Structural studies of influenza A virus by cryo-electron tomography

Influenza A virus (IAV) is a pleomorphic, enveloped virus known for its yearly epidemics and occasional, but fatal pandemics. The outer surface glycoprotein hemagglutinin (HA) together with the matrix protein 1 (M1) are the most abundant protein components of assembled virions. HA, located at the outside of virions, is involved in cell receptor recognition, membrane fusion and is the most relevant protein for antibody binding. Therefore the structure of isolated HA has been extensively characterised by X-ray crystallography. However, it remains unclear to which extent the structure of isolated HA corresponds to the in situ HA structure on the surface of IAV. M1 determines the morphology of the virus by forming a matrix layer underneath the viral membrane. A high resolution structure of full length M1 is missing and the lack of information about the in situ arrangement of the M1 matrix layer currently limits our understanding of how M1 functions. Here, I set out to determine the structures of HA and M1 directly from IAV particles using high resolution cryo-electron tomography (cryoET) and subtomogram averaging. I found that virus purification can affect the integrity of the virus HA glycoprotein layer and the morphology of virus particles. I therefore adapted a workflow which allows studying the structure of viral proteins directly from viruses in the vicinity of virus-producing cells. Biosafety regulations required inactivation of IAV samples by chemical fixation prior to cryoEM imaging. To assess effects of fixation, I complemented structural studies of HA from pathogenic, fixed IAV particles with studies of HA from non-infectious, unfixed virus-like particles (VLPs). These studies revealed that fixation captures HA in an open conformation while HA structures determined from unfixed samples perfectly match the closed conformation observed in the trimeric crystal structure. In concordance with recent work by others, this observation suggests that fixation captures HA in a an open, otherwise transient conformation, which is part of a constant opening and closing motion known as breathing motion. To characterise the in situ structure and arrangement of M1, I established a subtomogram averaging workflow to cope with the challenges presented by the small size of M1. I successfully obtained two independent structures of M1 directly from viruses and VLPs. Comparisons of my structures to existing high resolution models of the N-terminal domain (NTD) of M1 revealed that M1 monomers arrange as parallel strands, with a helical propensity and directly underneath the membrane. For the first time, my data allow to describe the M1-membrane interface as well as relevant M1-M1 interfaces within the matrix layer. Finally, I have gained first structural insights into the M1 C-terminal domain (CTD). I further combined the obtained structural information for M1 with a theoretical model of the mechanics of M1 polymerization and membrane deformation during virus assembly. The obtained results suggest that linear polymerization of M1 into multiple parallel strands efficiently provides energy to drive assembly of new virus particles. The results presented in this thesis improve our understanding of the arrangement and structure of the two influenza proteins HA and M1 in situ which has implications for current models of HA-mediated membrane fusion, virus architecture and virus assembly

New structural insights into the multifunctional influenza A matrix protein 1

Influenza A virus matrix protein 1 (M1) is the most abundant protein within virions and functions at multiple steps of the virus life cycle, including nuclear RNA export, virus particle assembly, and virus disassembly. Two recent publications have presented the first structures of full-length M1 and show that it assembles filaments in vitro via an interface between the N- and C-terminal domains of adjacent monomers. These filaments were found to be similar to those that form the endoskeleton of assembled virions. The structures provide a molecular basis to understand the functions of M1 during the virus life cycle. Here, we compare and discuss the two structures, and explore their implications for the mechanisms by which the multifunctional M1 protein can mediate virus assembly, interact with viral ribonucleoproteins and act during infection of a new cell

Environmental impacts of heating and cooling upgrades, considering the decarbonisation of the electricity grid

Rising temperatures and higher energy costs have created the imperative for Australian households to reduce energy consumption via more efficient heating and cooling systems. These energy efficient systems could lower the overall environmental impacts as heating and cooling accounts for around 20 % to 50 % of total energy usage of residential buildings. Nonetheless, the often overlooked aspect of efficiency upgrades pertains to the embodied energy and whether it is offset by operational savings as well as decarbonisation of the electricity grid. This research endeavours to determine the environmental impacts of 61 ducted gas heating to more efficient ducted gas heating upgrades and 59 gas heating to electric reverse cycle air conditioning upgrades via a life cycle assessment. A reference ducted gas heater, gas heater and air conditioner were deconstructed and assessed on their material composition. The operational energy savings of one year prior and after were collected for all upgrades.The results showed that there were differences between the chosen impact assessment methods and that for the ducted gas heating upgrades the operational energy savings had offset the embodied energy for all assessed impact categories except freshwater ecotoxicity and human toxicity (non-cancer). For the gas heating to reverse cycle air conditioning, the following impact categories were offset: climate change (short-term), fossil and nuclear energy use, mineral resources use and ozone layer depletion. However, climate change (long-term), photochemical oxidant formation, human toxicity (cancer) freshwater and terrestrial acidification, marine eutrophication, particulate matter formation, ionising radiation, water scarcity, land occupation and transformation (biodiversity) could only be offset with the decarbonisation of the electricity grid. Not-withstanding, human toxicity (non-cancer), freshwater ecotoxicity and eutrophication was not offset even with a complete decarbonisation of the electricity grid, as the impacts from the production stage were too high. Notwithstanding the limited effectiveness of full decarbonisation, it is worthwhile considering the materials used in the appliance to further lower the environmental impact

Life cycle assessment of 61 ducted gas heating upgrades in Australia

Operational energy use in buildings, accounts for 28% of global energy demand. One method to reduce operational energy is upgrading old appliances to more efficient ones. In Australia, the most common residential heating type is reverse-cycle heating, followed by gas heating. This research paper aims to determine the energy balance resulting from a gas heating upgrade through a life cycle assessment (LCA). Extensive primary data was collected for operational energy performance of 61 ducted gas heating upgrades. To address the scarcity of data on material composition, one ducted gas heater was deconstructed and assessed in terms of material composition (types and weights). The comparison between embodied energy and operational energy savings allows us to establish whether operational energy savings offset the embodied energy incurred with the upgrade. The end of life stage of the old appliance, as well as the production, construction and use stage of the new appliance were assessed. Results show that operational energy savings offset the following impact categories: global warming, ozone layer depletion, aquatic acidification, non-renewable energy, and carcinogens. Only the mineral extraction is not offset by the operational energy savings. Results clearly demonstrate that operational energy savings outweigh the embodied energy and therefore contribute positively to the environment. This study is the first to focus on the LCA of building services through extensive primary data collection and a focus on a high number of appliances. This supports ongoing energy efficient upgrades in Australia and pave the way for further, similar studies to confirm or disprove these findings in other parts of the world

Membranes by the Numbers

Many of the most important processes in cells take place on and across membranes. With the rise of an impressive array of powerful quantitative methods for characterizing these membranes, it is an opportune time to reflect on the structure and function of membranes from the point of view of biological numeracy. To that end, in this article, I review the quantitative parameters that characterize the mechanical, electrical and transport properties of membranes and carry out a number of corresponding order of magnitude estimates that help us understand the values of those parameters.Comment: 27 pages, 12 figure

Fluctuations in active membranes

Active contributions to fluctuations are a direct consequence of metabolic energy consumption in living cells. Such metabolic processes continuously create active forces, which deform the membrane to control motility, proliferation as well as homeostasis. Membrane fluctuations contain therefore valuable information on the nature of active forces, but classical analysis of membrane fluctuations has been primarily centered on purely thermal driving. This chapter provides an overview of relevant experimental and theoretical approaches to measure, analyze and model active membrane fluctuations. In the focus of the discussion remains the intrinsic problem that the sole fluctuation analysis may not be sufficient to separate active from thermal contributions, since the presence of activity may modify membrane mechanical properties themselves. By combining independent measurements of spontaneous fluctuations and mechanical response, it is possible to directly quantify time and energy-scales of the active contributions, allowing for a refinement of current theoretical descriptions of active membranes.Comment: 38 pages, 9 figures, book chapte

Environmental impacts of upgrading gas to electric heating and cooling, considering decarbonisation of the electricity grid

Rising temperatures and higher energy costs have created the imperative for Australian households to reduce energy consumption via more efficient heating and cooling systems. These energy efficient systems could lower the overall environmental impacts as heating and cooling accounts for around 20%–50% of total energy usage of residential buildings. Nonetheless, the embodied energy of efficiency upgrades and whether it is offset by operational savings as well as decarbonisation of the electricity grid, is often overlooked. This research aims to determine the environmental impacts of 59 gas heater to electric reverse cycle air conditioner upgrades via a life cycle assessment. A reference gas heater and air conditioner were deconstructed and assessed on their material compositions. The operational energy savings of one year prior and after were collected for all upgrades.The results show that operational energy savings offset the embodied energy for the following impact categories: climate change (short-term), fossil and nuclear energy use, mineral resources use and ozone layer depletion. However, climate change (long-term), photochemical oxidant formation, human toxicity (cancer) freshwater and terrestrial acidification, marine eutrophication, particulate matter formation, ionizing radiation, water scarcity, land occupation and transformation (biodiversity) can only be offset with the decarbonisation of the electricity grid. Notwithstanding, human toxicity (non-cancer), freshwater ecotoxicity and eutrophication cannot be offset even with a complete decarbonisation of the electricity grid, as the impacts from the production stage are too high. Notwithstanding the limited effectiveness of full decarbonisation, it is worthwhile considering the materials used in the appliance to further lower the environmental impact

Enhancing Neuraminidase Immunogenicity of Influenza A Viruses by Rewiring RNA Packaging Signals

Humoral immune protection against influenza virus infection is mediated largely by antibodies against hemagglutinin (HA) and neuraminidase (NA), the two major glycoproteins on the virus surface. While influenza virus vaccination efforts have focused mainly on HA, NA-based immunity has been shown to reduce disease severity and provide heterologous protection. Current seasonal vaccines do not elicit strong anti-NA responses-in part due to the immunodominance of the HA protein. Here, we demonstrate that by swapping the 5' and 3' terminal packaging signals of the HA and NA genomic segments, which contain the RNA promoters, we are able to rescue influenza viruses that express more NA and less HA. Vaccination with formalin-inactivated "rewired" viruses significantly enhances the anti-NA antibody response compared to vaccination with unmodified viruses. Passive transfer of sera from mice immunized with rewired virus vaccines shows better protection against influenza virus challenge. Our results provide evidence that the immunodominance of HA stems in part from its abundance on the viral surface, and that rewiring viral packaging signals-thereby increasing the NA content on viral particles-is a viable strategy for improving the immunogenicity of NA in an influenza virus vaccine. IMPORTANCE Influenza virus infections are a major source of morbidity and mortality worldwide. Increasing evidence highlights neuraminidase as a potential vaccination target. This report demonstrates the efficacy of rewiring influenza virus packaging signals for creating vaccines with more neuraminidase content which provide better neuraminidase (NA)-based protection