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Inhibition of pyrimidine synthesis reverses viral virulence factor-mediated block of mRNA nuclear export.

Zhang L, Das P, Schmolke M, Manicassamy B, Wang Y, Deng X, Cai L, Tu BP, Forst CV, Roth MG, Levy DE, García-Sastre A, de Brabander J, Phillips MA, Fontoura BM - J. Cell Biol. (2012)

Bottom Line: This quinoline carboxylic acid directly inhibited dihydroorotate dehydrogenase (DHODH), a host enzyme required for de novo pyrimidine biosynthesis, and partially reduced pyrimidine levels.This effect induced NXF1 expression, which promoted mRNA nuclear export in the presence of NS1.Thus, pyrimidines play a necessary role in the inhibition of mRNA nuclear export by virulence factors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

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DHODH inhibitor induces nuclear export and expression of mRNAs encoding antiviral factors via increasing NXF1 levels. (A) 293T cells transfected with a plasmid encoding NS1 were untreated or treated with 1-14. RNA was isolated from nuclear and cytoplasmic (cyto) fractions, and the indicated mRNA species were quantified by real-time RT-PCR. Controls for fraction purity are shown by immunoblot analysis, as indicated. (B) 293T cells were transfected with a control plasmid or a plasmid encoding NS1 and untreated or treated with 1-14. Total cell extracts were subjected to immunoblot analysis with antibodies against the indicated proteins. (C) 293T cells were transfected with a control plasmid or a plasmid encoding NS1 and untreated or treated with 1 or 1-14. Total cell extracts were subjected to immunoblot analysis with antibodies against the indicated proteins. (D) 293T cells were cotransfected with a control plasmid or a plasmid encoding NS1 and control siRNA oligonucleotide (SCR) or siRNA that target NXF1. RNA was isolated from nuclear and cytoplasmic fractions, and the indicated mRNA species were quantified by real-time RT-PCR. Controls for NXF1 knockdown are shown by immunoblot analysis, as indicated. Results represent mean values ± SD; **, P < 0.01. N/C, nuclear/cytoplasmic.
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fig5: DHODH inhibitor induces nuclear export and expression of mRNAs encoding antiviral factors via increasing NXF1 levels. (A) 293T cells transfected with a plasmid encoding NS1 were untreated or treated with 1-14. RNA was isolated from nuclear and cytoplasmic (cyto) fractions, and the indicated mRNA species were quantified by real-time RT-PCR. Controls for fraction purity are shown by immunoblot analysis, as indicated. (B) 293T cells were transfected with a control plasmid or a plasmid encoding NS1 and untreated or treated with 1-14. Total cell extracts were subjected to immunoblot analysis with antibodies against the indicated proteins. (C) 293T cells were transfected with a control plasmid or a plasmid encoding NS1 and untreated or treated with 1 or 1-14. Total cell extracts were subjected to immunoblot analysis with antibodies against the indicated proteins. (D) 293T cells were cotransfected with a control plasmid or a plasmid encoding NS1 and control siRNA oligonucleotide (SCR) or siRNA that target NXF1. RNA was isolated from nuclear and cytoplasmic fractions, and the indicated mRNA species were quantified by real-time RT-PCR. Controls for NXF1 knockdown are shown by immunoblot analysis, as indicated. Results represent mean values ± SD; **, P < 0.01. N/C, nuclear/cytoplasmic.

Mentions: To determine whether cells treated with 1-14 or 1 would revert the mRNA export block during influenza virus infection, cells were simultaneously subjected to oligo (dT) in situ hybridization to detect poly(A)+ RNA and immunofluorescence with antiinfluenza protein antibodies to label infected cells. Uninfected cells treated with 1-14 or 1 showed no effects on the intracellular distribution of mRNA, which was widely distributed in the cytoplasm and in the nucleus (Fig. 3, A and B; and Fig. S2, A and B). Infection with A/WSN/33 influenza virus resulted in poly(A)+ RNA retention in the nucleus, whereas infected cells that were also treated with 1-14 or 1 showed a normal distribution of mRNA in the nucleus and in the cytoplasm (Fig. 3, A and B; and Fig. S2, A and B). These results are consistent with the inhibition of viral replication by 1-14 or 1, which reduced NS1 levels and, therefore, prevented NS1-mediated mRNA export block. This release of mRNA export block is also consistent with a second mechanism in which effects of 1-14 on NS1 function in mRNA export allows expression of host mRNAs that encode antiviral factors (see Fig. 5 and Fig. S2). It should be noted that treatment of cells with 1-14 or 1 alone did not significantly alter the intensity of labeling of host poly(A)+ RNA. Although NS1 is known to inhibit mRNA export when expressed in mammalian cells (Nemeroff et al., 1998; Satterly et al., 2007), mRNA export has not been assessed in a virus lacking NS1. Here, we generated an A/WSN/33 lacking NS1 (WSN-ΔNS1) and showed that, in the absence of NS1, the virus does not inhibit mRNA export (Fig. 3, A and B). This result demonstrates the function of NS1 as an inhibitor of host mRNA export in the context of viral infection.


Inhibition of pyrimidine synthesis reverses viral virulence factor-mediated block of mRNA nuclear export.

Zhang L, Das P, Schmolke M, Manicassamy B, Wang Y, Deng X, Cai L, Tu BP, Forst CV, Roth MG, Levy DE, García-Sastre A, de Brabander J, Phillips MA, Fontoura BM - J. Cell Biol. (2012)

DHODH inhibitor induces nuclear export and expression of mRNAs encoding antiviral factors via increasing NXF1 levels. (A) 293T cells transfected with a plasmid encoding NS1 were untreated or treated with 1-14. RNA was isolated from nuclear and cytoplasmic (cyto) fractions, and the indicated mRNA species were quantified by real-time RT-PCR. Controls for fraction purity are shown by immunoblot analysis, as indicated. (B) 293T cells were transfected with a control plasmid or a plasmid encoding NS1 and untreated or treated with 1-14. Total cell extracts were subjected to immunoblot analysis with antibodies against the indicated proteins. (C) 293T cells were transfected with a control plasmid or a plasmid encoding NS1 and untreated or treated with 1 or 1-14. Total cell extracts were subjected to immunoblot analysis with antibodies against the indicated proteins. (D) 293T cells were cotransfected with a control plasmid or a plasmid encoding NS1 and control siRNA oligonucleotide (SCR) or siRNA that target NXF1. RNA was isolated from nuclear and cytoplasmic fractions, and the indicated mRNA species were quantified by real-time RT-PCR. Controls for NXF1 knockdown are shown by immunoblot analysis, as indicated. Results represent mean values ± SD; **, P < 0.01. N/C, nuclear/cytoplasmic.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig5: DHODH inhibitor induces nuclear export and expression of mRNAs encoding antiviral factors via increasing NXF1 levels. (A) 293T cells transfected with a plasmid encoding NS1 were untreated or treated with 1-14. RNA was isolated from nuclear and cytoplasmic (cyto) fractions, and the indicated mRNA species were quantified by real-time RT-PCR. Controls for fraction purity are shown by immunoblot analysis, as indicated. (B) 293T cells were transfected with a control plasmid or a plasmid encoding NS1 and untreated or treated with 1-14. Total cell extracts were subjected to immunoblot analysis with antibodies against the indicated proteins. (C) 293T cells were transfected with a control plasmid or a plasmid encoding NS1 and untreated or treated with 1 or 1-14. Total cell extracts were subjected to immunoblot analysis with antibodies against the indicated proteins. (D) 293T cells were cotransfected with a control plasmid or a plasmid encoding NS1 and control siRNA oligonucleotide (SCR) or siRNA that target NXF1. RNA was isolated from nuclear and cytoplasmic fractions, and the indicated mRNA species were quantified by real-time RT-PCR. Controls for NXF1 knockdown are shown by immunoblot analysis, as indicated. Results represent mean values ± SD; **, P < 0.01. N/C, nuclear/cytoplasmic.
Mentions: To determine whether cells treated with 1-14 or 1 would revert the mRNA export block during influenza virus infection, cells were simultaneously subjected to oligo (dT) in situ hybridization to detect poly(A)+ RNA and immunofluorescence with antiinfluenza protein antibodies to label infected cells. Uninfected cells treated with 1-14 or 1 showed no effects on the intracellular distribution of mRNA, which was widely distributed in the cytoplasm and in the nucleus (Fig. 3, A and B; and Fig. S2, A and B). Infection with A/WSN/33 influenza virus resulted in poly(A)+ RNA retention in the nucleus, whereas infected cells that were also treated with 1-14 or 1 showed a normal distribution of mRNA in the nucleus and in the cytoplasm (Fig. 3, A and B; and Fig. S2, A and B). These results are consistent with the inhibition of viral replication by 1-14 or 1, which reduced NS1 levels and, therefore, prevented NS1-mediated mRNA export block. This release of mRNA export block is also consistent with a second mechanism in which effects of 1-14 on NS1 function in mRNA export allows expression of host mRNAs that encode antiviral factors (see Fig. 5 and Fig. S2). It should be noted that treatment of cells with 1-14 or 1 alone did not significantly alter the intensity of labeling of host poly(A)+ RNA. Although NS1 is known to inhibit mRNA export when expressed in mammalian cells (Nemeroff et al., 1998; Satterly et al., 2007), mRNA export has not been assessed in a virus lacking NS1. Here, we generated an A/WSN/33 lacking NS1 (WSN-ΔNS1) and showed that, in the absence of NS1, the virus does not inhibit mRNA export (Fig. 3, A and B). This result demonstrates the function of NS1 as an inhibitor of host mRNA export in the context of viral infection.

Bottom Line: This quinoline carboxylic acid directly inhibited dihydroorotate dehydrogenase (DHODH), a host enzyme required for de novo pyrimidine biosynthesis, and partially reduced pyrimidine levels.This effect induced NXF1 expression, which promoted mRNA nuclear export in the presence of NS1.Thus, pyrimidines play a necessary role in the inhibition of mRNA nuclear export by virulence factors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

Show MeSH
Related in: MedlinePlus