Evolutionary and Functional Studies of the Mouse Retroviral Restriction Gene, Fvl Scott Anthony Ellis A thesis submitted in part fulfilment of the requirements of the University of London for the degree of Doctor of Philosophy March 2000 Virology Division National Institute for Medical Research The Ridgeway Mm Hill London NW71AA ProQuest Number: 10015911 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10015911 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract Fvl is a gene of mice known to restrict the replication of Murine Leukaemia virus (MLV) by blocking integration by an unknown mechanism. The gene itself is retroviral in origin, and is located on the distal part of chromosome 4. The sequence of markers known to flank Fvl in the mouse was used to identify sequence from the human homologues of these 2 genes. The construction of primers to these sequences permitted the screening of 2 YAC and a PAC human genomic libraries for clones containing either of these genes. The YAC libraries were negative for both markers. The human region was finally cloned as 2 overlapping PAC clones. This region was sequenced, and the comparison of this sequence and the analogous region in rat to the Fvl region in mice allowed the determination of what sequence had been lost and gained during the formation of the gene. The Fvl ORF of mice from across the Mus genus was PCR-amplified, cloned and sequenced. The analysis of this sequence has shown how Fvl has evolved during the spéciation of the Mus genus. By combining this data with what is known of the distribution of endogenous MLV and Fvl activity among the Mus genus, a scheme of how and why Fvl has evolved activity has been proposed. The mouse genome was screened for a 'progenitori sequence(s) that may have given rise to Fvl during its germline infection of Mus. A mouse 129/SvJ genomic library was screened by hybridisation for the sequences bearing homology to Fvl. Sequences obtained were shown to be the Fvl gene itself, members of the murine endogenous retroviral-L (MERV-L) family, or had no known sequence homology as determined by BLAST searching. A polymorphism at an EcoRI site was identified in the Fvl gene of the 129 mouse that appears to be responsible for the presence of 2 bands that hybridise with an Fi?!-specific probe in a 129 genomic southern blot. FvV"^^ cell lines expressing m utant Fvl ORFs were established and assayed for Fvl activity in an assay b^s^d pseudotyped MLV. This allowed the contribution of the 3 changes known to be important between the 2 main alleles to be assessed. The first amino add change in the Fvl ORF was found to be the major determinant of Fvl phenotype, with the difference in the C-terminus contributing to a lesser extent. Fvl^ allele, a less common allele of Fvl, was shown to be mediated by a single amino add change and not by a change in the level of Fvl expression. Acknowledgements I would like to thank my supervisor. Dr. Jonathan Stoye, for his help and advice both during the course of this project and in the preparation of this thesis. I am grateful to Paul Le Tissier and Greg Towers for their help and support in all aspects of research. I would like to thank Paul Wilson and Tony Stevens for their technical help and support in the lab. I would also like to acknowledge Nigel Douglas for his assistance in the construction of the phylogenetic trees. Lastly, I would like to thank Lisa Patel for her support and limitless patience throughout the duration of this project. Contents Abstract 2 Acknowledgements 4 Contents 5 List of Figures 12 List of Tables 14 Abbreviations 15 Chapter 1 Introduction 1.1 General Retroviral Biology 17 1.1.1 Taxonomy 17 1.1.2 Virion structure 18 1.1.3 The retroviral genome 20 1.1.4 Retroviral genes 21 1.1.5 The retroviral lifecycle 22 1.2 The Murine Leukaemia viruses 23 1.2.1 Host range of the MLV 23 1.2.2 Entry of the virus into the cytoplasm 24 1.2.3 The reverse transcription reaction 26 1.2.4 Entry of the virus into the nucleus 29 1.2.5 Integration of the retroviral genome 30 1.2.6 Expression of the MLV genome 32 1.2.7 Virion assembly 34 1.3 Endogenous retroviruses 35 1.3.1 Discovery of endogenous retroviruses 35 1.3.2 Presence in the genome 36 1.3.3 Significance of germ line infection 37 1.3.4 Human endogenous retroviruses 37 1.3.5 Mouse endogenous retroviruses 38 1.3.6 Distribution of endogenous MLV in Mus 38 1.3.7 Endogenous retroviruses and disease 39 1.3.8 The Friend virus complex 40 1.4 Evolution of host responses to retroviral infection 42 1.4.1 Cell entry and replication 42 1.4.2 Target cell 44 1.4.3 Immune response 45 1.5 The F vl MLV-restriction gene 47 1.5.1 The discovery of Fvl 47 1.5.2 The main Fvl alleles 47 1.5.3 The cloning of Fvl 48 1.5.4 Structure of the gene 49 1.5.5 Other alleles of Fvl 49 1.5.6 Fvl interaction with virus 51 1.5.6.1 Entry of the virus into the cell 51 1.5.6.2 Fvl and events after integration 52 1.5.6.3 Viral DNA levels in restrictive infections 52 1.5.7 Fvl sensitive steps 53 1.5.7.1 Viral determinants of N -o r B-tropism 53 1.5.7.2 Interaction with the preintegration complex 54 1.5.7.3 Involvement of cellular factors 54 1.6 Reverse Transcriptase and its significance to eukaiyotic genomes and the evolution of retroviruses 56 1.6.1 The origin of RT 56 1.6.2 Where do retroviruses come from? 58 1.6.3 The role of reverse transcriptase in the eukaryotic genome 61 1.6.4 The Retrogenes 61 1.6.5 Retrogenes and Exaptation 63 1.6.6 Intronless genes: an indicator of retroposition? 64 1.6.7 Retroposition and chromosome structure/function 64 1.7 Evolution of the Mus genus 66 1.7.1 Phytogeny of Mus 66 1.7.2 Partitioning in Mus musculus 68 1.7.3 Origin of Mus musculus 70 1.7.4 Hybrid zones 70 1.7.5 Laboratory mice 73 1.8 Aims of the Project 74 Chapter 2 Materials and Methods 2.1 Solutions, enzymes and buffers 76 2.2 List of Bacterial genotypes 81 2.3 Mouse strains 81 2.4 Genomic DNA samples 81 2.4.1 Mouse genomic DNA 81 2.4.2 Hum an genomic DNA 83 2.5 Genomic DNA libraries 83 2.5.1 The ICI human genomic YAC Library 83 2.5.2 The ICRF hum an genomic YAC Library 83 2.5.3 The HGMP human genomic PAC Library 83 2.5.4 The mouse 129 SvJ lambda genomic library 83 2.6 Preparation of DNA 84 2.6.1 Preparation of plasmid DNA 84 2.6.2 Preparation of PAC DNA 84 2.6.3 Preparation of single-stranded M13 DNA 85 2.6.4 Preparation of lambda phage DNA 86 2.6.5 Preparation of genomic DNA 87 2.6.6 Spectrophotometric determination of DNA 87 2.7 Preparation of total RNA 87 2.8 Subcloning of DNA 88 2.8.1 Digestion of DNA 88 2.8.2 Purification and recovery of DNA 88 2.8.3 Dephosphorylation of cohesive termini 89 2.8.4 Ligation of DNA 89 2.9 Bacterial transformation with DNA 89 2.9.1 Heat-shock transformation of CaClg-competent cells 89 2.9.2 Electrotransformation of electrocompetent cells 90 2.10 DNA electrophoresis 90 2.11 Fixation of DNA to membrane 90 2.11.1 Southern Blots 90 2.11.2 Lambda phage plaque lifts 91 2.11.3 Bacterial colony lifts 92 2.12 Hybridisation 92 2.12.1 Southern analysis 93 2.12.2 Lambda phage plaque lifts 93 2.12.3 Bacterial colony filter hybridisation 93 2.13 Production of radiolabelled probes 94 2.13.1 DNA Probes 94 2.13.2 Production of RNA probes (riboprobes) 94 2.14 Ribonudease protection assay 95 2.15 Library screening 96 2.15.1 YACHbraries 96 2.15.2 PAG libraries 96 2.15.3 Lambda phage library 96 2.16 Polymerase chain reaction 97 2.16.1 PGR conditions for standard reactions 97 2.16.2 PGR conditions for library screens 97 2.16.3 List of PGR primers 97 2.16.4 Direct doning of PGR-amplified products 98 2.17 Automated DNA sequendng 98 2.18 Transposon-based strategy to rapidly sequence large DNA fragments 99 2.19 Analysis of sequence data 100 2.19.1 Editing of raw sequence (Sequencing Analysis software) 100 2.19.2 Gontig assembly and construction of consensus sequences 100 2.19.3 Multiple sequence alignments (GDE) 100 2.19.4 Phytogeny analysis (Phylip) 100 2.19.5 Galculating substitution rates (PAML) 101 2.19.6 Sequence similarity searches (BGM) 101 2.20 Mouse cell culture 101 2.20.1 Maintenance of ceU lines 101 2.20.2 Transfection of Mus dunni ceUs and selection for donal cell lines 101 2.20.3 Storage and reviving of stable cell lines 102 2.20.4 Virus infection of cell cultures 103 8 2.21 Biological Assays for F vl phenotype 103 2.21.1 Reverse Transcriptase assay 103 2.21.2 LacZ assay using Pseudotyped MLV 104 2.21.3 Virus stocks 104 Chapter 3 The Cloning of the Human FVl Region 3.1 Overview of the cloning strategy 105 3.2 Construction of the human primers for screening 107 3.2.1 BLAST searches to identify human NFVl and NFV2 ESTs 107 3.2.2 Testing the primers at reduced template concentrations 107 3.3 Screening large-insert human genomic libraries by PCR 108 3.3.1 YAC libraries 108 3.3.2 PAC libraries 108 3.4 Analysis of PAC clones containing NFVl and NFV2 human homologous sequence 112 3.4.1 Analysis of PACs by hybridisation 112 3.4.2 Probing PACs with mouse Fvl ORF sequence 115 3.4.3 Probing PAC clones with mouse Nppfl sequence 115 3.5 Subcloning F vl