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Resolution of the cellular proteome of the nucleocapsid protein from a highly pathogenic isolate of porcine reproductive and respiratory syndrome virus identifies PARP-1 as a cellular target whose interaction is critical for virus biology.

Liu L, Lear Z, Hughes DJ, Wu W, Zhou EM, Whitehouse A, Chen H, Hiscox JA - Vet. Microbiol. (2014)

Bottom Line: The primary function of this protein is to encapsidate the viral RNA genome, and it is also thought to participate in the modulation of host cell biology and recruitment of cellular factors to facilitate virus infection.In order to the better understand these latter roles the cellular interactome of PRRSV N protein was defined using label free quantitative proteomics.This identified several cellular factors that could interact with the N protein including poly [ADP-ribose] polymerase 1 (PARP-1), a cellular protein, which can add adenosine diphosphate ribose to a protein.

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Affiliation: College of Life Sciences, Northwest A&F University, Yangling, China.

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Validation of the antiviral effect of PARP-1 inhibitor in Marc-145. Cells were inoculated with 1 MOI of PRRSV and treated with 20 mM or without 3-AB. After incubation for the indicated time, cells were lysed and analyzed by Western blotting with swine anti-PRRSV antibody (A), and rabbit anti-PARP-1 antibody (B). Protein bands on the western blot were labeled according to previous reported studies (Li et al., 2012; Music and Gagnon, 2010). β-Actin was also detected by anti-β-actin antibody to normalize the protein amount of cell lysates loaded in lanes (lower panel in A & B). (C) Quantization of PRRSV genomic (Nsp1) and subgenomic (N) RNA in infected Marc-145 cells. Total RNA was extracted at 24hpi and subjected to real-time RT-PCR analysis. Relative levels of N and Nsp1 were calculated using GAPDH as an internal control. Data were shown as means ±SD of three independent experiments. One-way ANOVA test was done to compare N and Nsp1 RNA level in 3-AB treated cells with untreated control (*P < 0.05; **P < 0.01).
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fig0030: Validation of the antiviral effect of PARP-1 inhibitor in Marc-145. Cells were inoculated with 1 MOI of PRRSV and treated with 20 mM or without 3-AB. After incubation for the indicated time, cells were lysed and analyzed by Western blotting with swine anti-PRRSV antibody (A), and rabbit anti-PARP-1 antibody (B). Protein bands on the western blot were labeled according to previous reported studies (Li et al., 2012; Music and Gagnon, 2010). β-Actin was also detected by anti-β-actin antibody to normalize the protein amount of cell lysates loaded in lanes (lower panel in A & B). (C) Quantization of PRRSV genomic (Nsp1) and subgenomic (N) RNA in infected Marc-145 cells. Total RNA was extracted at 24hpi and subjected to real-time RT-PCR analysis. Relative levels of N and Nsp1 were calculated using GAPDH as an internal control. Data were shown as means ±SD of three independent experiments. One-way ANOVA test was done to compare N and Nsp1 RNA level in 3-AB treated cells with untreated control (*P < 0.05; **P < 0.01).

Mentions: There are several stages to a virus life cycle, and the plaque assay provided a measure of the amount of virus being released into the supernatant. To investigate whether the PARP-1 inhibitor 3-AB affected virus biology in the cell, the abundance of viral proteins were compared using western blot at 24, 48 and 72 h post-infection between treated and untreated PRRSV-infected cells (Fig. 6A). The data indicated that at 24, 48 and 72 h post-infection nsp2, GP2, GP4, GP5, M and N proteins appeared less abundant in 3-AB treated cells than the non-treated cells (Fig. 6A). Western blot analysis indicated that in untreated and uninfected cells, PARP-1 was present in its native state. However, in cells treated with 20 mM 3-AB, PARP-1 is present as a cleavage product as well as native protein (Fig. 6B). This is consistent with the cell viability assay and that PARP-1 is cleaved during apoptosis. PARP-1 is present in the native and cleaved state in infected and treated cells at 24, 48 and 72 h post-infection. However, in untreated cells, PARP-1 is present in the native state at 24 h post-infection and in the native and cleaved state at 48 and 72 h post-infection. This is consistent with cleavage of PARP-1 previously reported in PRRSV infected cells (Lee and Kleiboeker, 2007).


Resolution of the cellular proteome of the nucleocapsid protein from a highly pathogenic isolate of porcine reproductive and respiratory syndrome virus identifies PARP-1 as a cellular target whose interaction is critical for virus biology.

Liu L, Lear Z, Hughes DJ, Wu W, Zhou EM, Whitehouse A, Chen H, Hiscox JA - Vet. Microbiol. (2014)

Validation of the antiviral effect of PARP-1 inhibitor in Marc-145. Cells were inoculated with 1 MOI of PRRSV and treated with 20 mM or without 3-AB. After incubation for the indicated time, cells were lysed and analyzed by Western blotting with swine anti-PRRSV antibody (A), and rabbit anti-PARP-1 antibody (B). Protein bands on the western blot were labeled according to previous reported studies (Li et al., 2012; Music and Gagnon, 2010). β-Actin was also detected by anti-β-actin antibody to normalize the protein amount of cell lysates loaded in lanes (lower panel in A & B). (C) Quantization of PRRSV genomic (Nsp1) and subgenomic (N) RNA in infected Marc-145 cells. Total RNA was extracted at 24hpi and subjected to real-time RT-PCR analysis. Relative levels of N and Nsp1 were calculated using GAPDH as an internal control. Data were shown as means ±SD of three independent experiments. One-way ANOVA test was done to compare N and Nsp1 RNA level in 3-AB treated cells with untreated control (*P < 0.05; **P < 0.01).
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fig0030: Validation of the antiviral effect of PARP-1 inhibitor in Marc-145. Cells were inoculated with 1 MOI of PRRSV and treated with 20 mM or without 3-AB. After incubation for the indicated time, cells were lysed and analyzed by Western blotting with swine anti-PRRSV antibody (A), and rabbit anti-PARP-1 antibody (B). Protein bands on the western blot were labeled according to previous reported studies (Li et al., 2012; Music and Gagnon, 2010). β-Actin was also detected by anti-β-actin antibody to normalize the protein amount of cell lysates loaded in lanes (lower panel in A & B). (C) Quantization of PRRSV genomic (Nsp1) and subgenomic (N) RNA in infected Marc-145 cells. Total RNA was extracted at 24hpi and subjected to real-time RT-PCR analysis. Relative levels of N and Nsp1 were calculated using GAPDH as an internal control. Data were shown as means ±SD of three independent experiments. One-way ANOVA test was done to compare N and Nsp1 RNA level in 3-AB treated cells with untreated control (*P < 0.05; **P < 0.01).
Mentions: There are several stages to a virus life cycle, and the plaque assay provided a measure of the amount of virus being released into the supernatant. To investigate whether the PARP-1 inhibitor 3-AB affected virus biology in the cell, the abundance of viral proteins were compared using western blot at 24, 48 and 72 h post-infection between treated and untreated PRRSV-infected cells (Fig. 6A). The data indicated that at 24, 48 and 72 h post-infection nsp2, GP2, GP4, GP5, M and N proteins appeared less abundant in 3-AB treated cells than the non-treated cells (Fig. 6A). Western blot analysis indicated that in untreated and uninfected cells, PARP-1 was present in its native state. However, in cells treated with 20 mM 3-AB, PARP-1 is present as a cleavage product as well as native protein (Fig. 6B). This is consistent with the cell viability assay and that PARP-1 is cleaved during apoptosis. PARP-1 is present in the native and cleaved state in infected and treated cells at 24, 48 and 72 h post-infection. However, in untreated cells, PARP-1 is present in the native state at 24 h post-infection and in the native and cleaved state at 48 and 72 h post-infection. This is consistent with cleavage of PARP-1 previously reported in PRRSV infected cells (Lee and Kleiboeker, 2007).

Bottom Line: The primary function of this protein is to encapsidate the viral RNA genome, and it is also thought to participate in the modulation of host cell biology and recruitment of cellular factors to facilitate virus infection.In order to the better understand these latter roles the cellular interactome of PRRSV N protein was defined using label free quantitative proteomics.This identified several cellular factors that could interact with the N protein including poly [ADP-ribose] polymerase 1 (PARP-1), a cellular protein, which can add adenosine diphosphate ribose to a protein.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Northwest A&F University, Yangling, China.

Show MeSH
Related in: MedlinePlus