvest. (A) The position of the primers (arrow points) used in qRT-PCR. (B) Abundance of HIV-1 unspliced (US, gray), singly spliced (SS, white), and multiply spliced (MS, black) mRNAs are shown relative to DMSO (+) controls. The housekeeping gene β-actin served as an internal loading control for the normalization of these data. Shown are data averaged from ≥ 5 experiments, error bars are SEM, and significant changes from control indicated by asterisks as described in ‘Materials and Methods’ section. (C-E) Changes in HIV-1 splice site use were assayed by RT-PCR of the 2 kb, MS class of HIV-1 mRNA (as outlined in ‘Materials and Methods’ section). (C) The position of the primers (arrow points) used to amplify MS mRNA species. (D) Representative RT-PCR gel of the levels of each MS mRNA species (arrows) from HeLa rtTA-HIV-ΔMls cells treated with doxycycline and 45 µM 8-Aza, 2 µM 5350150 or DMSO as performed in Figure 1. See Supplementary Figure S1 for a description of the PCR products generated. (E) Graph summarizing the effects of 8-Aza (white), 5350150 (black), and control treatments (gray) on the level of each MS mRNA species (x-axis) relative to the total HIV-1 MS mRNA (y-axis), displayed as percentage (%) of the total HIV-1 MS RNA. Data were averaged from ≥ 6 experiments, error bars are SEM, and significant differences from control indicated by asterisks as described in ‘Materials and Methods’ section. (F and G) RNAs from treatments outlined earlier in the text were subsequently used to analyse for changes in host RNA splicing. Primer pairs spanning host RNA alternative splicing event were used to amplify from cDNA and amplicons quantitated following resolution by capillary electrophoresis. Shown is a comparison of the level of alternative exon inclusion following treatment with (F) 5350150 or (G) 8-Aza relative to DMSO-treated cells. Values are the average of three independent trials, and events significantly different from control (DMSO) at P < 0.05 are indicated by open diamonds. The dotted line (grey) reflects the anticipated results if the drugs had no effect on host RNA splicing. The solid line (black) is linear regression of the data with strength of the correlation indicated.
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gkt727-F2: Effect of 8-Aza and 5350150 on HIV-1 and host RNA processing. The effects of 8-Aza or 5350150 on HIV-1 mRNA levels were assessed by qRT-PCR (as described in ‘Materials and Methods’ section) of HeLa rtTA-HIV-ΔMls cells treated with 45 µM 8-Aza, 2 µM 5350150 or DMSO (control) for 24 h with doxycycline before harvest. (A) The position of the primers (arrow points) used in qRT-PCR. (B) Abundance of HIV-1 unspliced (US, gray), singly spliced (SS, white), and multiply spliced (MS, black) mRNAs are shown relative to DMSO (+) controls. The housekeeping gene β-actin served as an internal loading control for the normalization of these data. Shown are data averaged from ≥ 5 experiments, error bars are SEM, and significant changes from control indicated by asterisks as described in ‘Materials and Methods’ section. (C-E) Changes in HIV-1 splice site use were assayed by RT-PCR of the 2 kb, MS class of HIV-1 mRNA (as outlined in ‘Materials and Methods’ section). (C) The position of the primers (arrow points) used to amplify MS mRNA species. (D) Representative RT-PCR gel of the levels of each MS mRNA species (arrows) from HeLa rtTA-HIV-ΔMls cells treated with doxycycline and 45 µM 8-Aza, 2 µM 5350150 or DMSO as performed in Figure 1. See Supplementary Figure S1 for a description of the PCR products generated. (E) Graph summarizing the effects of 8-Aza (white), 5350150 (black), and control treatments (gray) on the level of each MS mRNA species (x-axis) relative to the total HIV-1 MS mRNA (y-axis), displayed as percentage (%) of the total HIV-1 MS RNA. Data were averaged from ≥ 6 experiments, error bars are SEM, and significant differences from control indicated by asterisks as described in ‘Materials and Methods’ section. (F and G) RNAs from treatments outlined earlier in the text were subsequently used to analyse for changes in host RNA splicing. Primer pairs spanning host RNA alternative splicing event were used to amplify from cDNA and amplicons quantitated following resolution by capillary electrophoresis. Shown is a comparison of the level of alternative exon inclusion following treatment with (F) 5350150 or (G) 8-Aza relative to DMSO-treated cells. Values are the average of three independent trials, and events significantly different from control (DMSO) at P < 0.05 are indicated by open diamonds. The dotted line (grey) reflects the anticipated results if the drugs had no effect on host RNA splicing. The solid line (black) is linear regression of the data with strength of the correlation indicated.
Mentions: Based on the effect of these compounds on HIV-1 gene expression and recently reported modification of RNA splicing (Percifield et al., unpublished data), we examined whether either compound altered HIV-1 RNA accumulation or splice site selection. As indicated in Figure 2A and B, qRT-PCR analyses determined that 8-Aza treatment significantly altered HIV-1 RNA accumulation (similar to chlorhexidine) (24). Treatment with this compound reduced both US and SS viral RNA abundance to ∼20% of control (+Dox) levels, whereas MS RNA accumulation was increased by ∼1.5-fold. In contrast, 5350150 treatment led to only modest changes, US RNA being reduced to ∼60% of control, no effect on SS RNA accumulation, and a ∼1.5-fold increase in MS RNA abundance. In addition, there were no significant effects on the overall RNA concentration or copies of the housekeeping gene, actin (data not shown). In light of these alterations, we also investigated whether either compound elicited any changes in the frequency of splice site use. To this end, RT-PCR was performed to amplify viral MS RNAs, and the products were analysed by separation on denaturing gels. As seen in Figure 2C–E, treatment with 5350150 had little effect on the frequency of splice site usage within MS viral RNAs. In contrast, 8-Aza induced a marked alteration in splice site usage, strongly promoting the generation of Nef1 product [generated by the joining of the first 5′splice site of HIV (SD1) with the last 3′splice site (SA7)] with a comparable reduction in Nef2 RNA. In addition, there was a modest reduction in the level of Rev1/2 and increase of Tat1 and Tat2 RNAs.Figure 2.

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