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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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CSNK1G1 depletions.As illustrated, 293T cells were transfected with either control siRNAs or siRNAs against CSNK1G1. One day later, cells were co-transfected with the pNL4-3-Luc and pVSV-G plasmids for virus production. Supernatants were collected 48 hours later and p24 levels were measured by an ELISA assay. Infectivity of supernatants containing reporter the NL-4-3-Luc reporter viruses were measured by infecting HeLa cells with equal volume of supernatants, and Luciferase expression was measured 48 hours post-infection. Luciferase expression was normalized to the amount of p24 in the input supernatants.
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pone-0003146-g005: CSNK1G1 depletions.As illustrated, 293T cells were transfected with either control siRNAs or siRNAs against CSNK1G1. One day later, cells were co-transfected with the pNL4-3-Luc and pVSV-G plasmids for virus production. Supernatants were collected 48 hours later and p24 levels were measured by an ELISA assay. Infectivity of supernatants containing reporter the NL-4-3-Luc reporter viruses were measured by infecting HeLa cells with equal volume of supernatants, and Luciferase expression was measured 48 hours post-infection. Luciferase expression was normalized to the amount of p24 in the input supernatants.

Mentions: In an experimental strategy illustrated in Figure 5, cultures of 293T cells were transfected with siRNAs against CSNK1G1 (or control siRNAs), and then transfected with a HIV-1 pNL4-3-Luc proviral plasmid (deleted for Envelope) and a plasmid that expresses the VSV G glycoprotein to allow production of infectious pseudotyped viruses. This HIV-1 provirus contains Luciferase in place of Nef. Culture supernatants were collected 48 hours post-transfection with the proviral and VSV G plasmids. The amount of p24 in culture supernatants was reduced ∼20% in the CSKN1G1-depleted cells relative to the control siRNA cells (Fig. 5). Equal volumes of the supernatants from 293T cells containing the HIV-1 reporter viruses were used to infect HeLa cells, and Luciferase expression was measured 48 hours later. Luciferase expression was then normalized to the amount of p24 in the 293T supernatant inoculum (Fig. 5). The data indicate that the infectivity of viruses produced in the CSNK1G1-depleted cells was reduced five-fold relative to that produced in the control siRNA cells.


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)

CSNK1G1 depletions.As illustrated, 293T cells were transfected with either control siRNAs or siRNAs against CSNK1G1. One day later, cells were co-transfected with the pNL4-3-Luc and pVSV-G plasmids for virus production. Supernatants were collected 48 hours later and p24 levels were measured by an ELISA assay. Infectivity of supernatants containing reporter the NL-4-3-Luc reporter viruses were measured by infecting HeLa cells with equal volume of supernatants, and Luciferase expression was measured 48 hours post-infection. Luciferase expression was normalized to the amount of p24 in the input supernatants.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0003146-g005: CSNK1G1 depletions.As illustrated, 293T cells were transfected with either control siRNAs or siRNAs against CSNK1G1. One day later, cells were co-transfected with the pNL4-3-Luc and pVSV-G plasmids for virus production. Supernatants were collected 48 hours later and p24 levels were measured by an ELISA assay. Infectivity of supernatants containing reporter the NL-4-3-Luc reporter viruses were measured by infecting HeLa cells with equal volume of supernatants, and Luciferase expression was measured 48 hours post-infection. Luciferase expression was normalized to the amount of p24 in the input supernatants.
Mentions: In an experimental strategy illustrated in Figure 5, cultures of 293T cells were transfected with siRNAs against CSNK1G1 (or control siRNAs), and then transfected with a HIV-1 pNL4-3-Luc proviral plasmid (deleted for Envelope) and a plasmid that expresses the VSV G glycoprotein to allow production of infectious pseudotyped viruses. This HIV-1 provirus contains Luciferase in place of Nef. Culture supernatants were collected 48 hours post-transfection with the proviral and VSV G plasmids. The amount of p24 in culture supernatants was reduced ∼20% in the CSKN1G1-depleted cells relative to the control siRNA cells (Fig. 5). Equal volumes of the supernatants from 293T cells containing the HIV-1 reporter viruses were used to infect HeLa cells, and Luciferase expression was measured 48 hours later. Luciferase expression was then normalized to the amount of p24 in the 293T supernatant inoculum (Fig. 5). The data indicate that the infectivity of viruses produced in the CSNK1G1-depleted cells was reduced five-fold relative to that produced in the control siRNA cells.

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