Plasmid-based human norovirus reverse genetics system produces reporter-tagged progeny virus containing infectious genomic RNA

Human norovirus (HuNoV) is the leading cause of gastroenteritis worldwide. HuNoV replication studies have been hampered by the inability to grow the virus in cultured cells. The HuNoV genome is a positive-sense single-stranded RNA (ssRNA) molecule with three open reading frames (ORFs). We established a reverse genetics system driven by a mammalian promoter that functions without helper virus. The complete genome of the HuNoV genogroup II.3 U201 strain was cloned downstream of an elongation factor-1α (EF-1α) mammalian promoter. Cells transfected with plasmid containing the full-length genome (pHuNoV_U201F) expressed the ORF1 polyprotein, which was cleaved by the viral protease to produce the mature nonstructural viral proteins, and the capsid proteins. Progeny virus produced from the transfected cells contained the complete NoV genomic RNA (VP1, VP2, and VPg) and exhibited the same density in isopycnic cesium chloride gradients as native infectious NoV particles from a patient’s stool. This system also was applied to drive murine NoV RNA replication and produced infectious progeny virions. A GFP reporter construct containing the GFP gene in ORF1 produced complete virions that contain VPg-linked RNA. RNA from virions containing the encapsidated GFP-genomic RNA was successfully transfected back into cells producing fluorescent puncta, indicating that the encapsidated RNA is replication-competent. The EF-1α mammalian promoter expression system provides the first reverse genetics system, to our knowledge, generalizable for human and animal NoVs that does not require a helper virus. Establishing a complete reverse genetics system expressed from cDNA for HuNoVs now allows the manipulation of the viral genome and production of reporter virions.Human noroviruses (HuNoVs) belong to the genus Norovirus of the family Caliciviridae and are the predominant cause of epidemic and sporadic cases of acute gastroenteritis worldwide (1, 2). HuNoVs are spread through contaminated water or food, such as oysters, shellfish, or ice, and by person-to-person transmission (3, 4). Although HuNoVs were identified more than 40 y ago, our understanding of the replication cycle and mechanisms of pathogenicity is limited, because these viruses remain noncultivatable in vitro, a robust small animal model to study viral infection is not available, and reports of successful passage of HuNoVs in a 3D cell culture system have not been reproduced (5–7). Recently, a murine model for HuNoV infection was described that involves intraperitoneal inoculation of immunocompromised mice (8); its generalizability and robustness for studying individual HuNoVs and many aspects of HuNoV biology remain to be established. Gnotobiotic pigs can support replication of a HuNoV genogroup II (GII) strain with the occurrence of mild diarrhea, fecal virus shedding, and immunofluorescent (IF) detection of both structural and nonstructural proteins in enterocytes (9). Previous systems to express the HuNoV genome from cloned DNA using T7/vaccinia systems showed that mammalian cells can produce progeny virus (10, 11), but these systems are not sufficiently efficient to be widely used to propagate HuNoVs in vitro. The factors responsible for the block(s) of viral replication using standard cell culture systems remain unknown.The HuNoV genome is a positive-sense ssRNA of ∼7.6 kb that is organized in three ORFs: ORF1 encodes a nonstructural polyprotein, and ORF2 and ORF3 encode the major and minor capsid proteins VP1 and VP2, respectively. Because of the lack of an in vitro system to propagate HuNoV, features of their life cycle have been inferred from studies using other animal caliciviruses and murine NoV (MNV) that can be cultivated in mammalian cell cultures (12). A 3′ coterminal polyadenylated subgenomic RNA is produced within infected cells. Both genomic and subgenomic RNAs have the same nucleotide sequence motif at their 5′ ends, and they are believed for HuNoVs and shown for MNV to be covalently linked to the nonstructural protein VPg at the 5′ ends (10, 13). During MNV infection of cells, nonstructural proteins are expressed from genomic RNA and form an RNA replication complex that generates new genomic RNA molecules as well as subgenomic RNAs encoding VP1, VP2, and the unique protein called VF1 (14). After expression of the structural proteins from subgenomic RNA molecules, the capsid is assembled, and viral RNA is encapsidated before progeny release. Previous reverse genetics systems for HuNoV used helper vaccinia MVA/T7 virus-based systems. Although helper virus-free systems have been developed for MNV (15, 16), no such system is available for HuNoVs. To overcome these problems, we established a reverse genetics system driven by a mammalian elongation factor-1α (EF-1α) promoter without helper virus and then modified this system to package a reporter gene (GFP) into ORF1.