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Porcine HMGCR Inhibits Porcine Circovirus Type 2 Infection by Directly Interacting With the Viral Proteins

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Porcine HMGCR Inhibits Porcine Circovirus Type 2 Infection by Directly Interacting With the Viral Proteins

Ting Ouyang et al. Viruses.

Abstract

Porcine circovirus type 2 (PCV2) is the etiological agent of porcine circovirus diseases and porcine circovirus-associated diseases (PCVDs/PCVADs). However, the pathogenesis of PCV2 is not fully understood. We previously found that 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) is negatively associated with PCV2 infection in vitro and in vivo. HMGCR inhibits the early stages of PCV2 infection, while PCV2 infection induces the phosphorylation of HMGCR to inactivate the protein. In this study, we investigated the possibility that adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK), and protein phosphatase 2 (PP2A) participate in HMGCR-mediated inhibition of PCV2 infection and the interaction of porcine HMGCR with PCV2 proteins. The results showed that AMPK activity fluctuated in cells during the early stage of PCV2 infection, while PP2A had little effect on PCV2 infection and HMGCR activity. Furthermore, PCV2 infection may enhance or maintain the level of phosphorylated HMGCR by directly interacting with the protein in PK-15 cells. These findings may provide a better understanding of PCV2 pathogenesis, and HMGCR may be a novel PCV2 antiviral target.

Keywords: 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR); adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK); interaction; porcine circovirus type 2 (PCV2); protein phosphatase 2 (PP2A).

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
PCV2 infection increases phosphorylation of PP2A, AMPK and HMGCR. Cells were infected with PCV2 and examined using western blot and real-time PCR at the indicated time points. The experiments were repeated at least three times. All data are the means ± standard error (SD) of three independent experiments. *, p < 0.05, **, p < 0.01, ***, p < 0.001, and ****, p < 0.0001. (A) Western blot analysis and densitometric quantification of the indicated proteins. The graph (lower panel) represents the relative quantification (arbitrary unit) of each protein normalized to β-actin. The bar represents the mean of three independent experiments. (B) Real-time PCR of PCV2.
Figure 2
Figure 2
Inhibition of HMGCR by lovastatin has no effect on the activity of AMPK and PP2A during PCV2 infection. Cells were treated with lovastatin (20 μM) or 0.5% DMSO and infected with PCV2, then analyzed by western blot at the indicated time points. The experiments were repeated at least three times.
Figure 3
Figure 3
PP2A has little effect on PCV2 infection and HMGCR activity. Cells were treated with FTY720 (PP2A activator) or okadaic acid (PP2A inhibitor) and infected with PCV2, and analyzed by western blot and real-time PCR at the indicated time points. The results are expressed as the mean ± SD of three independent experiments. The experiments were repeated at least three times. **, p < 0.01, ***, p < 0.001, and ****, p < 0.0001. (A) Cytopathic effects of drugs. (B) Real-time PCR. (C) Western blot.
Figure 4
Figure 4
HMGCR activity is mainly regulated by AMPK during PCV2 infection. Cells were treated with compound C (AMPK inhibitor) or metformin (AMPK activator) and infected with PCV2, and analyzed by western blot at the indicated time points. The results are expressed as the mean ± SD of three independent experiments. The experiments were repeated at least three times. *, p < 0.05, ***, p < 0.001, and ****, p < 0.0001. (A) Cytopathic effects of drugs. (B) Western blot.
Figure 5
Figure 5
Construction scheme of the plasmids used in this study. The Cap and Rep genes of PCV2 were cloned into Lenti-CAG-WPRE by MluI and AgeI to generate recombinant expression plasmids pHA-Rep (A,D) and pMyc-Cap (B,E), respectively and were further verified by PCR. The porcine HMGCR gene was cloned into pCDNA3.1(+) by BamH I and EcoR I to generate the expression plasmid pFlag-HMGCR (C) and confirmed by double digestion with BamH I and EcoR I (F).
Figure 6
Figure 6
Interaction between HMGCR and PCV2 Cap protein. (A) Colocalization of HMGCR and PCV2 Cap protein. HMGCR (Cy3; red), PCV2 Cap protein (FITC; green), DAPI (blue) and overlap (yellow), scale bar: 10 nm. (B) Co-IP of HMGCR and PCV2 Cap protein. Flag (HMGCR), Myc (Cap).
Figure 7
Figure 7
Interaction between HMGCR and PCV2 Rep protein. (A) Colocalization of HMGCR and PCV2 Rep protein. HMGCR (Cy3; red), PCV2 Rep protein (FITC; green), DAPI (blue) and overlap (yellow), scale bar: 10 nm. (B) Co-IP of HMGCR and PCV2 Rep protein. Flag (HMGCR), HA (Rep).

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