Chinese giant salamander Bcl-w: An inhibitory role in iridovirus-induced mitochondrial apoptosis and virus replication.

B-cell lymphoma-2 (BCL-2) superfamily molecules play crucial roles in mitochondrial apoptosis induced by Chinese giant salamander iridovirus (GSIV). As an anti-apoptotic molecule in the BCL-2 family, the molecular mechanism of Bcl-w during GSIV infection remains unknown. In this study, we characterized for the first time an amphibian Bcl-w from Chinese giant salamander Andrias davidianus (AdBcl-w), and its function and regulatory mechanism during GSIV infection were investigated. AdBcl-w possesses the conserved structural features of Bcl-w and shares 35-54% sequence identities with other Bcl-w. mRNA expression of AdBcl-w was most abundant in liver and muscle. The AdBcl-w mRNA expression was regulated during GSIV infection. Western blotting assays revealed that the level of Bcl-w protein was downregulated markedly as the infection progresses. Confocal microscopy showed that overexpressed AdBcl-w was translocated to the mitochondria after infection with GSIV. Flow cytometry analysis demonstrated that compared with control, the apoptotic progress in cells transfected with AdBcl-w was reduced while that in cells transfected with AdBcl-w siRNA was enhanced. The number of virus major capsid protein gene copies was lower and protein synthesis was reduced in AdBcl-w overexpressing cells. In addition, AdBcl-w could bind directly to the pro-apoptotic molecule AdBak, while this interaction was weakened with GSIV infection. Moreover, p53 level was reduced and the mRNA expression levels of crucial regulatory molecules in the p53 pathway were regulated in AdBcl-w overexpressing cells during GSIV infection. These results suggested that AdBcl-w inhibit GSIV replication by regulating the virus induced mitochondrial apoptosis.

Nonstructural protein NS17 of grass carp reovirus Honghu strain promotes virus infection by mediating cell-cell fusion and apoptosis.

Fusion-associated small transmembrane (FAST) proteins can promote cell fusion, alter membrane permeability and trigger apoptosis to promote virus proliferation in orthoreoviruses. However, it is unknown whether FAST proteins perform these functions in aquareoviruses (AqRVs). Non-structural protein 17 (NS17) carried by grass carp reovirus Honghu strain (GCRV-HH196) belongs to the FAST protein family, and we preliminarily explored its relevance to virus infection. NS17 has similar domains to FAST protein NS16 of GCRV-873, comprising a transmembrane domain, a polybasic cluster, a hydrophobic patch and a polyproline motif. It was observed in the cytoplasm and the cell membrane. Overexpression of NS17 enhanced the efficiency of cell-cell fusion induced by GCRV-HH196 and promoted virus replication. Overexpression of NS17 also led to DNA fragmentation and reactive oxygen species (ROS) accumulation, and it triggered apoptosis. The findings illuminate the functions of NS17 in GCRV infection, and provide a reference for the development of novel antiviral strategies.

Molecular mechanism of Cuscutae semen-radix rehmanniae praeparata in relieving reproductive injury of male rats induced with tripterygium wilfordii multiglycosides: A tandem mass tag-based proteomics analysis.

Background: We determined the effects of Cuscutae semen (Cuscuta chinensis Lam. or Cuscuta australis R. Br.)-Radix rehmanniae praeparata (Rehjnannia glutinosa Libosch.) on the protein levels in testicular tissues of rats gavaged with tripterygium wilfordii multiglycosides (GTW) and elucidated the molecular mechanism underlying Cuscutae semen-Radix rehmanniae praeparata for relieving GTW-induced reproductive injury. Methods: A total of 21 male Sprague-Dawley rats were randomly divided into the control group, model group, and Cuscutae semen-Radix rehmanniae praeparata group based on their body weights. The control group was given 10 mLkg-1 of 0.9% normal saline by gavage daily. The model group (GTW group) was administered with 12 mg kg-1 GTW by gavage daily. Cuscutae semen-Radix rehmanniae praeparata group (the TSZSDH group) was administered with 1.56 gkg-1 of Cuscutae semen-Radix rehmanniae praeparata granules daily according to their model group dosing. The serum levels of luteinizing hormone, follicle-stimulating hormone, estradiol, and testosterone were measured after 12 weeks of continuous gavage, and the pathological lesion of testicular tissues was observed. Differentially expressed proteins were evaluated by quantitative proteomics and verified by western blotting (WB) and Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR). Results: Cuscutae semen-Radix rehmanniae praeparata can effectively relieve pathological lesions of GTW-induced testicular tissues. A total of 216 differentially expressed proteins were identified in the TSZSDH group and model group. High-throughput proteomics revealed that differentially expressed proteins are closely associated with the peroxisome proliferator-activated receptor (PPAR) signaling pathway, protein digestion and absorption, and protein glycan pathway in cancer. Cuscutae semen-Radix rehmanniae praeparata can significantly upregulate the protein expressions of Acsl1, Plin1, Dbil5, Plin4, Col12a1, Col1a1, Col5a3, Col1a2, Dcn, so as to play a protective role on testicular tissues. Acsl1, Plin1, and PPARγ on the PPAR signaling pathway were verified by WB and RT-qPCR experiments, which were found to be consistent with the results of proteomics analysis. Conclusion: Cuscutae semen and Radix rehmanniae praeparata may regulate the PPAR signaling pathway mediated Acsl1, Plin1 and PPARγ to reduce the testicular tissue damage of male rats caused by GTW.