- 作者: Yi-Ju Han, Kuo-Ming Lee, Guan-Hong Wu, Yu-Nong Gong, Avijit Dutta & Shin-Ru Shih
- 作者服務機構: 1.Department of Laboratory Science, Linkou Chang Gung Memorial Hospital, Taoyuan, Taiwan 2.Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, Taoyuan, Taiwan 3.Division of Infectious Diseases, Department of Medicine, Linkou Chang Gung Memorial Hospital, Taoyuan, Taiwan 4.Graduate Institute of Biomedical Science, College of Medicine, Chang Gung University, Taoyuan, Taiwan 5.Graduate Institute of Health Industry Technology, College of Human Ecology, Chang Gung University of Science and Technology, Taoyuan, Taiwan 6.International Master Degree Program for Molecular Medicine in Emerging Viral Infections, College of Medicine, Chang Gung University, Taoyuan, Taiwan 7.Research Center for Chinese Herbal Medicine, Chang Gung University of Science and Technology, Taoyuan, Taiwan 8.Research Center for Food and Cosmetic Safety, Chang Gung University of Science and Technology, Taoyuan, Taiwan 9.Research Center of Emerging Virus Infection, Division of Biotechnology, College of Medicine, Chang Gung University, Taoyuan, Taiwan
- 中文摘要:
- 英文摘要:
Background
The association between M segment splicing and pathogenicity remains ambiguous in human influenza A viruses. In this study, we aimed to investigate M splicing in various human influenza A viruses and characterize its physiological roles by applying the splicing inhibitor, herboxidiene.
Methods
We examined the M splicing of human H1N1 and H3N2 viruses by comparing three H1N1 and H3N2 strains, respectively, through reverse transcriptase-polymerase chain reaction (RT-PCR) analyses. We randomly selected M sequences of human H1N1, H2N2, and H3N2 viruses isolated from 1933 to 2020 and examined their phylogenetic relationships. Next, we determined the effects of single nucleotide variations on M splicing by generating mutant viruses harboring the 55C/T variant through reverse genetics. To confirm the importance of M2 splicing in the replication of H1N1 and H3N2, we treated infected cells with splicing inhibitor herboxidiene and analyzed the viral growth using plaque assay. To explore the physiological role of the various levels of M2 protein in pathogenicity, we challenged C57BL/6 mice with the H1N1 WSN wild-type strain, mutant H1N1 (55T), and chimeric viruses including H1N1 + H3wt and H1N1 + H3mut. One-tailed paired t-test was used for virus titer calculation and multiple comparisons between groups were performed using two-way analysis of variance.
Results
M sequence splice site analysis revealed an evolutionarily conserved single nucleotide variant C55T in H3N2, which impaired M2 expression and was accompanied by collinear M1 and mRNA3 production. Aberrant M2 splicing resulted from splice-site selection rather than a general defect in the splicing process. The C55T substitution significantly reduced both M2 mRNA and protein levels regardless of the virus subtype. Consequently, herboxidiene treatment dramatically decreased both the H1N1 and H3N2 virus titers. However, a lower M2 expression only attenuated H1N1 virus replication and in vivo pathogenicity. This attenuated phenotype was restored by M replacement of H3N2 M in a chimeric H1N1 virus, despite low M2 levels.
Conclusions
The discrepancy in M2-dependence emphasizes the importance of M2 in human influenza A virus pathogenicity, which leads to subtype-specific evolution. Our findings provide insights into virus adaptation processes in humans and highlights splicing regulation as a potential antiviral target. - 中文關鍵字:
- 英文關鍵字: Infuenza A virus, Pathogenicity, Splicing, Herboxidiene, Subtype-specifc signature, M2 protein