The role of host cell ether lipids in influenza virus infection

THE ROLE OF HOST CELL ETHER LIPIDS IN INFLUENZA VIRUS INFECTION CHARMAINE CHNG XUEMEI B.Sc (Hons.) National University of Singapore A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF BIOCHEMISTRY YONG LOO LIN SCHOOL OF MEDICINE, NATIONAL UNIVERSITY OF SINGAPORE 2012 Acknowledgments I would like to thank my supervisor, Dr Markus Wenk for the opportunity to complete my Master’s programme in his laboratory, and also for the support and advice he has given me I would like to express my gratitude to my mentors, Lukas Tanner and Dr Xueli Guan for their advices and guidance Especially to Lukas, who was always so willing to discuss and share his ideas with me, who has guided me through my project, and made the learning experience a very rewarding and exciting one for me I would like to thank Lukas also, for critically reading through this manuscript and for providing valuable suggestions To all members of Markus Wenk’s lab, thank you for your suggestions and help during my time in the lab, and thank you for making these few years memorable and fruitful ii Table of Contents Acknowledgments ii Table of Contents iii Summary vii List of Tables ix List of Figures x List of Abbreviations xii Introduction .1 1.1 Influenza virus 1.1.1 Epidemiology of influenza 1.1.2 Structure and function of influenza virus 1.2 Involvement of host factors in influenza virus infection 1.2.1 Genome studies identifying host factors involved in influenza virus infection… 1.2.2 Host proteins and their involvement in influenza virus infection .8 1.2.3 Involvement of host metabolism in influenza virus infection 11 1.2.3.1 Glycolytic flux in influenza virus infection 11 1.2.3.2 Lipid metabolism in influenza virus infection 12 1.2.3.2.1 Sphingolipids 17 1.2.3.2.2 Neutral lipids 18 1.2.3.2.3 Glycerophospholipids 20 1.3 Aims 24 Materials & Methods 25 iii 2.1 Cells, siRNAs, virus and antibodies .26 2.2 Influenza virus infection 27 2.3 Lipid Profiling of influenza virus-infected cells .28 2.3.1 Collection of cells 28 2.3.2 Lipid extraction .28 2.3.3 Quantitative analysis of lipids by HPLC/MS 29 2.3.4 Analysis of MS raw data 30 Effect of ether lipid-deficient cell lines on influenza virus infection – Plaque 2.4 assay & Western blot 30 2.4.1 Plaque assay to determine virus release 30 2.4.2 Viral protein accumulation observed by Western blot 31 2.5 siRNA reverse transfection .32 2.5.1 2.6 Validation of siRNA transfection 33 2.5.1.1 Real-time PCR 33 2.5.1.2 MTT cell viability assay .34 2.5.1.3 Immunoblotting 34 2.5.1.4 Lipid profiling of metabolite levels 34 Effect of siRNA transfection on influenza virus infection – Plaque assay & Western blot 35 2.7 Rescue of reduced ether lipid levels by addition of metabolites 35 Results 36 3.1 Lipid profile of wild-type and ether lipid-deficient CHO cells infected with influenza virus .37 3.2 Ether lipid-deficient cell line, NRel-4 affects influenza virus infection 43 3.3 AGPS knockdown using siRNA changes ether lipid levels in A549 cells 44 iv 3.3.1 AGPS siRNA treatment reduced AGPS gene expression levels, without compromising cell viability .45 3.3.2 AGPS siRNA treatment successfully reduced AGPS protein expression47 3.3.3 AGPS siRNA treatment reduced ether lipid levels 48 3.3.4 Effect of AGPS knockdown on influenza virus infection 51 Discussion .54 4.1 Insights from lipid profile of infected cells 55 4.1.1 The decrease in GM3 possibly reflects neuraminidase activity 55 4.1.2 Sphingomyelin species are increased in infected cells: Possible implications for virus budding and particle functionality .57 4.1.2.1 The up-regulation of saturated, long chain lipids might be implicated in the remodelling of functional ordered domains 57 4.1.3 Ether lipids, specifically ether PCs could play key roles in influenza virus infection 60 4.2 Host cell ether lipids in influenza virus infection 60 4.2.1 Ether lipid-deficient cell line, NRel-4 significantly impaired influenza virus infection 60 4.2.2 siRNA knockdown of AGPS observed reduction in influenza virus infection 63 4.2.3 The up-regulation of ePCs might be hypothetically linked to an important role in influenza virus life cycle .65 4.2.3.1 A possible role of ether lipids in influenza virus trafficking, assembly and budding 65 4.2.3.2 The up-regulation of ether lipids could be indirectly linked to an increase in glycolysis in infected cells 68 4.3 Conclusion 72 v Bibliography 73 Appendix 87 vi Summary There is substantial lack in the understanding of the role of host lipids in virus infections, and this project aimed to identify possible lipid candidates that could be crucial during influenza virus replication Based on our previous experiments identifying an enrichment of ether lipids in influenza virus particles and influenza virus-infected human alveolar adenocarcinoma cells (A549) (our lab’s unpublished data), I established the lipid profiles of influenza virus-infected Chinese hamster ovary (CHO-K1) wild-type (WT) cells and its ether lipid-deficient derivatives, NRel4, using high performance liquid chromatography (HPLC) mass spectrometry Similar to the observations in influenza virus-infected A549 cells, many choline containing lipids such as phosphatidylcholine (PC) and sphingomyelin (SM) lipid species were significantly different in infected cells, compared to mock-infected cells There was a specific increase in ether phosphatidylcholine (ePC) species in influenza virusinfected CHO-K1 cells, but a decrease in ester phosphatidylcholine (aPC) species This trend in PC lipid species however, was not distinct in NRel-4, indicating the misregulation of ether lipids in these NRel-4 cells Based on this observation, I postulated that ePCs might play important roles in influenza virus infection and hence, designed functional assays to further elucidate their roles For this purpose, NRel-4 cells were infected with influenza virus and a decrease in influenza virus titers were observed, as compared to the CHO-K1 wild-type cells To further confirm the importance of ether lipids during influenza virus infection, I harnessed a siRNA knockdown approach targeting alkylglycerone phosphate synthase (AGPS), the enzyme catalyzing the second step of ether lipid biosynthesis, in A549 cells Intriguingly, a 60 to 70% reduction in virus titer was also observed in AGPS vii knockdown cells Based on these data and other published literature, it was hypothesized that ePCs could play important roles in the completion of influenza virus life cycle stages, especially at later stages of infection, including trafficking, assembly and budding This hypothesis is mainly attributed to the observation that 1) trafficking vesicles like synaptic vesicles are enriched in ether lipids, 2) ether lipids are involved in cholesterol homeostasis and protein trafficking, and 3) M2-mediated membrane scission of influenza virus, is regulated by cholesterol levels at the budding site It was further postulated that ether lipid biosynthesis could be regulated by the glycolytic flux in host cells, since the ether lipid biosynthetic pathway branches from dihydroxyacetone phosphate (DHAP) in the glycolytic pathway, and glycolytic flux has been implicated in many virus infections, including influenza virus viii List of Tables Table List of standards and concentrations used in MS profiling 88 Table m/z value of lipid species analyzed in this study 89 Table Knockdown effect of AGPS siRNA and the off-target effects 90 Table Knockdown effect of Rab11a siRNA and the off-target effects 92 ix List of Figures Figure 1.1 Estimates of the transmission of influenza virus strains in a given week in 2012 Figure 1.2 Structure of an influenza virion Figure 1.3 Structures of main lipid classes 14 Figure 1.4 Structures of ester-linked and ether-linked PC (18:0/20:4) lipid species 16 Figure 3.1 The number of lipid species in each lipid class that were significantly different between infected and mock-infected (A) CHO-K1 cells and (B) NRel-4 cells 39 Figure 3.2 Heat plot of 68 lipid species that were previously identified to change significantly in influenza virus-infected A549 cells, and the observed trend in infected CHO-K1 and NRel-4 cells 40 Figure 3.3 Lipid classes significantly different in infected and mock-infected cells 41 Figure 3.4 NRel-4 cells showed decreased virus titer and viral protein expression compared to CHO-K1 WT cells 43 Figure 3.5 AGPS gene expression levels decreased after siRNA treatment 46 Figure 3.6 A549 cells were viable after AGPS siRNA knockdown 47 Figure 3.7 AGPS protein expression decreased after AGPS siRNA knockdown 48 Figure 3.8 Total ester and ether lipids after AGPS siRNA knockdown 50 Figure 3.9 ePC/aPC ratio decreased in AGPS knockdown cells 51 Figure 3.10 Virus titer and viral protein expression levels after AGPS and Rab11a siRNA knockdown 53 Figure 4.1 Ether lipid biosynthesis pathway 62 Figure 4.2 Triglyceride synthesis pathway (Hajra et al., 2000) 71 Figure 6.1 PE and PC lipid species were significantly changed in AGPS knockdown cells 91 Figure 6.2 Rab11a protein expression decreased after 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Burke, D.S (2009) Historical perspective Emergence of influenza A (H1N1) viruses N Engl J Med 361, 279-285 86 Appendix 87 Appendix Table List of standards and concentrations used in MS profiling 88 Appendix Table m/z value of lipid species analyzed in this study 89 Appendix Table Knockdown effect of AGPS siRNA and the off-target effects The percentage knockdown effect after treatment with AGPS s16248 and s16249 siRNA were calculated relative to the –ve ctrl siRNA The off-target effect of the siRNA was also briefly examined by probing for another randomly chosen primer, PGK, and the percentage off-target effects were measured relative to the –ve ctrl 90 Appendix Figure 6.1 PE and PC lipid species were significantly changed in AGPS knockdown cells PE and PC lipid species were included in the plot above, based on significant difference (p