Potential Role Of HMGB1 In PRRSV Infection And Antiviral Activity Of Glycyrrhizin Against PRRSV

Porcine reproductive and respiratory syndrome virus (PRRSV) is a non-segmented, single-stranded positive-sense RNA virus, which is classified within the Arteriviridae Arterivirus family, and cause porcine reproductive and respiratory syndrome (PRRS). PRRS is an economically important infectious disease characterized by severe interstitial pneumonia. High mobility group box 1 (HMGB1) is an endogenous damage-associated molecular pattern molecule involved in the pathogenesis of various infectious agents. Badaoui et al proposed that HMGB1 might be a possible target of PRRSV in the manipulation of the host immune response, while the function of HMGB1 in PRRSV pathogenesis is unclear. In this study, we found that HMGB1 has no effect on PRRSV replication, but enhances the efficiency of virus-induced inflammatory responses. The major studies include:1. HMGB1 levels are significantly increased in PRRSV-challenged pigletsPRRSV-negative pigs were challenged with PRRSV, real-time RT-PCR and ELISA assay showed that pigs inoculated with PRRSV showed a significant elevation in HMGB1 levels. Moreover, a positive correlation was observed between HMGB1 level and viral load and serum HMGB1 was also positively associated with TNF-α, IFN-γ, IL-1β, IL-6 and IL-8 expression in serum.2. PRRSV infection triggers HMGB1 releaseIndirect immunofluorescence assay showed that HMGB1 translocates from the nucleus to cytoplasm in both PRRSV infected MARC-145 cells and PAMs. ELISA assay showed that PRRSV triggers the secretion of HMGB1 to the extracellular milieu in MARC-145 cells and PAMs in dose-dependent manner. Furthermore, the release of HMGB1 induced by PRRSV was likely replication-dependent because UV-irradiated PRRSV did not significantly induce HMGB1 release.3. HMGB1 does not significantly affect PRRSV proliferationIn our current study, we used three different techniques to investigate the role of HMGB1 in PRRSV replication:knockdown of endogenous HMGB1 with specific siRNAs; blocking extracellular HMGB1 with an HMGB1 neutralizing antibody; and the extracellular addition of purified recombinant HMGB1 protein. Results from the three approaches clearly showed that HMGB1 does not significantly affect PRRSV proliferation under our experimental conditions.4. HMGB1 enhances PRRSV-induced NF-κB activationTo examine whether HMGB1 participates in PRRSV-induced NF-κB activation, MARC-145 cells were transfected with HMGBl-specific siRNAs. The rusults showed that knockdown of HMGB1 inhibited PRRSV-induced NF-κB promoter activity, and phosphorylation levels of p65 in MARC-145 cells. In contrast, addition of recombinant HMGB1 enhanced PRRSV-induced NF-κB activation and phosphorylation levels of p65.5. HMGBl promotes PRRSV-induced expression of inflammatory cytokinesTo further explore the effects of HMGB1 on PRRSV-induced inflammatory cytokines expression, PRRSV-infected MARC-145 cells and PAMs were transfected with HMGB1-specific siRNAs or treated with purified HMGB1 protein. Cells were collected for detecting cytokine message expression by real-time RT-PCR. The results showed that knockdown of HMGB1 inhibited PRRSV-induced IL-1β,IL-6, IL-8, RANTES and TNF-a mRNA expression levels. In contrast, addition of recombinant HMGB1 enhanced these effects, suggesting that it plays an important role in enhancing expression of inflammatory cytokines.6. HMGB1 receptors are involved in PRRSV-induced inflammatory responseTo dissect the signal pathways involved in HMGB1-promoted inflammatory effect during PRRSV infection, specific siRNA targeting RAGE, TLR2, TLR4 and TLR9 were synthesized respectively. Then the siRNAs targeting RAGE or TLRs (TLR2,4,9) were transfected in MARC-145 cells before PRRSV infection. The results showed that specific siRNAs targeting RAGE, TLR2 and TLR4 significantly inhibited NF-κB transcriptional activity, and IL-1β, IL-6, IL-8, RANTES and TNF-a mRNA expression in PRRSV-infected MARC-145 cells. Collectively, these findings suggested that HMGB1 may act as a mediator for NF-κB activation and inflammatory cytokine production through RAGE, TLR2 and TLR4 receptors in PRRSV-infected MARC-145 cells.7. Glycyrrhizin inhibits PRRSV proliferationGlycyrrhizin is a natural component extracted from the roots of Glycyrrhiza glabra. We treated the cells with different concentrations of glycyrrhizin, and found that glycyrrhizin significantly reduced PRRSV replication and PRRSV-encoded protein expression in a dose-dependent manner. Furthermore, glycyrrhizin mainly inhibits the penetration stage, rather than the step of adsorption and release of PRRSV replication cycle, and a direct inhibitory action of glycyrrhizin on PRRSV particles could also be excluded. Animal studies found that glycyrrhizin reduced virus load in serum, and delayed the process of PRRS in vivo. Together, glycyrrhizin may be a candidate component for a novel PRRS control strategy.