Human norovirus cultivation systems and their use in antiviral research

Abstract

Human norovirus (HuNoV) is a major cause of acute gastroenteritis and foodborne diseases, affecting all age groups. Despite its clinical needs, no approved antiviral therapies are available. Since the discovery of HuNoV in 1972, studies on anti-norovirals, mechanism of HuNoV infection, viral inactivation, etc., have been hampered by the lack of a robust laboratory-based cultivation system for HuNoV. A recent breakthrough in the development of HuNoV cultivation systems has opened opportunities for researchers to investigate HuNoV biology in the context of de novo HuNoV infections. A tissue stem cell-derived human intestinal organoid/enteroid (HIO) culture system is one of those that supports HuNoV replication reproducibly and, to our knowledge, is most widely distributed to laboratories worldwide to study HuNoV and develop therapeutic strategies. This review summarizes recently developed HuNoV cultivation systems, including HIO, and their use in antiviral studies.

Keywords: antiviral study; human norovirus; intestinal organoid; laboratory cultivation system.

Fig 1

The HuNoV genome and its surrogate system. (A) HuNoV RNA genome. HuNoV possesses a single-stranded, positive-sense RNA genome encoding three ORFs, which are attached to the VPg protein and poly(A) at the 5′- and 3′-ends, respectively. ORF1 encodes a polyprotein containing six nonstructural proteins (p48/N-terminal protein [NS1/2], NTPase [NS3], p22 [NS4], VPg [NS5], protease [NS6], and RdRp [NS7]), whereas ORF2 and ORF3 encode structural proteins VP1 and VP2, respectively. Polyproteins derived from the ORF1 region in genomic RNA are translated and subsequently cleaved into six nonstructural proteins by NS6. In infected cells, shorter genomic RNA, called subgenomic RNA, is transcribed and used as a template to translate VP1 and VP2. (B) HuNoV (human Norwalk) replicon system established by Chang et al. (16). The RNA possesses HuNoV genomic RNA where the coding regions of VP1 (ORF2) are replaced with a neomycin-resistant gene (Neo) and a T7 promoter sequence at the 5′-end so that the RNA can be transcribed by T7 polymerase in vitro. Neomycin-resistant cells harboring this RNA produce HuNoV RNA without progeny virus production due to lack of VP1 protein/capsid. (C) Virus-like particle (VLP), structurally and antigenically indistinguishable from naïve HuNoV virion, can be generated by expressing VP1 in insect cells using a baculovirus expression system.

Fig 2

Proposed model for HuNoV life cycle. Certain HuNoVs (e.g., GII.4 genotype) attach to the host cell membrane through interaction between the VP1 P2 domain and HBGA and invade the cell possibly via an unknown cellular receptor(s). The viral particles are then uncoated and disassembled by endosome acidification. The incoming viral (+) RNA genome is translated by the host translational machinery recruited by VPg, which is covalently bound to the 5’ end of viral RNA in the host cytoplasm. The translated polyprotein encoded by ORF1 is co- or post-translationally cleaved into p48/N-terminal protein (NS1/2), NTPase (NS3), p22 (NS4), VPg (NS5), protease (NS6), and RdRp (NS7) by NS6. For genomic replication, NS7 first uses positive-sense viral RNA to transcribe negative-sense RNA intermediates and then transcribes them into positive-sense (+) genomic RNA and (+) subgenomic RNA. Subgenomic RNA (+) contains only ORF2 and ORF3, which are used to synthesize VP1 and VP2 proteins. Genomic RNAs are then packed into the capsid, mainly comprising VP1 proteins, and viral particles are released from the infected cells.