The Study On The Role Of Cdc42on Modification And Transport NA Protein Of Influenza A Virus

Influenza virus neuraminidase (NA) plays an important role in the invasion and the release of viruses and is considered as the most suitable target of anti-influenza drugs. Although NA inhibitors have been continuously developed, resistance to these drugs quickly emerges among influenza virus strains. Therefore, the need for developing new anti-flu drugs and quests of new targets are urgent. In the host cells, many host proteins have significant impacts on the replication of influenza virus. Three important members of small molecular G protein Rho GTPases family-Cdc42, RhoA and Racl, as molecular switches, regulate various cellular signaling pathways and are involved in various cellular biological activities, such as the regulation of intracellular transport. However, whether they participate in the intracellular transport of NA protein and which of the three members provides the function are still unknown. Therefore, we designed our experiments to address these questions.We established a system of intracellular transport of NA protein by observing the localization changes of NA and NA (H274Y) mutant protein in the cells and detecting reduced surface activity of NA (H274Y) mutant. The experiments of treatments of NA transfected cells with drugs Toxin B confirmed that Rho GTPases family members (Cdc42, RhoA and Racl) can promote NA protein transport. Then, we observed the co-localization of NA with Cdc42, RhoA and Rac1and their related constitutively activated mutants and inhibitory mutants, respectively. Together with the experiments with C3Transferase treatment, we concluded that Cdc42may have a facilitating function. A specific GTPase-activating protein,(GAP)-ARHGAP21, was transfected, resulting in inhibition of activity of Cdc42in host cells. We further confirmed the catalytic role played by Cdc42.We explored how Cdc42affects the transport of NA protein. The GST-pulldown experiments showed that there may be no direct interaction between Cdc42and NA. After the Golgi apparatus were labeled with TGN and GM130, respectively, the confocal microscopy showed that Cdc42influences the transport process of NA protein from the Golgi apparatus to the cytoplasm and the cell membrane. The Cytochalasin D treatment experiments revealed that Cdc42affects transport of NA protein through the regulation of actin.A series of mutants carrying deletions in the signal peptide region of NA have been constructed. We found that NA protein do not express, when more than10amino acids are deleted in the NA transmembrane domain. In addition, the NA mutant carrying11amino acids deletion did not show any changes in the sub-cellular localization in the host cells.The results above showed that Cdc42plays a significant role in promoting modification and transport of NA protein of Influenza A virus. Further studies are needed to elucidate the mechanism of regulation of NA protein transport by Cdc42.