The Study On Interaction Between NS2 Of Influenza A Virus And Host Factors CHD3 And Characterization Of The Genes Encoding Complete US10, Sorf3 And US2 Proteins From Duck Enteritis Virus

Influenza viruses belongs to Orthomyxoviridae virus family, flu virus. Until April 9, 2010, the global death caused by avian influenza virus has reached to 292 people. Although so far no report of bird flu virus that could transmitted from person to person, to control disasters causing by avian influenza virus and develope of new vaccines, studying the pathogenesis of avian influenza virus appear to be important. Researchers have proposed a new strategy for prevention and control:blocking the essential process of interaction which is necessary for influenza virus infection. This method will effectively prevent the escape of the virus. In view of this, understanding the interactions between influenza virus and host are very necessary.NS2 protein of influenza viruses is conversation among influenza A virus, since the initially establishing the roles of NS2 in exporting viral ribonucleoprotein (vRNP) from the nucleus, other functions for the NS2 has not been reported. Moreover, the role of NS2 in vRNP is in question by some scientists. Relative to other proteins of influenza virus, the report of NS2 is very rare. Discovering new NS2 interactions with host factors, not only expand the existing network between influenza virus and host, but also may open a new way for preventing and controlling disease.As a high-throughput method for studying protein interaction, Yeast two-hybrid has been used to study proteomics of various model organisms. In last two years, the measures have been used to study the interactions between influenza virus proteins and host proteins. In this thesis, yeast two-hybrid was used to study the interactions between the influenza virus NS2 protein and host protein. This study tries to explain. the significance of the NS2-CHD3 interaction in the proliferation of virus. The principal results were described as the following:1 Screening proteins that interact with from a human fetal brain cDNA library using NS2 protein of AIV (H5N1) as a bait38 candidates positive clones were selected. The 38 clones did no show self-activation. And the interactions can be reproduced in yeast. The 38 plasmids pGADT7-preys were extracted and sequenced, the results revealed 28 different clones.2 Validating the interactionsIn COS1 cells, the mammalian two-hybrid results show that, chromatin domain helicase DNA binding protein-3(C-terminal region (C-CHD3))and a protein similar to heat shock protein 70 binding protein (HSPBP1) (HSPBP1s) showed strong interaction with NS2. Using the co-immunoprecipitation and GST pull-down experiments, we showed that C-CHD3 and HSPBP1s could interact with NS2.Using indirect immunofluorescence experiments we could clearly find that C-CHD3 located in the nucleus and NS2 distributed throughout the cell. Co-expression of NS2 and the C-CHD3 led CHD3 relocate to the cytoplasm and performed co-location. 24 hours after transfection with C-CHD3 and the cells were infected for 4 hours by virus. The results indicated that virus infection can also change the C-CHD3 subcellμlar localization. However, the virus does not change the subcellμlar localization of the endogenous CHD3.3 The conservative of interactions between NS2-C-CHD3 and NS2-HSPBPlsTaking into account NS2 is conservative in the influenza A virus. But virus still exists the small amount of acid sequence differences. We cloned NS2 of A/WSN/1933 (H1N1), A/Mexico City/001/2009 (H1N1) and A/duck/Hubei/W1/2004 (H9N2). The results by mammalian two-hybrid proved that in spite of the acid sequence differences among these NS2, these NS2 could interacte with the C-CHD3 and HSPBP1s.4 Mapping the interaction domain of NS2 and HSPBPlsN-terminal domain and the C-terminal domain of NS2 and HSPBP1s were cloned. By mammalian two-hybrid experiments, we showed that, functional domain of NS2 locate at the N- terminal region. Either N-terminal domain or C-terminal domain of HSPBP1s alone did not interact with NS2. We made three mutations in the NS2 nuclear export signal (M16A, M19A, L21A). By mammalian two-hybrid experiments, we showed that the mutations did not affect its interaction with C-CHD3 and HSPBP1s.5 CHD3 impact the proliferation of the virusBy absolute quantitative PCR, we found that at 36 hours, C-CHD3 can inhibit virus multiplication (86 times); through RNA (siRNA)-mediated knockdown, we found that virus decreased 16 times at postinfection 36 hours.6 The mechanism of CHD3 interfereing the proliferation of the virusUsing luciferase reporter driven by GAL4, we indicated that NS2 can antagonize down-regμlation expression by endogenous CHD3. Therefore, this may be one reason of C-CHD3 interfering virus proliferation. In addition, the study found, C-CHD3 delay virus vRNP nuclear exporting.