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Ex vivo response to histone deacetylase (HDAC) inhibitors of the HIV long terminal repeat (LTR) derived from HIV-infected patients on antiretroviral therapy.

Lu HK, Gray LR, Wightman F, Ellenberg P, Khoury G, Cheng WJ, Mota TM, Wesselingh S, Gorry PR, Cameron PU, Churchill MJ, Lewin SR - PLoS ONE (2014)

Bottom Line: We found that in both cell lines, the HDACi panobinostat, trichostatin A, vorinostat and entinostat activated patient-derived HIV LTRs to similar levels seen with NL4-3 and all patient derived isolates had similar sensitivity to maximum HDACi stimulation.We observed a marked difference in the maximum fold induction of luciferase by HDACi in HeLa and SVG, suggesting that the effect of HDACi may be influenced by the cellular environment.Finally, we observed significant synergy in activation of the LTR with vorinostat and the viral protein Tat.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, Monash University, Melbourne, Victoria, Australia; Centre for Biomedical Research, Burnet Institute, Melbourne, Victoria, Australia.

ABSTRACT
Histone deacetylase inhibitors (HDACi) can induce human immunodeficiency virus (HIV) transcription from the HIV long terminal repeat (LTR). However, ex vivo and in vivo responses to HDACi are variable and the activity of HDACi in cells other than T-cells have not been well characterised. Here, we developed a novel assay to determine the activity of HDACi on patient-derived HIV LTRs in different cell types. HIV LTRs from integrated virus were amplified using triple-nested Alu-PCR from total memory CD4+ T-cells (CD45RO+) isolated from HIV-infected patients prior to and following suppressive antiretroviral therapy. NL4-3 or patient-derived HIV LTRs were cloned into the chromatin forming episomal vector pCEP4, and the effect of HDACi investigated in the astrocyte and epithelial cell lines SVG and HeLa, respectively. There were no significant differences in the sequence of the HIV LTRs isolated from CD4+ T-cells prior to and after 18 months of combination antiretroviral therapy (cART). We found that in both cell lines, the HDACi panobinostat, trichostatin A, vorinostat and entinostat activated patient-derived HIV LTRs to similar levels seen with NL4-3 and all patient derived isolates had similar sensitivity to maximum HDACi stimulation. We observed a marked difference in the maximum fold induction of luciferase by HDACi in HeLa and SVG, suggesting that the effect of HDACi may be influenced by the cellular environment. Finally, we observed significant synergy in activation of the LTR with vorinostat and the viral protein Tat. Together, our results suggest that the LTR sequence of integrated virus is not a major determinant of a functional response to an HDACi.

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Phylogenetic analyses of DNA sequences derived from integrated virus in CD4+ memory T-cells.Phylogenetic trees were constructed using a neighbour-joining method with sequences from nucleotide 6 to 548 of the LTR derived from memory CD4+ T-cells prior to the initiation of cART (open symbols), after at least 18 months of cART (closed symbols) in four participants and the consensus sequence from NL4-3 (square symbol). Arrows indicate clones selected at random for cloning into pCEP4. Scale-bars indicate genetic distance (e.g., 0.01 = 1% genetic distance). Bootstrap values of >75 are shown on branches. All hypermutated clones (P<0.05 analysed on Hypermut V2.0) were excluded from the analysis.
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pone-0113341-g002: Phylogenetic analyses of DNA sequences derived from integrated virus in CD4+ memory T-cells.Phylogenetic trees were constructed using a neighbour-joining method with sequences from nucleotide 6 to 548 of the LTR derived from memory CD4+ T-cells prior to the initiation of cART (open symbols), after at least 18 months of cART (closed symbols) in four participants and the consensus sequence from NL4-3 (square symbol). Arrows indicate clones selected at random for cloning into pCEP4. Scale-bars indicate genetic distance (e.g., 0.01 = 1% genetic distance). Bootstrap values of >75 are shown on branches. All hypermutated clones (P<0.05 analysed on Hypermut V2.0) were excluded from the analysis.

Mentions: To investigate whether there was selection or evolution of the HIV LTR following suppressive cART, integrated HIV LTR from memory CD4+ T-cells from HIV infected patients prior to and after suppressive cART were cloned and sequenced. The clinical details of these patients (n = 4) are summarised in Table 2. Sequence analysis of the integrated HIV LTRs showed no compartmentalisation between viral sequences in memory CD4+ T-cells prior to and following 18–24 months (Fig 2, P4–6) or up to 60 months of suppressive cART (Fig 2, P1).


Ex vivo response to histone deacetylase (HDAC) inhibitors of the HIV long terminal repeat (LTR) derived from HIV-infected patients on antiretroviral therapy.

Lu HK, Gray LR, Wightman F, Ellenberg P, Khoury G, Cheng WJ, Mota TM, Wesselingh S, Gorry PR, Cameron PU, Churchill MJ, Lewin SR - PLoS ONE (2014)

Phylogenetic analyses of DNA sequences derived from integrated virus in CD4+ memory T-cells.Phylogenetic trees were constructed using a neighbour-joining method with sequences from nucleotide 6 to 548 of the LTR derived from memory CD4+ T-cells prior to the initiation of cART (open symbols), after at least 18 months of cART (closed symbols) in four participants and the consensus sequence from NL4-3 (square symbol). Arrows indicate clones selected at random for cloning into pCEP4. Scale-bars indicate genetic distance (e.g., 0.01 = 1% genetic distance). Bootstrap values of >75 are shown on branches. All hypermutated clones (P<0.05 analysed on Hypermut V2.0) were excluded from the analysis.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4237424&req=5

pone-0113341-g002: Phylogenetic analyses of DNA sequences derived from integrated virus in CD4+ memory T-cells.Phylogenetic trees were constructed using a neighbour-joining method with sequences from nucleotide 6 to 548 of the LTR derived from memory CD4+ T-cells prior to the initiation of cART (open symbols), after at least 18 months of cART (closed symbols) in four participants and the consensus sequence from NL4-3 (square symbol). Arrows indicate clones selected at random for cloning into pCEP4. Scale-bars indicate genetic distance (e.g., 0.01 = 1% genetic distance). Bootstrap values of >75 are shown on branches. All hypermutated clones (P<0.05 analysed on Hypermut V2.0) were excluded from the analysis.
Mentions: To investigate whether there was selection or evolution of the HIV LTR following suppressive cART, integrated HIV LTR from memory CD4+ T-cells from HIV infected patients prior to and after suppressive cART were cloned and sequenced. The clinical details of these patients (n = 4) are summarised in Table 2. Sequence analysis of the integrated HIV LTRs showed no compartmentalisation between viral sequences in memory CD4+ T-cells prior to and following 18–24 months (Fig 2, P4–6) or up to 60 months of suppressive cART (Fig 2, P1).

Bottom Line: We found that in both cell lines, the HDACi panobinostat, trichostatin A, vorinostat and entinostat activated patient-derived HIV LTRs to similar levels seen with NL4-3 and all patient derived isolates had similar sensitivity to maximum HDACi stimulation.We observed a marked difference in the maximum fold induction of luciferase by HDACi in HeLa and SVG, suggesting that the effect of HDACi may be influenced by the cellular environment.Finally, we observed significant synergy in activation of the LTR with vorinostat and the viral protein Tat.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, Monash University, Melbourne, Victoria, Australia; Centre for Biomedical Research, Burnet Institute, Melbourne, Victoria, Australia.

ABSTRACT
Histone deacetylase inhibitors (HDACi) can induce human immunodeficiency virus (HIV) transcription from the HIV long terminal repeat (LTR). However, ex vivo and in vivo responses to HDACi are variable and the activity of HDACi in cells other than T-cells have not been well characterised. Here, we developed a novel assay to determine the activity of HDACi on patient-derived HIV LTRs in different cell types. HIV LTRs from integrated virus were amplified using triple-nested Alu-PCR from total memory CD4+ T-cells (CD45RO+) isolated from HIV-infected patients prior to and following suppressive antiretroviral therapy. NL4-3 or patient-derived HIV LTRs were cloned into the chromatin forming episomal vector pCEP4, and the effect of HDACi investigated in the astrocyte and epithelial cell lines SVG and HeLa, respectively. There were no significant differences in the sequence of the HIV LTRs isolated from CD4+ T-cells prior to and after 18 months of combination antiretroviral therapy (cART). We found that in both cell lines, the HDACi panobinostat, trichostatin A, vorinostat and entinostat activated patient-derived HIV LTRs to similar levels seen with NL4-3 and all patient derived isolates had similar sensitivity to maximum HDACi stimulation. We observed a marked difference in the maximum fold induction of luciferase by HDACi in HeLa and SVG, suggesting that the effect of HDACi may be influenced by the cellular environment. Finally, we observed significant synergy in activation of the LTR with vorinostat and the viral protein Tat. Together, our results suggest that the LTR sequence of integrated virus is not a major determinant of a functional response to an HDACi.

Show MeSH
Related in: MedlinePlus