Carbon Monoxide Inhibits Porcine Reproductive and Respiratory Syndrome Virus Replication by the Cyclic GMP/Protein Kinase G and NF-κB Signaling Pathway

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses to the pork industry worldwide each year. Our previous research demonstrated that heme oxygenase-1 (HO-1) can suppress PRRSV replication via an unknown molecular mechanism. In this study, inhibition of PRRSV replication was demonstrated to be mediated by carbon monoxide (CO), a downstream metabolite of HO-1. Using several approaches, we demonstrate that CO significantly inhibited PRRSV replication in both a PRRSV permissive cell line, MARC-145, and the predominant cell type targeted during in vivo PRRSV infection, porcine alveolar macrophages (PAMs). Our results showed that CO inhibited intercellular spread of PRRSV; however, it did not affect PRRSV entry into host cells. Furthermore, CO was found to suppress PRRSV replication via the activation of the cyclic GMP/protein kinase G (cGMP/PKG) signaling pathway. CO significantly inhibits PRRSV-induced NF-B activation, a required step for PRRSV replication. Moreover, CO significantly reduced PRRSV-induced proinflammatory cytokine mRNA levels. In conclusion, the present study demonstrates that CO exerts its anti-PRRSV effect by activating the cellular cGMP/PKG signaling pathway and by negatively regulating cellular NF-B signaling. These findings not only provide new insights into the molecular mechanism of HO-1 inhibition of PRRSV replication but also suggest potential new control measures for future PRRSV outbreaks.IMPORTANCE PRRSV causes great economic losses each year to the swine industry worldwide. Carbon monoxide (CO), a metabolite of HO-1, has been shown to have antimicrobial and antiviral activities in infected cells. Our previous research demonstrated that HO-1 can suppress PRRSV replication. Here we show that endogenous CO produced through HO-1 catalysis mediates the antiviral effect of HO-1. CO inhibits PRRSV replication by activating the cellular cGMP/PKG signaling pathway and by negatively regulating cellular NF-B signaling. These findings not only provide new insights into the molecular mechanism of HO-1 inhibition of PRRSV replication but also suggest potential new control measures for future PRRSV outbreaks.

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“…NF‐κB is a transcription factor playing a key role in inflammation, and several studies have demonstrated that it is regulated by the NO/cGMP/PKG signaling pathway . Therefore, we analyzed the phosphorylation of NF‐κB p65 subunit in the nuclear fraction of Molt‐3 cells treated with tadalafil/Sp‐8‐Br‐PET‐cGMP by Western blot analysis, which revealed that phospho‐NF‐κB p65 was downregulated by the drugs, but PKG inhibitor KT5823 (100 nM) reversed the effect (Figure ).…”

A novel intracellularly expressed NS5B-specific nanobody suppresses bovine viral diarrhea virus replication

“…Western blotting was performed as previously described (Zhang et al, 2017) with minor modifications. Cells were harvested and lysed using NP-40 lysis buffer (Beyotime, Shanghai, China) supplemented with protease inhibitor phenylmethanesulfonyl fluoride (PMSF), and the protein concentration was determined using a Bicinchoninic acid Protein Assay Kit (Thermo Fisher Scientific, Shanghai, China).…”

MYH9 Key Amino Acid Residues Identified by the Anti-Idiotypic Antibody to Porcine Reproductive and Respiratory Syndrome Virus Glycoprotein 5 Involve in the Virus Internalization by Porcine Alveolar Macrophages

“…Cell samples were harvested using NP-40 cell lysis buffer 24 h post-PRRSV inoculation unless noted otherwise. Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) and samples were mixed with 5× SDS sample loading buffer before samples containing equal amounts of total protein were loaded onto 12% SDS-PAGE gels and separated proteins were transferred onto PVDF membranes as described previously [36]. Membranes were probed with Mab2-5G2 or 6D10 (against the PRRSV-N protein) or anti-β-actin mAb (Abcam, Cambridge, MA, USA).…”

“…Total RNA was extracted from cells using TRizol reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer's instructions. Reverse transcription and qPCR were conducted using PrimeScript RT reagent Kit (TaKaRa) as previously described [36]. Transcripts of GAPDH were also amplified to normalize the total RNA input.…”