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Cyclin T1-dependent genes in activated CD4 T and macrophage cell lines appear enriched in HIV-1 co-factors.

Yu W, Ramakrishnan R, Wang Y, Chiang K, Sung TL, Rice AP - PLoS ONE (2008)

Bottom Line: Notably, 10 of these CTDGs have been reported to be involved in HIV-1 replication, a significant over-representation of such genes when compared to randomly generated lists of 54 genes (p value<0.00021).The results of siRNA depletion and dominant-negative protein experiments with two CTDGs identified here, CDK11 and Casein kinase 1 gamma 1, suggest that these genes are involved either directly or indirectly in HIV-1 replication.The presence of CTDGs in the protein space that was available for HIV-1 to sample during its evolution and acquisition of Tat function may provide an explanation for why CTDGs are enriched in viral co-factors.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, United States of America.

ABSTRACT
HIV-1 is dependent upon cellular co-factors to mediate its replication cycle in CD4(+) T cells and macrophages, the two major cell types infected by the virus in vivo. One critical co-factor is Cyclin T1, a subunit of a general RNA polymerase II elongation factor known as P-TEFb. Cyclin T1 is targeted directly by the viral Tat protein to activate proviral transcription. Cyclin T1 is up-regulated when resting CD4(+) T cells are activated and during macrophage differentiation or activation, conditions that are also necessary for high levels of HIV-1 replication. Because Cyclin T1 is a subunit of a transcription factor, the up-regulation of Cyclin T1 in these cells results in the induction of cellular genes, some of which might be HIV-1 co-factors. Using shRNA depletions of Cyclin T1 and transcriptional profiling, we identified 54 cellular mRNAs that appear to be Cyclin T1-dependent for their induction in activated CD4(+) T Jurkat T cells and during differentiation and activation of MM6 cells, a human monocytic cell line. The promoters for these Cyclin T1-dependent genes (CTDGs) are over-represented in two transcription factor binding sites, SREBP1 and ARP1. Notably, 10 of these CTDGs have been reported to be involved in HIV-1 replication, a significant over-representation of such genes when compared to randomly generated lists of 54 genes (p value<0.00021). The results of siRNA depletion and dominant-negative protein experiments with two CTDGs identified here, CDK11 and Casein kinase 1 gamma 1, suggest that these genes are involved either directly or indirectly in HIV-1 replication. It is likely that the 54 CTDGs identified here include novel HIV-1 co-factors. The presence of CTDGs in the protein space that was available for HIV-1 to sample during its evolution and acquisition of Tat function may provide an explanation for why CTDGs are enriched in viral co-factors.

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A) MM6 cells: non-infected or MM6 cells infected at an m.o.i. of five with the indicated lentiviral vector were cultured for five days. Cells were then treated with LPS for 24 hours as indicated. Cell extracts were prepared and immunoblots performed to measure levels of Cyclin T1, CDK9, and β-actin. B) Jurkat cells: non-infected or Jurkat cells infected at an m.o.i. of five with the indicated lentiviral vector were cultured for five days. Cells were then treated with PMA+ ionomycin for 24 hours as indicated. Cell extracts were prepared to measure expression of Cyclin T1, CDK9, and β-actin.
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pone-0003146-g001: A) MM6 cells: non-infected or MM6 cells infected at an m.o.i. of five with the indicated lentiviral vector were cultured for five days. Cells were then treated with LPS for 24 hours as indicated. Cell extracts were prepared and immunoblots performed to measure levels of Cyclin T1, CDK9, and β-actin. B) Jurkat cells: non-infected or Jurkat cells infected at an m.o.i. of five with the indicated lentiviral vector were cultured for five days. Cells were then treated with PMA+ ionomycin for 24 hours as indicated. Cell extracts were prepared to measure expression of Cyclin T1, CDK9, and β-actin.

Mentions: Without LPS activation, the basal level of expression of Cyclin T1 in MM6 cells is low (Fig. 1A). LPS activation resulted in a strong induction of Cyclin T1 in both parental MM6 cells and cells transduced with the shRNA-Con vector. In contrast, Cyclin T1 expression was not induced in LPS-activated cells that were transduced with the shRNA-T1 vector. Flow cytometry analysis showed that >90% of cells in all transduced cultures expressed the eGFP marker protein (data not shown). We observed in real-time RT-PCR assays that Cyclin T1 mRNA levels were reduced approximately four-fold in cells infected with the shRNA-T1 vector (data not shown).


Cyclin T1-dependent genes in activated CD4 T and macrophage cell lines appear enriched in HIV-1 co-factors.

Yu W, Ramakrishnan R, Wang Y, Chiang K, Sung TL, Rice AP - PLoS ONE (2008)

A) MM6 cells: non-infected or MM6 cells infected at an m.o.i. of five with the indicated lentiviral vector were cultured for five days. Cells were then treated with LPS for 24 hours as indicated. Cell extracts were prepared and immunoblots performed to measure levels of Cyclin T1, CDK9, and β-actin. B) Jurkat cells: non-infected or Jurkat cells infected at an m.o.i. of five with the indicated lentiviral vector were cultured for five days. Cells were then treated with PMA+ ionomycin for 24 hours as indicated. Cell extracts were prepared to measure expression of Cyclin T1, CDK9, and β-actin.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2519787&req=5

pone-0003146-g001: A) MM6 cells: non-infected or MM6 cells infected at an m.o.i. of five with the indicated lentiviral vector were cultured for five days. Cells were then treated with LPS for 24 hours as indicated. Cell extracts were prepared and immunoblots performed to measure levels of Cyclin T1, CDK9, and β-actin. B) Jurkat cells: non-infected or Jurkat cells infected at an m.o.i. of five with the indicated lentiviral vector were cultured for five days. Cells were then treated with PMA+ ionomycin for 24 hours as indicated. Cell extracts were prepared to measure expression of Cyclin T1, CDK9, and β-actin.
Mentions: Without LPS activation, the basal level of expression of Cyclin T1 in MM6 cells is low (Fig. 1A). LPS activation resulted in a strong induction of Cyclin T1 in both parental MM6 cells and cells transduced with the shRNA-Con vector. In contrast, Cyclin T1 expression was not induced in LPS-activated cells that were transduced with the shRNA-T1 vector. Flow cytometry analysis showed that >90% of cells in all transduced cultures expressed the eGFP marker protein (data not shown). We observed in real-time RT-PCR assays that Cyclin T1 mRNA levels were reduced approximately four-fold in cells infected with the shRNA-T1 vector (data not shown).

Bottom Line: Notably, 10 of these CTDGs have been reported to be involved in HIV-1 replication, a significant over-representation of such genes when compared to randomly generated lists of 54 genes (p value<0.00021).The results of siRNA depletion and dominant-negative protein experiments with two CTDGs identified here, CDK11 and Casein kinase 1 gamma 1, suggest that these genes are involved either directly or indirectly in HIV-1 replication.The presence of CTDGs in the protein space that was available for HIV-1 to sample during its evolution and acquisition of Tat function may provide an explanation for why CTDGs are enriched in viral co-factors.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, United States of America.

ABSTRACT
HIV-1 is dependent upon cellular co-factors to mediate its replication cycle in CD4(+) T cells and macrophages, the two major cell types infected by the virus in vivo. One critical co-factor is Cyclin T1, a subunit of a general RNA polymerase II elongation factor known as P-TEFb. Cyclin T1 is targeted directly by the viral Tat protein to activate proviral transcription. Cyclin T1 is up-regulated when resting CD4(+) T cells are activated and during macrophage differentiation or activation, conditions that are also necessary for high levels of HIV-1 replication. Because Cyclin T1 is a subunit of a transcription factor, the up-regulation of Cyclin T1 in these cells results in the induction of cellular genes, some of which might be HIV-1 co-factors. Using shRNA depletions of Cyclin T1 and transcriptional profiling, we identified 54 cellular mRNAs that appear to be Cyclin T1-dependent for their induction in activated CD4(+) T Jurkat T cells and during differentiation and activation of MM6 cells, a human monocytic cell line. The promoters for these Cyclin T1-dependent genes (CTDGs) are over-represented in two transcription factor binding sites, SREBP1 and ARP1. Notably, 10 of these CTDGs have been reported to be involved in HIV-1 replication, a significant over-representation of such genes when compared to randomly generated lists of 54 genes (p value<0.00021). The results of siRNA depletion and dominant-negative protein experiments with two CTDGs identified here, CDK11 and Casein kinase 1 gamma 1, suggest that these genes are involved either directly or indirectly in HIV-1 replication. It is likely that the 54 CTDGs identified here include novel HIV-1 co-factors. The presence of CTDGs in the protein space that was available for HIV-1 to sample during its evolution and acquisition of Tat function may provide an explanation for why CTDGs are enriched in viral co-factors.

Show MeSH
Related in: MedlinePlus