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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.

View Article: PubMed Central - PubMed

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

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Related in: MedlinePlus

Immuno-fluorescence analysis of PRRSV infected Marc-145 with and without PARP inhibitor. Cells were incubated for indicated hours after infection and treatment with 20 mM 3-AB or DMSO as control. PRRSV infections were detected by IFA with swine anti-PRRSV serum and FlTC-conjugated rabbit anti-pig IgG antibody. (A) The immuno-fluorescence images were taken at an excitation wavelength of 495 nm with fluorescence microscope equipped with an appropriate filter. (B) Infection rate of Marc-145 determined by IFA. The percentage of infected cells were calculated by dividing the number of fluorescent cells by the total cells in the corresponding field. Each datum was obtained as the mean value from triplicate images.
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fig0025: Immuno-fluorescence analysis of PRRSV infected Marc-145 with and without PARP inhibitor. Cells were incubated for indicated hours after infection and treatment with 20 mM 3-AB or DMSO as control. PRRSV infections were detected by IFA with swine anti-PRRSV serum and FlTC-conjugated rabbit anti-pig IgG antibody. (A) The immuno-fluorescence images were taken at an excitation wavelength of 495 nm with fluorescence microscope equipped with an appropriate filter. (B) Infection rate of Marc-145 determined by IFA. The percentage of infected cells were calculated by dividing the number of fluorescent cells by the total cells in the corresponding field. Each datum was obtained as the mean value from triplicate images.

Mentions: Different doses of 3-AB were assessed for potential anti-viral activity by infecting cells with the same MOI of PRRSV and then treating cells with 5, 10, and 20 mM 3-AB over the infection period. Progeny virus production was compared by endpoint dilution assay to an untreated control at 12, 24, 48, 72 and 96 h post-infection. The data indicated that at the 12, 24 and 48 h time points, using a one-way ANOVA test, there was no significant difference (p > 0.05) in progeny virus production, however at 72 and 96 h post-infection there was a significant difference (p < 0.05) in progeny virus production between the treatments compared to the control non-treated infected cells (Fig. 4B). This was also confirmed using indirect immunofluorescence microscopy to detect viral antigens in virus-infected cells at 24, 48, 72 and 96 h post-infection compared to an untreated control (Fig. 5A). There were considerably less foci of infection in the treated cells compared to untreated control infected cells (Fig. 5B) (data shown for the 20 mM concentration of 3-AB). This confirmed the titration data that virus biology was adversely affected by 3-AB, with virus output being reduced to 1–2 log10 at 96 h post-infection compared to 7 log10 for the infected, but untreated control (Fig. 4B).


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)

Immuno-fluorescence analysis of PRRSV infected Marc-145 with and without PARP inhibitor. Cells were incubated for indicated hours after infection and treatment with 20 mM 3-AB or DMSO as control. PRRSV infections were detected by IFA with swine anti-PRRSV serum and FlTC-conjugated rabbit anti-pig IgG antibody. (A) The immuno-fluorescence images were taken at an excitation wavelength of 495 nm with fluorescence microscope equipped with an appropriate filter. (B) Infection rate of Marc-145 determined by IFA. The percentage of infected cells were calculated by dividing the number of fluorescent cells by the total cells in the corresponding field. Each datum was obtained as the mean value from triplicate images.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4414928&req=5

fig0025: Immuno-fluorescence analysis of PRRSV infected Marc-145 with and without PARP inhibitor. Cells were incubated for indicated hours after infection and treatment with 20 mM 3-AB or DMSO as control. PRRSV infections were detected by IFA with swine anti-PRRSV serum and FlTC-conjugated rabbit anti-pig IgG antibody. (A) The immuno-fluorescence images were taken at an excitation wavelength of 495 nm with fluorescence microscope equipped with an appropriate filter. (B) Infection rate of Marc-145 determined by IFA. The percentage of infected cells were calculated by dividing the number of fluorescent cells by the total cells in the corresponding field. Each datum was obtained as the mean value from triplicate images.
Mentions: Different doses of 3-AB were assessed for potential anti-viral activity by infecting cells with the same MOI of PRRSV and then treating cells with 5, 10, and 20 mM 3-AB over the infection period. Progeny virus production was compared by endpoint dilution assay to an untreated control at 12, 24, 48, 72 and 96 h post-infection. The data indicated that at the 12, 24 and 48 h time points, using a one-way ANOVA test, there was no significant difference (p > 0.05) in progeny virus production, however at 72 and 96 h post-infection there was a significant difference (p < 0.05) in progeny virus production between the treatments compared to the control non-treated infected cells (Fig. 4B). This was also confirmed using indirect immunofluorescence microscopy to detect viral antigens in virus-infected cells at 24, 48, 72 and 96 h post-infection compared to an untreated control (Fig. 5A). There were considerably less foci of infection in the treated cells compared to untreated control infected cells (Fig. 5B) (data shown for the 20 mM concentration of 3-AB). This confirmed the titration data that virus biology was adversely affected by 3-AB, with virus output being reduced to 1–2 log10 at 96 h post-infection compared to 7 log10 for the infected, but untreated control (Fig. 4B).

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