High-resolution functional profiling of the norovirus genome.
Human noroviruses (HuNoV) are a major cause of nonbacterial gastroenteritis worldwide, yet details of the life cycle and replication of HuNoV are relatively unknown due to the lack of an efficient cell culture system. Studies with murine norovirus (MNV), which can be propagated in permissive cells, have begun to probe different aspects of the norovirus life cycle; however, our understanding of the specific functions of the viral proteins lags far behind that of other RNA viruses. Genome-wide functional profiling by insertional mutagenesis can reveal protein domains essential for replication and can lead to generation of tagged viruses, which has not yet been achieved for noroviruses. Here, transposon-mediated insertional mutagenesis was used to create 5 libraries of mutagenized MNV infectious clones, each containing a 15-nucleotide sequence randomly inserted within a defined region of the genome. Infectious virus was recovered from each library and was subsequently passaged in cell culture to determine the effect of each insertion by insertion-specific fluorescent PCR profiling. Genome-wide profiling of over 2,000 insertions revealed essential protein domains and confirmed known functional motifs. As validation, several insertion sites were introduced into a wild-type clone, successfully allowing the recovery of infectious virus. Screening of a number of reporter proteins and epitope tags led to the generation of the first infectious epitope-tagged noroviruses carrying the FLAG epitope tag in either NS4 or VP2. Subsequent work confirmed that epitope-tagged fully infectious noroviruses may be of use in the dissection of the molecular interactions that occur within the viral replication complex.
Calicivirus Research Group, Section of Virology, Department of Medicine, Imperial College London, London, United Kingdom.
Lucy
L
Thorne
Dalan
D
Bailey
Ian
I
Goodfellow
D000818:Animals; D002460:Cell Line; D004251:DNA Transposable Elements; D055786:Gene Knockout Techniques / Q000379:methods; D005814:Genes, Viral; D005827:Genetics, Microbial / Q000379:methods*; D008264:Macrophages / Q000821:virology; D051379:Mice; D016254:Mutagenesis, Insertional / Q000379:methods*; D029322:Norovirus / Q000235:genetics* / Q000502:physiology; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication
D004251:DNA Transposable Elements; D014764:Viral Proteins