From the Institute of Biochemistry of the University of Lübeck Director: Prof. Dr. Rolf Hilgenfeld Analysis of overlapping reading frames in viral genomes DissertAtion in Fulfillment of the Requirements for the DoctorAl Degree of the University of Lübeck from the DepArtment of NAturAl Sciences Submitted by Aditi Shukla from RAnchi, IndiA Lübeck, 2015 1. First Referee: Prof. Dr. Rolf Hilgenfeld 2. Second Referee: Prof. Dr. Bernhard Haubold DAte of OrAl ExAminAtion: 20.10.2015 Approved for printing: Lübeck, on 20.10.2015 Acknowledgements I thank, Prof. Hilgenfeld with deep gratitude for providing me with this reseArch opportunity And making me A pArt of your esteemed Institute. I thAnk you for sharing your knowledge And ideAs, providing support And encourAgement throughout, and for An extensive review of this work. Your attention to detail towArds every project, every proposAl, every manuscript, Amazes me. I wish to imbibe this immaculAteness in my professionAl life. Prof. MArtinetz And Prof. MAmlouk for interesting discussions, useful critiques for this research work, And for helping to keep my progress on schedule. The friendly manAgement of the grAduAte school for your sustAined support throughout. Special thanks to Olga and Katja for organizing interesting workshops, seminArs And get-togethers. Achim for your technical AssistAnce; Mrs. Rosenfeld for making my initiAl ArrivAl in Germany very eAsy And pleAsAnt And Mrs. SchwAb for work behind the scenes! Raffaele, Monarin, Raspudin, Zhenggang, Linlin, Friedrich And Caroline for your kind fraternity; Jeroen and Ksenia for your suggestions And encourAgements whenever required; for being there as a support! Other stAff members at the institute for making the institute so interesting. UpAsAnA And SusmitA for your friendship. My fAmily for immense patience, undying love and constant support. Contents Abstract Declaration 1. Introduction ....................................................................................................................... 1 1.1 Overlapping reading frames ........................................................................................ 1 1.1.1 Types of overlap ....................................................................................................... 2 1.2 Direction and phase of overlapping reading frames ................................................. 3 1.2.1 Direction of overlap .................................................................................................. 3 1.2.2 Phase of overlap in overlapping reading frames ...................................................... 4 1.3 Selection pressure on overlapping reading frames .................................................. 5 1.3.1 Degeneracy of the genetic code .............................................................................. 5 1.3.2 Synonymous and non-synonymous substitutions .................................................... 7 1.3.3 Different kinds of evolutionary strategy adopted by overlapping genes ................... 7 1.4 Viruses with overlapping reading frames ................................................................... 8 1.5 Objective of this study ................................................................................................. 8 2. Biological Background .................................................................................................... 11 2.1 Accessory proteins of coronaviruses ....................................................................... 11 2.1.1 Alphacoronaviruses ................................................................................................ 13 2.1.2 Betacoronaviruses ................................................................................................. 16 2.1.3 Gammacoronaviruses ............................................................................................ 19 2.1.4 Deltacoronaviruses ................................................................................................ 19 2.1.5 SARS-coronavirus: a member of betacoronavirus lineage b; genome organization and overlapping proteins .............................................................. 21 2.2 Overlapping protein in Murine norovirus ................................................................. 27 2.3 Overlapping proteins in Hepatitis B virus ................................................................ 27 3. Special sequence properties of overlapping genes ...................................................... 29 3.1 Codon usage bias ....................................................................................................... 29 3.2 Methods ....................................................................................................................... 30 3.2.1 Relative Synonymous Codon Usage (RSCU) ........................................................ 30 3.2.2 Correlation analysis and codon usage ................................................................... 31 3.2.3 Datasets ................................................................................................................. 31 3.2.4 Comparison of codon usage of overlapping and non-overlapping gene sets ........ 33 3.3 Results ......................................................................................................................... 34 3.4 Discussion ................................................................................................................... 37 4. Effect of overlaps on the protein products .................................................................... 39 4.1 Intrinsically disordered proteins ............................................................................... 39 4.2 Disorder predictors ..................................................................................................... 40 4.3 Methods ....................................................................................................................... 41 4.3.1 Comparison of disorder content of overlapping proteins sets ................................ 41 4.4 Results ......................................................................................................................... 42 4.5 Discussion ................................................................................................................... 46 5. Prediction of RNA secondary structure at the site of initiation of alternative reading frames ...................................................................................................................... 49 5.1 Molecular mechanism for the initiation of the alternative reading frame in overlapping genes ............................................................................................................ 49 5.1.1 Leaky scanning ...................................................................................................... 50 5.1.2 Internal Ribosomal Entry ........................................................................................ 52 5.2 Methods ....................................................................................................................... 53 5.2.1 Vienna RNA package ............................................................................................. 53 5.2.2 RNAComposer ....................................................................................................... 54 5.2.3 Computer prediction of RNA secondary and tertiary structural elements .............. 54 5.3 Results ......................................................................................................................... 56 5.4 Discussion ................................................................................................................... 57 6. Mutational model for the evolution of SARS-CoV overlapping accessory protein 9b .............................................................................................................................. 59 6.1 Introduction ................................................................................................................. 59 6.2 Methods ....................................................................................................................... 59 6.2.1 Dataset ................................................................................................................... 59 6.2.2 Mutation rate analysis ............................................................................................ 60 6.2.3 Entropy-plotting ...................................................................................................... 60 6.3 Results ......................................................................................................................... 62 6.3.1 The effect of the overlap on the mutation rate in the N and orf9b genes ............... 62 6.3.2 Evolutionary strategy adopted by the overlapping N and orf9b gene set ............... 63 6.3.3 Effect of mutations on the three-dimensional structures of the overlapping proteins ........................................................................................................................... 67 6.4 Discussion ................................................................................................................... 68 7. Conclusions ...................................................................................................................... 70 Bibliography .........................................................................................................................