| Influenza A virus is still one of the most devastating pathogens which causes threats to humans and animals. Influenza A virus has a wide variety of adaption host and poses great disasters among three subtypes. Influenza A virus belongs to Orthomyxoviridae Family, type A influenza virus. According to antigenic variations of hemagglutinin (HA) and neuraminidase (NA), influenaza A virus can be divided into 18 HA subtypes and 11 NA subtypes. Influenza A virus has a genome of 8 segments, single stranded, negative sense RNA, with a wide variety of infection host, all these characterizations facilitate influenza A virus easy to undergo variation and reassortment. As a non-structural protein, NS1 can interact with many cellular proteins during influenza A virus infection, regulate virus replication cycle and antagonise interferon system of the host. But there are relative few reports about these studies, the regulation mechanism of NS1 is still not very clear. So, different origins of NS1 proteins were studied how to regulate the replication and virulence of influenza A virus, the purpose of which is to provide some theory datas for further study of influenza A virus and developing novel anti-viral drugs.1. Isolation and identification of different subtypes influenza viruses and its evolutionIn February 2013, a novel H7N9 subtype avian influenza virus "jumped" from chickens to humans and caused death to the patients. In order to investigate the origin of human-infecting H7N9, we examined poultry specimens from live bird markets linked to human H7N9 infection in Hangzhou, China. These specimens are five environmental samples, one quail pharyngeal swab, one duck cloacal swab, eight chicken pharyngeal swabs, and three chicken cloacal swabs. Metagenomic sequencing revealed mixed subtypes (H5, H7, H9, N1, N2, and N9). Subsequently, avian influenza virus (AIV) H5N9, H7N9, and H9N2 were isolated from these specimens. Evolutionary analysis showed that the hemagglutinin gene of the novel H5N9 virus originated from A/Muscovy duck/Vietnam/LBM227/2012 (H5N1), which belongs to clade 2.3.2.1. The neuraminidase gene of the novel H5N9 virus originated from human-infecting A/Hangzhou/1/2013 (H7N9). The six internal genes were similar to those of other H5N1, H7N9, and H9N2 virus strains. The novel H5N9 virus harbored the PQRERRRKR/GL motif at the HA cleavage site, which is the characteristic of highly pathogenic AIV. Receptor-binding experiments demonstrated that the virus binds to a-2,3 sialic acid but not a-2,6 sialic acid. This newly isolated H5N9 virus is a highly pathogenic reassortant virus originating from H5N1, H7N9, and H9N2 subtypes. Live bird markets represent a potential transmission risk to public health and the poultry industry.2. Functional analysis and interaction study of heterogeneous nuclear ribonucleoprotein C2 (hnRNP C2) with NS1 protein of influenza A virusAs a regulatory protein, NS1 can interact with multiple cellular proteins during influenza A virus infection. HnRNP C2 is one of the most abundant nuclear proteins, which plays important roles in cleaving, processing, and transporting of cellular pre-mRNAs. This essay has proved that NS1 co-localizes and aggregates with hnRNP C2 in the nucleus, during different subtypes of influenza A virus infection (H3N2, H7N9, H9N2, H1N1 and H5N9). A series of experiments including Co-IP, RNA immunoprecipitation (RNA-IP) and GST Pull-Down demonstrated that RNA is a necessary for the interaction, but it has no specificity to the RNA species. The interaction between NS1 and hnRNP C2 is a common phenomenon among influenza A virus.Fine mapping of hnRNP C2 indicated that the interaction domain is from amino acid (aa) residue 107 to 119, which has additional 13 amino acids compared with hnRNP Cl. Fine mapping of NS1 indicated that the interaction domain is RNA binding domain, which harbors 73 amino acids in the N-terminal.of NS1 protein. Analysis of NS1 1-73 amino acid sequence and dimer structure indicated that this domain was divided into three a helixes:a helix-1, aa.3-25; a helix-2, aa.30-50; a helix-3, aa.54-69. We use "Alanine Scanning" to substitute the basic amino acid with alanine, and construct the corresponding mutation vectors. The Co-IP results demonstrated that multi-mutations including R19AK20AR21A and R35AR37AR38AK41AR44AR46A; single-mutation including R35A, R37A, R38A, K41 A, R46A, and double mutations R38AK41A could not interact with Myc-hnRNP C2, which meant these amino acids played important roles in the interaction.In order to determine whether these mutations in NS1 protein will affect the interaction of mutant virus with endogeneous hnRNP C2, eight plasmids reverse genetic system was used to rescue all the mutant viruses which could not interact with hnRNP C2 in this essay. Co-IP results indicated that the NS1 proteins of all the mutant viruses could not interact with endogenous hnRNP C2. The mutant virus Mut.35-46 replicates inefficiently in Specific Pathogen Free (SPF) chicken embryonated eggs. All the viruses replicate to a constant level when passaged to F7 in SPF chicken embryonated eggs. The HA titer, TCID50, and ELD50 of Mut.35-46 was 6 log2,1045 and 1065, which were much lower than the corresponding values of 9 log2,107.63 and 1085 compared with the H5N9 wild type (WT) virus. The lethal time of the SPF chicken embryonated eggs was delayed to 40 h, however, the WT virus can cause death to SPF chicken embryonated eggs in a short time of 24 h. The pathogenicity of these viruses to BALB/c mice indicated that there were only mild symptom and partial mortality in the group of mutant viruses inoculated mice. In contrast, the mice inoculated with WT virus exhibited signs of illness, anorexia, and dyspnea. The body temperature of the mice infected with WT or mutant virus decreased sharply and reached to the lowest level on the six day post infection (dpi). These mice had almost lost 40% of their body weights by 7 dpi, and died off during the observation period. In contrast, the negative control PBS was not lethal to mice.All together, the basic amino acids 35R,37R,38R,41K,44R,and 46R in the RNA binding domain of NS1 protein play important roles in the interaction with cellular protein hnRNP C2, and further to regulate virus life cycles and the virulence of influenza A virus.3. Functional study of hnRNP C2 during influenza A virus infectionThe genome of H5N9 virus RNA (vRNA), complementary RNA (cRNA), and message RNA (mRNA) were inhibited to replicate in 293T cells over-expression with Flag-hnRNP C2, viral proteins were further inhibited to translate. The virus titer was decreased to 104.62 TCID50 at 40 h post infection compared with negative control. Similarly, the genome of H5N9 virus RNA (vRNA), complementary RNA (cRNA), and message RNA (mRNA) were promoted to replicate in the hnRNP C2 knock down 293T cells, and the viral proteins level was also promoted. The virus titer was raised by nearly 104 TCID50 at 48 h post infection compared with negative control. |
PDF Full Text Request