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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: Porcine reproductive and respiratory syndrome virus (PRRSV) is a major threat to the swine industry and food security worldwide.The nucleocapsid (N) protein is a major structural protein of PRRSV.Use of the PARP-1 small molecule inhibitor, 3-AB, in PRRSV infected cells demonstrated that PARP-1 was required and acted as an enhancer factor for virus biology.

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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: Porcine reproductive and respiratory syndrome virus (PRRSV) is a major threat to the swine industry and food security worldwide.The nucleocapsid (N) protein is a major structural protein of PRRSV.Use of the PARP-1 small molecule inhibitor, 3-AB, in PRRSV infected cells demonstrated that PARP-1 was required and acted as an enhancer factor for virus biology.

View Article: PubMed Central - PubMed

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

Show MeSH
Related in: MedlinePlus