Characterization of Recombinant Flaviviridae Viruses Possessing a Small Reporter Tag

Abstract

The family consists of four genera,, ,, and , and comprises important pathogens of human and animals. Although the construction of recombinant viruses carrying reporter genes encoding fluorescent and bioluminescent proteins has been reported, the stable insertion of foreign genes into viral genomes retaining infectivity remains difficult. Here, we applied the 11-amino-acid subunit derived from NanoLuc luciferase to the engineering of the viruses and then examined the biological characteristics of the viruses. We successfully generated recombinant viruses carrying the split-luciferase gene, including dengue virus, Japanese encephalitis virus, hepatitis C virus (HCV), and bovine viral diarrhea virus. The stability of the viruses was confirmed by five rounds of serial passages in the respective susceptible cell lines. The propagation of the recombinant luciferase viruses in each cell line was comparable to that of the parental viruses. By using a purified counterpart luciferase protein, this split-luciferase assay can be applicable in various cell lines, even when it is difficult to transduce the counterpart gene. The efficacy of antiviral reagents against the recombinant viruses could be monitored by the reduction of luciferase expression, which was correlated with that of viral RNA, and the recombinant HCV was also useful to examine viral dynamics Taken together, our findings indicate that the recombinant viruses possessing the split NanoLuc luciferase gene generated here provide powerful tools to understand viral life cycle and pathogenesis and a robust platform to develop novel antivirals against viruses. The construction of reporter viruses possessing a stable transgene capable of expressing specific signals is crucial to investigations of viral life cycle and pathogenesis and the development of antivirals. However, it is difficult to maintain the stability of a large foreign gene, such as those for fluorescence and bioluminescence, after insertion into a viral genome. Here, we successfully generated recombinant viruses carrying the 11-amino-acid subunit derived from NanoLuc luciferase and demonstrated that these viruses are applicable to and experiments, suggesting that these recombinant viruses are powerful tools for increasing our understanding of viral life cycle and pathogenesis and that these recombinant viruses will provide a robust platform to develop antivirals against viruses.

A Sensitive Yellow Fever Virus Entry Reporter Identifies Valosin-Containing Protein (VCP/p97) as an Essential Host Factor for Flavivirus Uncoating

“…Although we have illustrated free nucleocapsids within the cytosol, we cannot exclude the possibility that fusion is tightly coupled to capsid ubiquitylation and disassembly, such that nucleocapsids may be ubiquitylated and disassembled as they are exposed to the cytosol. Consistent with our model, VCP/p97 activity was previously shown to be important for WNV, JEV, and DENV infection, although specific role(s) for VCP/p97 in virus entry were not determined (53,83). Similarly, VCP/p97 is also important for an early step, upstream of N-protein degradation, during the entry of infectious bronchitis virus, a coronavirus (84).…”

“…Given these considerations, we chose to pursue a function-based approach to study the productive entry of a recombinant YFV that expresses a reporter enzyme only after viral entry and translation. Several flavivirus reporter systems have been developed, mainly for high-throughput screening of viral replication (17,26,(48)(49)(50)(51)(52)(53)(54)(55). In one remarkable study, Byk et al adapted a Renilla luciferase-expressing DENV reporter virus to show that the incoming DENV capsid protein must be ubiquitylated prior to viral gene expression (17).…”

“…These constructs were originally designed to reduce biosafety risks for high-throughput screening; our data suggest that these constructs should also be useful in dissecting early, prereplication events in the life cycle of these flaviviruses. Further improvements to our design are also possible; for instance, smaller tags, such as a split Nluc reporter (53), could improve viral titers or allow postfusion events to be monitored prior to viral genome translation.…”

Protein-Protein Interaction Assays Using Split-NanoLuc

“…Sasaki et al developed a quantitative detection system of viral entry and release using HiBiT fused to subviral particles and flavivirus-like particles of West Nile virus [64]. Tamura et al constructed recombinant viruses carrying HiBiT [65]. Viral amplification and propagation were rapid and comparable with the parental viruses, due to the small size of HiBiT.…”

Real Time Analysis of Bovine Viral Diarrhea Virus (BVDV) Infection and Its Dependence on Bovine CD46

“…Members of this genus are pathogens of cloven-hoofed animals, including the highly economically relevant pathogens bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV). In recent years, genetically labelled BVDV and CSFV clones have been constructed based on N-terminal fusion of luciferase or fluorophores to either E rns or E2 [10][11][12][13]. For a BVDV E2-fluorophore fusion protein, visualization of purified particles in fluorescence microscopy based on the specific fluorescence signal has been reported [13].…”