Production of infectious reporter murine norovirus by VP2 trans -complementation

Author:

Ishiyama Ryoka1ORCID,Yoshida Kazuhiro2,Oikawa Kazuki1,Takai-Todaka Reiko1ORCID,Kato Akiko2,Kanamori Kumiko2,Nakanishi Akira23,Haga Kei1ORCID,Katayama Kazuhiko1ORCID

Affiliation:

1. Department of Infection Control and Immunology, Laboratory of Viral Infection, Ōmura Satoshi Memorial Institute & Graduate School of Infection Control Sciences, Kitasato University, Tokyo, Japan

2. Department of Aging Intervention, National Center for Geriatrics and Gerontology, Laboratory of Gene Therapy, and Laboratory for Radiation Safety, Aichi, Japan

3. Department of Biology-Oriented Science and Technology, Kindai University, Wakayama, Japan

Abstract

ABSTRACT Human norovirus (HuNoV) causes gastroenteritis, a disease with no effective therapy or vaccine, and does not grow well in culture. Murine norovirus (MNV) easily replicates in cell cultures and small animals and has often been used as a model to elucidate the structural and functional characteristics of HuNoV. An MNV plasmid-based reverse genetics system was developed to produce the modified recombinant virus. In this study, we attempted to construct the recombinant virus by integrating a foreign gene into MNV ORF3, which encodes the minor structural protein VP2. Deletion of VP2 expression abolished infectious particles from MNV cDNA clones, and supplying exogenous VP2 to the cells rescued the infectivity of cDNA clones without VP2 expression. In addition, the coding sequence of C-terminal ORF3 was essential for cDNA clones compensated with VP2 to produce infectious particles. Furthermore, the recombinant virus with exogenous reporter genes in place of the dispensable region of ORF3 was propagated when VP2 was constitutively supplied. Our findings indicate that foreign genes can be transduced into the norovirus ORF3 region when VP2 is supplied and that successive propagation of modified recombinant norovirus could lead to the development of norovirus-based vaccines or therapeutics. IMPORTANCE In this study, we revealed that some of the coding regions of ORF3 could be replaced by a foreign gene and infectious virus could be produced when VP2 was supplied. Propagation of this virus depended on VP2 being supplied in trans , indicating that this virus could infect only once. Our findings help to elucidate the functions of VP2 in the virus lifecycle and to develop other caliciviral vectors for recombinant attenuated live enteric virus vaccines or therapeutics tools.

Funder

Japan Agency for Medical Research and Development

MEXT | Japan Society for the Promotion of Science

Publisher

American Society for Microbiology

Subject

Virology,Insect Science,Immunology,Microbiology

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