| Although the H9N2 subtype avian influenza virus is a low pathogenic influenza virus,since the H9N2 subtype influenza virus was isolated from poultry flocks in China in 1994,this subtype avian influenza has appeared in many places and continued to spread,which has seriously affected the healthy development of poultry industry.At the same time,the host infection spectrum of this subtype of avian influenza virus is wide and still expanding,causing serious harm to public health.The internal proteins of the influenza A virus(IAV)have evolved slowly compared to the HA and NA proteins exposed on the viral surface,with matrix protein 1(M1)being one of the slowest evolving proteins.As the most abundant IAV protein,the low mutation tolerance of M1 implies that its role in maintaining viral structure and function should not be underestimated.By comparing the full-length gene sequences of H9N2 virus M1(n=2,201),we found that there are five main evolutionary patterns of the M1 gene during the evolution of our H9N2 virus,and more importantly,the pattern differences of M1 correspond to the adaptation and epidemic advantage of the virus.Further analysis found that the differential sites existing between different patterns were mainly located in the binding region of M1 and NEP,the transcriptional repression region of M1,or the binding region of M1 and v RNP,suggesting that these differential sites may have convergent effects on biological functions related to the life cycle of H9N2 viruses.Therefore,starting from the adaptability of M1 protein,this study firstly synthesized 5 kinds of M1 gene sequences,connected with the p HW2000 vector,and applied reverse genetic technology to rescue 5 viruses with different M1 patterns.According to the one-step and multi-step growth kinetic curves of the virus in MDCK cells,it was found that the virus titer of the P5 group was significantly higher than that of the P3 group(p<0.05?0.0001),while the P2,P4,and P1 groups had no significant difference compared with the P3 group(p>0.05).The results of the growth competition test for viruses showed that the differential site of P5 group accounted for74% of SNP sequencing.Therefore,among the five M1 patterns,the proliferation ability and growth competition ability of the P5 group were significantly higher than those of the other groups.In order to explore the internal reasons for the differences in the proliferation ability and growth competition ability of different M1 patterns,we started from the virus replication cycle and further explored the biological effects of M1 in different stages of virus replication.Firstly,we used the acid bypass assay to directly induce virus uncoating and combined it with the dual-luciferase activity assay to determine the difference in uncoating ability among the five M1 patterns.The results showed that the shelling ability of the P5,P4,and P2 groups were significantly higher than that of the P3 group(p<0.05?0.0001),while there was no significant difference between the P1 group and the P3 group(p>0.05).Viral polymerase activity is a key factor determining the efficiency of vRNA replication and transcription,and M1 after entering the nucleus plays a role in regulating polymerase activity.Therefore,we measured viral polymerase activity using a dual-luciferase activity assay.The results showed that the polymerase activity of the P5 and P4 groups was significantly higher than that of the P3 group(p<0.001),while there was no significant difference between the P2 and P1 groups and the P3 group(p>0.05).After M1 enters the nucleus,it can timely terminate the continued transcription of viral mRNA and assist v RNP to exit the nucleus.Therefore,we detected the mRNA levels by q PCR,and the results showed that at 4-10 hpi,the mRNA levels of the P5 and P4 groups were significantly higher than those of the P3 group(p<0.01?0.0001),while at 10-12 hpi,the mRNA levels of the P5 and P4 groups decreased.The trend was more obvious than that in the P3 group.Therefore,it is speculated that the transcription efficiency of the P5 and P4 groups is higher than that of the P3 group in the early stage,and the transcription of the virus is timely terminated in the late stage so that the v RNP can be rapidly exported from the nucleus after assembly.To examine the ability of M1 to assist in the nuclear export of v RNP,we isolated nuclear and cytoplasmic fractions and examined the levels of vRNA in the nucleus and cytoplasm by q PCR.The results showed that at4-8 hpi,the levels of vRNA in the nuclei of the P5,P4,and P2 groups were extremely significantly higher than those of the P3 group(p<0.001?0.0001),and there was no significant difference between the P1 group and the P3 group(p>0.05).Then at8-12 hpi,the vRNA levels of the P5,P4,and P2 groups gradually flattened,and the P1 and P3 groups still showed an upward trend.In the cytoplasm,the vRNA levels of the P5,P4,and P2 groups at 8-12 hpi were significantly higher than P3 group(p<0.0001),there was no significant difference between P1 group and P3 group(p>0.05).This proves that in the process of v RNP nuclear export,M1 in P5,P4,and P2 groups can assist the rapid nuclear export of vRNA.During the virus assembly and budding process,we quantitatively detected the content of M1 protein in the cytoplasm and cytoplasmic membrane fractions by Western blot and further detected the effect of different patterns of M1 on the virus budding ability by in vitro budding assay.The results showed that the binding ability of M1 to the cytoplasmic membrane and the budding ability of the P5,P2,and P4 groups were significantly higher than those of the P3 group(p<0.0001),while there was no significant difference between the P1 group and the P3 group(p>0.05).In conclusion,by examining the biological effects of five different M1 patterns on virus uncoating,viral polymerase activity,viral genome transcription,exit of v RNP from the nucleus,virus assembly,and budding,we found that there are five major evolutionary patterns of the M1 gene of the H9N2 virus in China.There is a strict correspondence between adaptation and epidemic dominance,especially the P5 group showed a very significant replication dominance in the virus replication cycle,indicating that M1/P5 is the dominant gene pattern in the evolutionary adaptation of the H9N2 subtype avian influenza virus.Therefore,this study revealed the role of the genetic polymorphism of the M1 gene in the replication of the H9N2 virus and provided a basis for the scientific evaluation of the molecular mechanism of H9N2 virus adaptive differences and the reasons for the change of epidemic dominance. |
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