Influenza B virus causes a respiratory disease in people with a compromised immune system. The NS1B protein of influenza B virus is essential for virus growth and plays a crucial role in inhibiting the anti-viral responses mounted by the infected host cell. The N terminal 104 amino acids of NS1B bind a cellular protein called ISG15. ISG15 is an interferon induced 'ubiquitin-like' protein, and upon interferon induction, is conjugated to hundreds of targets. It has been found that both ISG15 and its conjugation inhibit many viruses. The focus of the current study was to characterize the interaction between NS1B and ISG15. Study of a recombinant influenza B virus which encoded a mutant NS1B protein that is unable to bind ISG15 revealed that ISG15 is mis-localized in cells infected with wild type but not the mutant influenza B virus. Further, such a mutant virus is attenuated in growth as compared to wild type virus in human cell lines but is not attenuated in canine cell lines. This result led to the discovery of the species specific nature of the interaction between NS1B and ISG15. Specifically, NS1B was found to bind ISG15 homologs from human and old world monkeys like Rhesus macaques and African green monkeys but not those from mouse or canines. These findings were extended by identifying the hinge between the N and C terminal domains of ISG15 as one of the major determinants of species specificity. These results highlight the importance of using human or primate cell culture models to study the effect of ISG15 on influenza B virus, and raises new possibilities on differences in the function of the ISG15 system in different species. / text

The poultry-to-human transmission of the influenza virus and the recent H1Nl influenza pandemic have become major concerns worldwide. The nucleoprotein (NP) of influenza virus binds the RNA genome and plays essential role in transcription and replication during the virus life cycle. / The study leads to a better understanding towards the RNP organization of influenza virus and provides information for the future design of anti-influenza agents. / We have also shown, by RNP reconstitution assay and co-immunoprecipitation, that the interaction between NP and PB2 is crucial for the proper functioning of the RNP. The functional association of NP and PB2 requires either the PB2 host-determining residue lysine-627 or arginine-630 with the latter involving NP arginine-150 also. Using SPR, we have demonstrated that both residues take part in the direct protein-protein interaction, without the involvement of RNA. These results suggest a dual interaction mechanism between NP and PB2. This may confer replication advantages to the virus, as either one can give an active RNP and explains the increased virulence of avian influenza viruses carrying the E627K mutation in mammalian cells. In addition, our findings identify the NP-PB2 interacting surface, with the PB2 627/630 region facing the RNA binding groove of NP. / We have determined the 3.3 A crystal structure of H5N1 NP, which is composed of head and body domains and a tail loop. Using surface plasmon resonance (SPR), we found the basic loop (residues 73-91) and arginine-rich groove, but mostly a protruding element centering at R174 and R175, to be important in RNA binding. Ribonucleoprotein (RNP) reconstitution assay with these multiple-point and deletion mutants indicate their functional importance towards the transcription-replication activities of the virus polymerase. Single-point mutations at these concerned regions do not have a significant effect on their RNP activities, suggesting that NP mediates RNA-binding through multiple residues. / Ng, Ka Leung. / Adviser: Pang Chui Shaw. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 121-136). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.