Gene expression analysis during recovery process indicates the mechanism for innate immune injury and repair from Coxsackievirus B3-induced myocarditis.

To investigate the innate immune injury and repair mechanism during recovery from Coxsackievirus B3 (CVB3) induced myocarditis, we established an acute viral myocarditis recovery model by infecting BALB/c mice with CVB3. Histopathological examination of cardiac tissues after infection showed a gradual increase of myocardial injury to the maximum degree at 8 dpi (days post infection), followed by a recovery process with reduced viral replication. We also measured expression changes of innate immune genes in heart after 4, 8 and 12 days of infection using innate immune real-time PCR array. The results showed expression alterations in many Pattern Recognition Receptors (PRRs) genes upon CVB3 infection, which activated multiple important signaling pathways during recovery process. The expression of TLRs, RLRs, PKR and cytokines were strongly induced and reached the peak at 4 dpi in early myocarditis stage, followed by a gradual reduction in recovery stage, during which the levels were even lower than normal at 12 dpi. The strong correlation between cardiac histopathology score and chemokine expression level suggested that the chemokines might play a role in pathological changes during early myocarditis stage. In addition, we also found that both cell survival signaling pathways (AKT1, p38MAPK) and antiviral signaling pathways (IKKα/ß/ε) were activated and promoted the recovery during late myocarditis stage. Altogether, our observations improved the understanding of formation and progression of the pathological lesions, as well as the repair mechanism for acute viral myocarditis.

Coxsackievirus B3 2A protease promotes encephalomyocarditis virus replication.

To determine whether 2A protease of the enterovirus genus with type I internal ribosome entry site (IRES) effect on the viral replication of type II IRES, coxsackievirus B3(CVB3)-encoded protease 2A and encephalomyocarditis virus (EMCV) IRES (Type II)-dependent or cap-dependent report gene were transiently co-expressed in eukaryotic cells. We found that CVB3 2A protease not only inhibited translation of cap-dependent reporter genes through the cleavage of eIF4GI, but also conferred high EMCV IRES-dependent translation ability and promoted EMCV replication. Moreover, deletions of short motif (aa13-18 RVVNRH, aa65-70 KNKHYP, or aa88-93 PRRYQSH) resembling the nuclear localization signals (NLS) or COOH-terminal acidic amino acid motif (aa133-147 DIRDLLWLEDDAMEQ) of CVB3 2A protease decreased both its EMCV IRES-dependent translation efficiency and destroy its cleavage on eukaryotic initiation factor 4G (eIF4G) I. Our results may provide better understanding into more effective interventions and treatments for co-infection of viral diseases.

Infection of prions and treatment of PrP106-126 alter the endogenous status of protein 14-3-3 and trigger the mitochondrial apoptosis possibly via activating Bax pathway.

The 14-3-3 proteins are a family of highly homologous and ubiquitously expressed isoforms that are involved in a wide variety of physiological processes. 14-3-3 have showed actively molecular interaction with PrP and positive 14-3-3 is frequently observed in the cerebrospinal fluid (CSF) samples of the patients with sporadic Creutzfeldt-Jakob disease (CJD). However, the alterations of 14-3-3 in the brain tissues of patients with prion diseases remain little addressed. To address the possible change of brain 14-3-3 during prion infection, we firstly tested the levels of 14-3-3 in the brain tissues of scrapie agent 263 K infected hamsters. Obviously decreased 14-3-3 were observed in the samples of the infected animals, showing time-dependent reduction in the incubation period, while the amounts of S-nitrosylated 14-3-3 were increased in the brains collected at the late stage. A low level of 14-3-3 was also observed in the scrapie infectious cell line SMB-S15, accompanied with up-regulated Bax and down-regulated Bcl-2. Moreover, we found that treatment of PrP106-126 on the cultured cells decreased the cellular 14-3-3 and caused translocations of cellular Bax to the membrane fractions. Knockdown of cellular 14-3-3 sensitized the cultured cells to the challenge of PrP106-126. These data illustrate that significant down-regulation of brain 14-3-3 levels during prion infection may not only be a scenario of the terminal consequence of interacting with abnormal PrP(Sc) but may also participate in the pathogenesis of neuronal damage.

[Study on PrP106-126 peptide disturbed dimeration of 14-3-3beta].

OBJECTIVE: To investigate both PrP and PrP106-126 peptide effect on 14-3-3beta dimeration. METHODS: 14-3-3beta were incubated with different does recombinant PrP or PrP106-126 peptide, both 14-3-3beta dimer and polymer were separated 15% non-denaturing polyacrylamide gel electrophoresis (PAGE) and the 14-3-3 dimers were evaluated using 14-3-3beta-specific Western blotting. And then,14-3-3beta dimeration buffer were incubated with different does recombinant PrP and 250 micromol/L PrP106-126 peptide, 14-3-3beta dimer and polymer were detected by above methods. Cellular 14-3-3 dimer were also detected after PrP106-126 peptide were added to HeLa cell for 8 hours. RESULTS: Recombinant full-length PrP facilitated the dimerization of 14-3-3beta and PrP106-126 disturbed 14-3-3beta dimeration as both have dose dependence effect. PrP antagonized PrP106-126-induced 14-3-3beta dimer with PrP protein increase in vitro. Cellular 14-3-3 dimerization also decreased after treatment of peptide PrP106-126 on HeLa cells for 8 hours. CONCLUSION: [corrected] Dimerization process of 14-3-3beta was promoted by full-length PrP (PrP23-231) but inhibited by peptide PrP106-126 in vitro. PrP agonized PrP106-126-induced inhibition of 14-3-3 dimeration. PrP106-126 inhibited cellular 14-3-3 dimerization.

[The 2A protease of enterovirus 71 cleaves nup62 to inhibit nuclear transport].

To study the impact of the enterovirus 71(EV71) on the nuclear transport mechanism,The pGFP-NLS vector with nuclear location signal(NLS) was constructed, RD cells transfected by the pGFP-NLS vector were inoculated with the EV71 or cotransfected by EV71-2A vector. The results showed that GFP protein with NLS was expressed in the cytoplasm due to the inhibition of nuclear transport. In order to further study the mechanism of the EV71 to prevent nuclear transport,Nup62 was detected by Western blotting after RD cells were infected with EV71 or transfected by EV71-2A vector. The results showed that decreased expression of Nup62 could be detected after infection with EV71 and transfection by EV71-2A vector. This study demonstrates that the cleavage of Nup62 by EV71 2A protease may be the mechanism of nuclear transport inhibition.