General information
Title of the offer: Thesis offer (M/F): Integrative Profiling of Viruses by Whole Genome Mutational Analysis
Reference: UMR5048-MANLEP-004
Number of Posts: 1
Workplace: MONTPELLIER
Publication date: Thursday October 3, 2024
Type of contract: Fixed-term doctoral student/Doctoral contract
Contract duration: 36 months
Thesis start date: January 1, 2025
Work quota: Full time
Remuneration: The remuneration is a minimum of €2,135.00 monthly
Section(s) CN: Organization, expression, evolution of genomes
Description of the thesis subject
Integrative Virus Profiling by Whole Genome Mutational Analysis
Work context
The objective of this thesis is to construct and characterize all possible variants of entire genomes in order to produce integrated functional descriptions of these organisms. The goals of this thesis are to develop deep sequencing screens for all of the phenotypes mentioned above (and others), process the acquired data into precise quantitative scores, perform cross-analysis, and integrate these results to gain a unique system-level understanding of the virus. These will be used to inform the design of safer and more effective alternatives to chemical pesticides and to explore more direct health applications.
The candidate will use synthetic biology methods to systematically construct densovirus variants and develop a variety of deep sequencing assays to assess numerous phenotypes of interest (gene expression, viral replication, virulence, specificity, etc.). The goal is to integrate these quantitative measurements to produce holistic models that link mechanistic, systemic, and evolutionary descriptions for entire organisms.
The candidate will perform molecular biology, cloning, deep sequencing (NGS), data analysis and statistical modeling.
The expected results are a large dataset linking hundreds of thousands of mutations to a dozen different phenotypes. This could lead to the development of viral variants with improved properties for biological control and gene therapy.
Constraints and risks
Synthetic genomics aims to elucidate genomic organizations using synthetic biology techniques. Given the size of the genomes, the modifications introduced are arbitrary and far from systematic. The deep mutagenic scanning technique consists of building libraries of systematic variants around out-of-context sequences and using deep sequencing for high-throughput phenotyping. This project focuses on a small viral genome as a model to combine and extend these two approaches.
Densoviruses are small viruses of the Parvoviridae family that infect invertebrates. Junonia Coenia densovirus (JcDV) can infect several Lepidoptera species and is highly pathogenic against fall armyworm (Spodoptera frugiperda), a multi-drug resistant crop pest invading the world. JcDV presents a 6 kb single-stranded DNA genome flanked by two telomeres. It produces two head-to-tail overlapping transcripts: one carries three nonstructural proteins, two of which overlap entirely on different reading frames and are only expressed after splicing, the other encodes four versions of a protein capsid carrying different N-terminal extensions, depending on the start codon used. The capsid, whose structure has been solved, is naked and directly involved in host recognition. Once better understood and controlled, JcDV could provide sustainable and programmable alternatives to chemical insecticides against major insect pests. It is also closely related to AAV gene therapy vectors and could offer interesting opportunities in this area as it presents lower antigenicity and more storage space. We have built a library of 300,000 mutants that span the entire genome and are all marked with a unique DNA barcode. Sequencing these barcodes allows for efficient measurement of changes in frequencies of viral mutants in response to various screens, thereby enabling the quantification of various phenotypes for all mutants in parallel across the entire viral life cycle. This generic approach makes it possible to quantify replication or pathogenicity using cellular viral DNA, determine the abundances, start, end and splice sites of viral transcripts, measure the assembly, encapsidation and stability of the capsid, as well as the abilities of mutants to enter cells and nuclei from virion DNA. The library includes all single nucleotide substitutions as well as all single nucleotide/amino acid deletions and insertions in non-coding/coding regions. These mutations can have direct or cascading effects on multiple phenotypes.
