published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Although influenza is usually acute self-limiting respiratory infection, influenza viruses are among the most common pathogens that threaten the health of humans and animals worldwide. Various anti-viral therapeutic agents are currently used for treatment and prophylaxis of influenza virus, but the problem is that the targets of these drugs are easily mutated and result in resistance. Therefore, medications that have broad spectrum coverage are urgently needed to combat with the disease. Since nucleoprotein (NP), which is encoded by influenza virus genome, is regarded as a druggable target due to its conserved sequence and important functions during influenza virus life cycle, numerous studies are focused on this protein in attempts to develop broad-spectrum anti-influenza therapeutics. Recently, Kao et al. found that the addition of a novel small molecule nucleozin could lead to large aggregates of NP, which in turn caused cessation of virus replication. Give that the interaction between NP and nucleozin is still not unveiled, it is crucial to identify the binding sites using X-ray crystallography. The full length influenza A/WSN/33 (H1N1) NP gene was cloned into pET28 vector, with His-tag in its C-terminus and overexpressed in E.coli strain Rosetta 2. Cell culture was purified by HisTrap HP and Superdex-200 16/60 gel filtration columns. Crystals were grown using the vapour diffusion method and the NP-nucleozin complex was prepared by soaking native crystal in solution containing 0.25 mM nucleozin for 2h. Crystals of the complex can diffract to 3.0 Å at the Shanghai Synchrotron Radiation Facility. The structure of NP was determined by molecular replacement and it belongs to space group C121 with two NP trimers per asymmetric unit. After further refinement, two nucleozin molecules were found in each asymmetric unit, and each of them could bind with two NP molecules at the same time. The ligand binding pockets were formed by the combination of Y289/N309 pocket from one NP molecule, and R382 pocket from another NP molecule. Therefore, the function of nucleozin is to bridge two NP molecules and lead to NP aggregation, which are in agreement with functional studies on nucleozin. Furthermore, computational models of the NP-nucleozin binding are provided to reveal the mechanism of nucleozin induced aggregation. In addition, recent work on interaction between NP and another novel molecule named compound A has also been briefly described and compared with NP-nucleozin complex at the end of this thesis. Collectively, this study presents a new paradigm for better understanding of how NP and nucleozin interact with each other and hence result in NP aggregates, which is envisaged to accelerate the development of anti-influenza therapeutic agents. / published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

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Influenza A virus (family Orthomyxoviridae) is one of the most important human pathogens, causing annual epidemics with significant worldwide mortality, and sporadic but potentially devastating pandemics. The influenza A viral genome encodes 14 proteins and consists of 8 segments of negative-stranded RNA. During infection, the virus exploits the host cell signaling machinery to ensure efficient replication. The PI3K/Akt and Ras/ERK are two of the signaling cascades that are induced for virus survival. Influenza A virus replicates in the nucleus, hence the newly synthesized RNPs must be exported from the nucleus and exported to the cell membrane. Although the detailed mechanism of vRNP nuclear export is not yet fully elucidated, several studies on this process have begun to emerge. Influenza A virus nucleoprotein nuclear export is CRM1-dependent. Ran-binding protein 3 (RanBP3) is a Ran-interacting protein that is best known for its role as a cofactor of CRM1-mediated cargo nuclear export. In this study, we investigated the role of RanBP3 during the influenza A virus life cycle. We found that RanBP3 was phosphorylated at Ser58 in early and late phases of infection. Knockdown of RanBP3 expression led to a vRNP nuclear retention, suggesting that RanBP3 is involved in vRNP nuclear export. Moreover, we demonstrated that RanBP3’s function during vRNP nuclear export is regulated by phosphorylation at Ser58, and the RanBP3 phosphorylation is modulated by both PI3K/Akt and Ras/ERK/RSK pathways in the late phase of viral infection. In conclusion, this study has shown that RanBP3 is a host factor that has a vital role during the late stage of influenza A virus replication, specifically as a co-factor in CRM1-mediated nuclear export. Identifying this host factor will contribute to the understanding of the mechanism of vRNP transport.

A fetus’ in utero environment has a profound effect on the individual’s development in postnatal life. Research has suggested that intrauterine growth restricted children have a less robust response to vaccination. Studies have confirmed similar results in animal models; however, the effect of low birth weight on clinical disease expression is unclear. This research aims to determine if pigs from low birth weight litters have increased severity of disease after experimental infection with influenza A virus (IAV) when compared to their counterparts from high birth weight litters, thus clarifying the effect of litter birth weight on disease expression. Pilot trials were conducted to determine the appropriate dose of virus to use and the optimal days post inoculation for necropsy to use for the main trial. The results indicated that the main trial should use an inoculation dose of 1 x 107 plaque forming units of IAV and the time of necropsy should be 48 hours post inoculation. In the main trial, male piglets (n=68) from parity one or two sows were identified at farrowing as coming from high or low birth weight litters. At four weeks of age, intratracheal IAV inoculation was performed (day 0) and pigs were euthanized at 48 hours post inoculation. Clinical signs were assessed prior to euthanasia. After euthanasia macroscopic and microscopic lesion severity were assessed, along with immunohistochemical staining intensity of IAV in lung tissue. SearchLight Chemiluminescent Array Technology was used to measure the concentration of the inflammatory cytokines interleukin 1 beta, interleukin 6, and interleukin 8 in bronchoalveolar lavage fluid. Interferon alpha was measured using fluorescent microsphere immunoassay. Fifty Percent Tissue Culture Infective Dose was used to measure influenza viral titers in lung tissue. The study found no differences in clinical scores or cytokine concentration between pigs from high and low birth weight litters. Gross, histopathological and immunohistochemical scores were significantly higher in piglets from high birth weight litters and viral titers trended higher in these piglets. These findings indicate that pathologic disease scores in piglets experimentally inoculated with IAV are more severe in piglets from high birth weight litters.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

Thesis (Ph.D.) - University of St Andrews, January 2008. / Restricted until 15th January 2009, during which time access is available with the consent of the Head of School.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1961. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 54-57).

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (p. 212-238). Also available in print.