Limits...
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.

Show MeSH

Related in: MedlinePlus

Suppression of HIV-1 replication in chronically infected PBMCs by 8-Aza and 5350150. CD8-depleted PBMCs from chronically HIV-1-infected patients were activated by treatment with anti-CD3 and anti-CD28 antibodies to induce HIV-1 growth in the culture. Cells were treated with 3-TC, 8-Aza, or 5350150 (triangles), or control (DMSO, diamonds). Equal concentrations of DMSO solvent were present at each concentration of compound/drug tested. Media were harvested at multiple times points (0-21 days) to assess virus replication by p24CA ELISA of Gag. Shown (A, B, C) are the viral growth curves using 5 nM 3-TC, 1 µM 8-Aza or 1 µM 5350150, and (D, E) the dose response effects of the indicated drugs after 14 days in culture. Results shown are derived from assays performed on four different patient samples. Effect of drugs on cell viability (Cell Viab.) was also tested by XTT assay and results expressed relative to DMSO-treated cells (grey circles). All error bars are SEM and statistical significance indicated by asterisks as detailed in ‘Materials and Methods’ section.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3814367&req=5

gkt727-F4: Suppression of HIV-1 replication in chronically infected PBMCs by 8-Aza and 5350150. CD8-depleted PBMCs from chronically HIV-1-infected patients were activated by treatment with anti-CD3 and anti-CD28 antibodies to induce HIV-1 growth in the culture. Cells were treated with 3-TC, 8-Aza, or 5350150 (triangles), or control (DMSO, diamonds). Equal concentrations of DMSO solvent were present at each concentration of compound/drug tested. Media were harvested at multiple times points (0-21 days) to assess virus replication by p24CA ELISA of Gag. Shown (A, B, C) are the viral growth curves using 5 nM 3-TC, 1 µM 8-Aza or 1 µM 5350150, and (D, E) the dose response effects of the indicated drugs after 14 days in culture. Results shown are derived from assays performed on four different patient samples. Effect of drugs on cell viability (Cell Viab.) was also tested by XTT assay and results expressed relative to DMSO-treated cells (grey circles). All error bars are SEM and statistical significance indicated by asterisks as detailed in ‘Materials and Methods’ section.

Mentions: To validate our findings in a more relevant setting, the ability of 8-Aza or 5350150 to suppress HIV-1 replication was examined in the context of CD8-depleted PBMCs obtained from treatment-naïve HIV-infected patients. Cells were incubated in the presence of lamivudine (3-TC) (a reverse transcriptase inhibitor), 8-Aza or 5350150 and media harvested at various days after cell activation. As shown in Figure 4A–C, both 8-Aza and 5350150 displayed significant suppression of viral growth at a dose of 1 µM. Subsequent dose response analysis (Figure 4D and E) determined that optimal suppression of HIV-1 growth was obtained at 10 µM of 8-Aza or 0.5 µM of 5350150.Figure 4.


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)

Suppression of HIV-1 replication in chronically infected PBMCs by 8-Aza and 5350150. CD8-depleted PBMCs from chronically HIV-1-infected patients were activated by treatment with anti-CD3 and anti-CD28 antibodies to induce HIV-1 growth in the culture. Cells were treated with 3-TC, 8-Aza, or 5350150 (triangles), or control (DMSO, diamonds). Equal concentrations of DMSO solvent were present at each concentration of compound/drug tested. Media were harvested at multiple times points (0-21 days) to assess virus replication by p24CA ELISA of Gag. Shown (A, B, C) are the viral growth curves using 5 nM 3-TC, 1 µM 8-Aza or 1 µM 5350150, and (D, E) the dose response effects of the indicated drugs after 14 days in culture. Results shown are derived from assays performed on four different patient samples. Effect of drugs on cell viability (Cell Viab.) was also tested by XTT assay and results expressed relative to DMSO-treated cells (grey circles). All error bars are SEM and statistical significance indicated by asterisks as detailed in ‘Materials and Methods’ section.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814367&req=5

gkt727-F4: Suppression of HIV-1 replication in chronically infected PBMCs by 8-Aza and 5350150. CD8-depleted PBMCs from chronically HIV-1-infected patients were activated by treatment with anti-CD3 and anti-CD28 antibodies to induce HIV-1 growth in the culture. Cells were treated with 3-TC, 8-Aza, or 5350150 (triangles), or control (DMSO, diamonds). Equal concentrations of DMSO solvent were present at each concentration of compound/drug tested. Media were harvested at multiple times points (0-21 days) to assess virus replication by p24CA ELISA of Gag. Shown (A, B, C) are the viral growth curves using 5 nM 3-TC, 1 µM 8-Aza or 1 µM 5350150, and (D, E) the dose response effects of the indicated drugs after 14 days in culture. Results shown are derived from assays performed on four different patient samples. Effect of drugs on cell viability (Cell Viab.) was also tested by XTT assay and results expressed relative to DMSO-treated cells (grey circles). All error bars are SEM and statistical significance indicated by asterisks as detailed in ‘Materials and Methods’ section.
Mentions: To validate our findings in a more relevant setting, the ability of 8-Aza or 5350150 to suppress HIV-1 replication was examined in the context of CD8-depleted PBMCs obtained from treatment-naïve HIV-infected patients. Cells were incubated in the presence of lamivudine (3-TC) (a reverse transcriptase inhibitor), 8-Aza or 5350150 and media harvested at various days after cell activation. As shown in Figure 4A–C, both 8-Aza and 5350150 displayed significant suppression of viral growth at a dose of 1 µM. Subsequent dose response analysis (Figure 4D and E) determined that optimal suppression of HIV-1 growth was obtained at 10 µM of 8-Aza or 0.5 µM of 5350150.Figure 4.

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