Development of an optimized RNA-based murine norovirus reverse genetics system

Abstract

Murine norovirus (MNV), identified in 2003, is the only norovirus which replicates efficiently in tissue culture and as a result has been used extensively as a model for human noroviruses, a major cause of acute gastroenteritis. The current report describes the generation of a new approach to reverse genetics recovery of genetically defined MNV that relies on the transfection of in vitro transcribed capped RNA directly into cells. The use of the recently developed ScriptCap post-transcriptional enzymatic capping system, followed by optimized Neon mediated electroporation of the highly permissive RAW 264.7 cells, resulted in the rapid and robust recovery of infectious MNV. Transfection of cells capable of supporting virus replication but not permissive to virus infection, namely human or hamster kidney cells, also resulted in robust recovery of infectious virus without subsequent amplification by multiple rounds of re-infection. This latter system may provide a reproducible method to measure the specific infectivity of mutant norovirus RNA allowing the accurate quantitation of the effect of mutations on norovirus replication.

Fig. 1

Murine norovirus full-length cDNA clone and translation of in vitro synthesised RNA. (A) Schematic of the infectious cDNA clone used in this study highlighting the positions of the four open reading frames, the T7 RNA polymerase promoter and the position of the 3′ ribozyme sequence. The asterisk highlights the position of the frame shift used to generate the mutated cDNA clone pT7:MNV-G 3′Rz F/S. (B) In vitro translation of in vitro transcribed murine norovirus genomic (G) or subgenomic RNA (SG). RNA was in vitro transcribed as described in Section 2.3 and in some cases enzymatically capped, highlighted by the prefix m⁷, prior to translation in rabbit reticulocyte lysates. Samples were resolved subsequently by 15% SDS-PAGE prior to exposure to a phosphoimager screen. Note that the protein assignments are based on the nomenclature proposed previously (Sosnovtsev et al., 2006). NS7* represents the truncated NS7 product generated as a result of the frame shift introduced in pT7:MNV-G 3′Rz F/S.

Fig. 2

Recovery of murine norovirus using a heterologous cell system.Western blot analysis of either baby hamster kidney cells (BSR-T7) or human embryonic kidney cells (293T) transfected in vitro transcribed murine norovirus RNA. Cells were transfected with in vitro transcribed uncapped (G, SG) or enzymatically capped RNA (m⁷G, m⁷SG or m⁷F/S) as described in Section 2.3. In vitro transcribed genomic (G) or a derivative containing a frame shift in the NS7 region was prepared using the plasmids pT7:MNV-G 3′Rz and pT7:MNV-G 3′Rz F/S respectively. Subgenomic RNA was prepared using a PCR product engineered to contain a truncated T7 RNA polymerase at the 5′ end as described in Section 2.2. Cells were transfected with RNA and samples prepared for western blot using antisera to the viral NS7 and VP2 proteins 24 h post-transfection. Duplicate samples were also harvested and analysed subsequently for the presence of infectious virus by TCID50 on RAW 264.7 cells. Virus yield is show as TCID50 per transfection (1.5 × 10⁶ cells).

Fig. 3

Neon mediated electroporation of RAW 264.7 cells. (A) Table depicting the results obtained by transfection of RAW 264.7 cells under various conditions with either 1 μg of GFP encoding DNA (pGFPmax) or 1 μg murine norovirus viral RNA (contained in a total RNA preparation isolated from infected cells). RAW 264.7 cells were transfected with 1 μg of plasmid using the conditions detailed in the table. The percentage of cells expressing GFP was determined at 48 h post-transfection using flow cytometry. Virus yield was determined as TCID50 per 6 × 10⁶ cells at 24 h post-transfection. (B) Bright field and fluorescence imaging of RAW 264.7 cells transfected with pGFPmax using condition 6 (1700 V, 25 ms) 48 h post-transfection.

Fig. 4

Optimization of murine norovirus recovery from RAW 264.7 cells. The virus yield obtained 24 h post-transfection of RAW 264.7 cells using varying amounts of in vitro transcribed post-transcriptionally capped MNV RNA. RAW 264.7 cells were transfected using the optimized conditions determined for virus recovery (1725 V, 25 ms). Virus yield is expressed as TCID50 per 6 × 10⁶ cells and represents the average of three repetitions. The asterisk highlights that no infectious virus was obtained but that the detection limit was ∼50 TCID50. Error bars represent the standard deviation.

Fig. 5

Time course analysis of murine norovirus recovery from RAW 264.7 cells. The virus yield from RAW 264.7 cells transfected with various amounts of either in vitro transcribed post-transcriptionally capped murine norovirus RNA or purified viral VPg-linked RNA. At various times post-transfection, cells were freeze–thawed to release infectious virus and the yield of virus determined by TCID50. Virus yield is expressed as TCID50 per 6 × 10⁶ cells and represents the average of three repetitions. Error bars represent the standard deviation.