Mechanisms Of Porcine Circovirus Type 2 Manipulation Of Host Cell Stress Responses In Favor Of Its Replication

Porcine circovirus type 2(PCV2)is an important pathogen responsible for porcine circovirus associated disease(PCVAD),which causes significant economic losses to the global pig industry.Not all pigs infected by PCV2 develop clinical PCVAD,while coinfection or secondary infection with other swine pathogens,or environmental stress can exacerbate PCVAD.Due to its small genome size,PCV2 proliferation relies heavily on the host cell machinery,thereby triggering cellular stresses,such as endoplasmic reticulum(ER)stress,oxidative stress and autophagy.Our laboratory has demonstrated that PCV2 could modulate cellular stress responses through interaction with host cellular factors to facilitate its replication.High mobility group box 1 protein(HMGB1)is a major chromatin-associated non-histone protein with multifunction in different cellular compartments.It is normally located in the nucleus to acts as a chromosome guardian and DNA chaperone.Reactive oxygen species(ROS)is known to promote HMGB1 translocation to the cytosol.Accumulating evidence has revealed that HMGB1translocation occurs in response to virus infection and plays an important role in the progression of viral infection.However,it remains unknow:(1)whether and how PCV2-induced ER stress triggered oxidative stress;(2)the relationship between increased ROS generation during PCV2 infection and distribution of HMGB1;(3)if and how HMGB1 impacts PCV2 replication.In this study,we conduct a systematic study on these scientific issues to elucidate the molecule mechanisms that PCV2manipulates host cell stress responses favoring its replication.Major research achievements are summarized as follows:1.Nuclear HMGB1 negatively regulated PCV2 replicationExpression and distribution of HMGB1 in PK-15 cells and 3D4/31 cells(porcine alveolar macrophage cell line)infected with PCV2 was analyzed by immunofluorescence and western blotting.We found that PCV2 infection could promote HMGB1 translocation from nuclei to cytoplasm without affecting its transcription and translation.To investigate the effects of HMGB1 on PCV2 replication,we regulated HMGB1 level or its distribution in infected cells by overexpression,RNA silencing or chemical inhibitors.HMGB1 over-expression resulted in marked reduction of PCV2-infected cells,viral genome copies and Cap expression.HMGB1 knockdown significantly facilitate replication and proliferation of PCV2.We treated PCV2-infected cells with HMGB1 inhibitor to examine if retention of HMGB1 in the nuclei would impact PCV2 replication,and found that ethyl pyruvate could effectively suppress HMGB1 migration and PCV2 replication.Inhibition of extracellular HMGB1 activity by glycyrrhizin did not affect viral replication.These results support the notion that nuclear HMGB1 contributes to repression of PCV2 replication.2.HMGB1 interacted with the Ori region of PCV2 genome to inhibit viral replicationTo characterize the critical domain of HMGB1 responsible for restriction of PCV2replication,full-length and truncated versions of HMGB1(A box?AB box and B box CT)were transiently expressed in PCV2-infected cells.Overexpression of the truncated peptides containing the B box domain led to marked inhibition of Cap expression and viral genome copies in levels comparable to that of full-length HMGB1,which indicates that HMGB1 B box domain is involved in repression of PCV2 replication.The full-length PCV2 genome was divided into several DNA fragments(Ori,orf1,orf2,Ori-orf1 and Ori-orf2)to identify the region of the PCV2 genome that might interact with HMGB1.We found that nuclear HMGB1 bound directly to the Ori region of the PCV2 genome in the infected cells through the gel-shift assay and immunoprecipitation coupled with quantitative PCR(q PCR).3.PCV2 infection increased ROS generation that facilitated nucleocytoplasmic translocation of HMGB1 and facilitated viral replicationFlow cytometric method was performed to measure cytosolic ROS levels in PK-15 cells.Distribution of HMGB1 was also examined after treatment of PCV2-infected cells with N-acetylcysteine(NAC)and H2O2.Increased ROS generation and nucleocytoplasmic migration of HMGB1 in PCV2 infected cells could be readily observed.Mitigation of ROS by the antioxidant NAC could effectively increase nuclear HMGB1 retention and inhibit PCV2 replication.Treatment with H2O2 further exacerbated nucleocytoplasmic translocation of HMGB1 and even facilitated viral replication.It is,therefore,clear that PCV2 infection induces elevation of cellular ROS and that increased ROS reduces the HMGB1 level in the nuclei by facilitating migration of nuclear HMGB1 into the cytoplasmic compartment,thus derepressing PCV2replication from suppressive nuclear HMGB1.4.PCV2 infection induced ER stress and oxidative stress to promote HMGB1migrationWe previously reported that PCV2 infection could selectively activates the PERK branch of the UPR via the PERK-e IF2?-ATF4-CHOP axis.ERO1?(Endoplasmic reticulum oxidoreductase 1?)that is transcriptionally regulated by CHOP catalyzes disulfide bond formation and transfers electrons on to molecular oxygen,leading to generation of H2O2.In order to prove that HMGB1 migration is due to ROS generated from ER via PERK branch of UPR with ERO1?activation,RNA silencing or GSK inhibition of PERK or EN460 inhibition of ERO1?activity was approached.Inhibition of either PERK or ERO1?led to reduced migration of HMGB1 out of the nuclei,decreased ROS accumulation and viral replication.Together,these data clearly reveal that PCV2 could activate PERK-dependent ERO1?induction with generation of ROS sufficient to promote nucleocytoplasmic translocation of HMGB1,thus favoring its replication by diminishing the restrictive effect of nuclear HMGB1 on viral DNA replication.5.Molecular mechanism that PCV2 disturbed redox homeostasis in the ER to promote HMGB1 migrationTo identify the viral proteins inducing ERO1?expression and generation of ROS,individual PCV2 proteins(Rep,Cap,ORF3,ORF4,and ORF5 protein)were expressed in PK-15 cells.Both Rep and Cap proteins were potent inducer of ERO1?activation with ROS accumulation and redistribution of HMGB1.Individual point substitutions were introduced into the proteins Cap at Cys108 and Rep at Cys107 or Cys305according to the analysis of cysteine residues.We found that the mutant proteins lost their ability to activate the PERK-ERO1?axis and failed to promote nucleocytoplasmic migration of HMGB1,while the wild-type proteins did.To further identify the roles of these three cysteine residues during infection,reverse genetic approach was used to construct mutant PCV2 strains with one or all three cysteine-to-serine substitutions[PCV2^Rep1(C107S),PCV2^Rep2(C305S),PCV2^Cap1(C108S)and PCV2^Rep1/2-Cap1].Notably,PCV2^Rep1/2-Cap1 failed to activate PERK and ERO1?with reduced ROS generation and increased retention of HMGB1 in the nuclei.These findings indicate that cysteine residues at the protein level are different from those at the virus level in activating PERK and ERO1?partly because of differences in the expression level of the proteins in two different systems.However,synergistic involvement of two or more cysteine residues at the virus level might have more contribution to increased ROS generation as a result of oxidative disulfide folding.In conclusion,this study provides a novel insight into the mechanism of PCV2evasion strategy:HMGB1 in the nuclei is an antiviral restriction factor against PCV2replication by binding of its B box domain to the Ori region of the PCV2 genome.PCV2manipulates ER to perturb its redox homeostasis via the PERK-ERO1?axis and the ER-sourced ROS from oxidative folding is sufficient to reduce HMGB1 retention in the nuclei,thus preventing viral genome from HMGB1 sequestration for its replication.This study will also help decision making on the use of antioxidants in the feeding regime on pig farms that suffer from PCVAD.