Study of the role of viral coat protein and host factor HSP70 homologs in the assembly and disassembly of Cucumber necrosis virus particles

Virion assembly and disassembly are crucial aspects of the virus multiplication cycle, however, relatively little is known about these processes in plant viruses. While the former helps to produce multiple copies of stable infectious progeny virions, the latter is required for release of the encapsidated viral genome into a host cell for initiating new rounds of virus multiplication. In this thesis, I aimed to study Cucumber necrosis virus (CNV) particle assembly and disassembly and the role of CNV coat protein (CP) and host HSP70 homologs in these processes. It was found that CNV infection of Nicotiana benthamiana causes a significant upregulation of HSP70 homologs, and that, in turn, HSP70 is co-opted by the virus at several stages of the multiplication cycle to promote various aspects of the infection cycle including viral RNA, CP and particle accumulation. HSP70 homologs were also found to assist CNV CP in chloroplast targeting possibly to attenuate chloroplast-mediated plant defence and thereby allow further spread of the virus. It was also determined that the HSP70 homologue, Hsc70-2 is bound to CNV virions and that this association appears to facilitate the uncoating efficiency of CNV particles likely via triggering a conformational change in particles. This is the first report that a plant virus utilizes HSP70 homologs for disassembly. A highly basic “KGRKPR” sequence in the ε-region of the CNV CP arm was also examined for its role in virion assembly and encapsidation of viral RNA. Through mutational analysis, it was found that the basic residues promote T=3 versus T=1 virion formation and encapsidation of full-length viral RNA in vivo. Moreover, mutants lacking 2-4 of the basic residues encapsidated proportionately greater amounts of host RNA suggesting the role of these basic residues in selection of viral RNA during assembly. It was also shown that heat shock enhances transcription of heat-inducible ONSEN-like retrotransposons known to be induced during CNV infection. Since retrotransposons are known to play an important role in genome variation, the described studies may be helpful in understanding the importance of plant viruses in inducing genome variation and perhaps adaptation of plants to changes in the environment.Land and Food Systems, Faculty ofGraduat

Apolipoprotein C3 facilitates internalization of cationic lipid nanoparticles into bone marrow-derived mouse mast cells

Abstract Mast cells (MCs), are hematopoetically-derived secretory immune cells that release preformed as well as de novo synthesized inflammatory mediators in response to activation by several stimuli. Based on their role in inflammatory responses, particularly in the lung and skin, MCs provide an effective target for anti-inflammatory therapeutic strategies. Drug-delivery of lipophilic payloads to MCs can be challenging due to their functionally distinct intracellular structures. In the present study, pH-sensitive cationic lipid-based nanoparticles (LNPs) composed of DODMA, DODAP or DOTAP lipids that encapsulated a GFP or eGFP plasmid were constructed using non-turbulent microfluidic mixing. This approach achieved up to 75–92% encapsulation efficiency. Dynamic light scattering revealed a uniformly sized and homogeneous dispersion of LNPs. To promote cellular internalization, LNPs were complexed with apolipoproteins, amphipathic proteins capable of binding lipids and facilitating their transport into cells. Cryo-TEM analysis showed that LNP structure was differentially modified when associated with different types of apolipoproteins. LNP preparations made up of DODMA or DODMA, DODAP and DOTAP lipids were coated with seven apolipoproteins (Apo A1, B, C3, D, E2, E4 and H). Differentiated bone-marrow derived mouse mast cells (BMMCs) were exposed to apolipoprotein-LNP and internalization was measured using flow cytometry. Out of all the apolipoproteins tested, ApoC3 most efficiently facilitated cellular internalization of the LNP into BMMCs as determined by GFP fluorescence using flow cytometry. These effects were confirmed in a less differentiated but also interleukin-3-dependent model of mouse mast cells, MC/9. ApoC3-LNP enhanced internalization by BMMC in a concentration-dependent manner and this was significantly increased when BMMC were pre-treated with inhibitors of actin polymerization, suggesting a dependence on intracellular shuttling. Activation of peroxisome proliferator-activated receptor gamma (PPARγ) decreased ApoC3-LNP internalization and reduced the expression of apolipoprotein E receptor 2 (ApoER2), suggesting that ApoC3-LNP binding to ApoER2 may be responsible for its enhanced internalization. Furthermore, ApoC3 fails to facilitate internalization of LNPs in Lrp8 −/− KO BMMC that do not express ApoER2 on their cell surface. Altogether, our studies reveal an important role of ApoC3 in facilitating internalization of cationic LNPs into MCs

A Canadian perspective on severe acute respiratory syndrome coronavirus 2 infection and treatment: how prevalent underlying inflammatory disease contributes to pathogenesis

The coronavirus disease 2019 (COVID-19), a serious respiratory illness caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has emerged as a global pandemic. Canada reported its first case of COVID-19 on 25th January 2020. By March 2020 the virus had spread within Canadian communities reaching the most frail and vulnerable elderly population in long-term care facilities. The majority of cases were reported in the provinces of Quebec, Ontario, Alberta and British Columbia and the highest mortality was seen among individuals aged 65 years or older. Canada has the highest prevalence and incidence rates of several chronic inflammatory diseases, such as multiple sclerosis, inflammatory bowel disease and Parkinson’s disease. Many elderly Canadians also live with comorbid medical illnesses, such as hypertension, diabetes, cardiovascular disease and chronic lung disease and are more likely to suffer from severe COVID-19 with a poor prognosis. It is becoming increasingly evident that underlying inflammatory disease contributes to SARS-CoV-2 pathogenesis. Here, we review the mechanisms of SARS-CoV-2 infection and the host inflammatory responses that lead to resolution or progression to severe COVID-19 disease. Furthermore, we discuss the landscape of COVID-19 therapeutics that are currently in development in Canada.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author