Reverse Genetics Mediated Recovery of Infectious Murine Norovirus

1. RNA Transcription and Capping for The Recovery of Infectious MNV

This protocol is designed to allow the efficient recovery of infectious MNV from cDNA via in vitro transcription and its subsequent in vitro capping (section 1.1). The resulting capped transcripts are then transfected into cells to recover infectious MNV (sections 1.2 and 1.3). This approach provides the most sensitive method for the recovery of MNV with typical yields in excess of 10⁵ infectious units per 35 mm (in diameter)-dish of cells for MNV. The protocol is detailed below:

1.1 Synthesis of infectious capped MNV transcripts:

1. Digest the plasmid containing the wild type MNV cDNA (pT7:MNV 3’Rz) with NheI to obtain linear DNA. NheI recognises a unique restriction site after the 3′ end polyA tail of MNV genome (Figure 2). Linearised plasmids are typically purified with using silica columns (e.g. GFX PCR DNA Gel Band Purification Kit from GE Healthcare) and eluted in H₂O.
2. In vitro transcribe the linearised vector using T7 RNA polymerase as previously described ¹⁷. Many commercial kits are available for this purpose and provide a reproducible method of large amounts of RNA synthesis such as MEGAScript (Life Technologies) and RiboMAX (Promega). Transcription reactions are typically DNAse digested prior to further analysis; however in many instances this is not required as lithium chloride purifications as described below do not precipitate DNA efficiently.
3. Analyze a small aliquot of the RNA transcription reaction, typically 0.5 μl or less, by agarose gel electrophoresis to ensure the transcription reaction has worked efficiently and RNA is full-length. Whilst many users may want to run denaturing gels to correctly size the RNA, we typically use non-denaturing agarose gel electrophoresis as a rapid method to analyse RNA integrity. The MNV genome as produced from the infectious cDNA clone pT7:MNV 3’Rz will run at approximately 3 Kbp relative to a dsDNA ladder on a non-denaturing agarose gel (Figure 3).
4. Consider that other methods are available as an alternative to obtain a fast analysis of RNA integrity such as using an Agilent bioanalyser (Figure 3).
5. Note that poor gel resolution may be encountered if too much RNA is loaded. Take great care to ensure the agarose gel is prepared using RNAse-free reagents to avoid RNA degradation during electrophoresis which may affect band resolution. Heating RNA to 65 °C followed by cooling on ice may also help in some instances.
6. Purify RNA sample to remove the unincorporated nucleotides. Many methods are available for this including silica column based approaches however in this protocol we typically use lithium chloride as a cost effective alternative. For this purpose, add H₂O to reach a final volume of 100 μl and then add 40 μl of Lithium chloride precipitation solution (7.5 M LiCl, 50 mM EDTA, pH 8.0, Ambion) and store the sample at -20 °C for at least 30 min.
7. Pellet the RNA by centrifugation at 12,000 X g at 4 °C for 15 min.
8. Remove the supernatant, taking care not to disturb the translucent RNA pellet and wash it in 150 μl of 70% ethanol. Centrifuge the tube at 12,000 X g at 4 °C for 15 min.
9. Remove the ethanol and air-dry the RNA, avoiding the pellet to completely dry out as this will make resuspension difficult.
10. Then, resuspend the MNV transcripts into 50-100 μl of RNA storage solution (Ambion). Care should be taken to ensure all the RNA has dissolved properly. Should the RNA appear difficult to dissolve fully, heating the sample to 60 °C may help to resuspend it. Any insoluble material should then be removed by centrifugation prior to RNA quantification. The purified transcripts are uncapped and require a subsequent in vitro capping step to be infectious (ScriptCap m7G Capping System, Epicentre Biotechnologies).
11. Quantify the RNA by spectrophotometry. Depending on the nature and scale of the transcription reaction, typical yields range from 50-150 μg of RNA per 100 μl transcription reaction. The integrity of RNA should be analysed before the capping reaction by running out 100-300 ng of sample into a 1% agarose gel (Figure 3).
12. To improve the efficiency of RNA capping, heat 60 to 70 μg of MNV RNA transcripts at 65 °C for 10 min and then place the tube immediately on ice. This step may reduce any inhibitory effect of RNA structure on the capping. Pulse the tube in a chilled microfuge to collect droplets formed during the heating step.
13. Prepare a capping reaction mixture as suggested by the manufacturer (ScriptCap m7G Capping System, Epicentre Biotechnologies). Briefly, add 60-70 μg of MNV RNA to a final reaction volume of 100 μl. The capping reaction mix may contain 10 μl of 10 x Capping buffer (500 mM TrisHCl pH 8.0, 60 mM KCl, 12.5 mM MgCl2), 10 μl of 10 mM GTP, 0.5 μl of 20 mM S-adenosyl methionine, 2.5 μl of Scriptguard (100 units), and 4 μl of Scriptcap enzyme (40 units).
14. During the reaction set up, keep RNA transcripts on ice to avoid degradation. Mix well the reaction mixture and then incubate it at 37 °C for 1 h. Note the reaction size may be scaled according to the amount of capped transcript required.
15. Purify the RNA by LiCl precipitation as explained above (see point 1.1.6). Dissolve the pellet in 50-100 μl of RNA storage solution (Ambion) and quantify the amount of RNA. Typically, RNA samples are subsequently normalised to 1 μg/μl. Again, check all the RNA has dissolved properly. If it has not been properly dissolved, heat the sample to 60 °C to allow its dissolution. Remove by centrifugation any insoluble material prior to RNA quantification.
16. Check the integrity of RNA again before proceeding with the transfection step. To this aim, run an amount of 100-300 ng of sample into a 1% agarose gel (Figure 3).

1.2 Recovery by Neon-mediated transfection of RNA into Raw264.7 cells:

For the recovery of MNV infectious virions in a permissive cell line it is possible to electroporate the capped MNV transcript into Raw264.7 cells using Neon transfection system (Invitrogen). Raw264.7 are cells susceptible to MNV infection, supporting multiple rounds of virus replication and subsequent re-infection. As a result, typical yields will approach in excess of 10⁵ infectious units per ml at 24 hours post transfection but peak at >10⁷ infectious units after 48 hours.

1. One day before transfection, seed Raw264.7 cells at an estimated 50% confluency. Typically two T75 flasks of cells are required for 3 transfections.
2. The day of transfection, scrape the cell monolayers into Dulbecco’s modified Eagle medium (DMEM) containing 10% foetal calf serum (FCS), ensuring you generate a single cell suspension by repeated pipetting.
3. Determine the concentration of viable cells in a haemocytometer using trypan blue exclusion to label the non-viable cells.
4. Pellet the cells at 1,200 X g for 5 min and resuspend them in DMEM containing 10% FCS at a final concentration of 8 x 10⁶ cells/ml.
5. Just prior to transfection, aliquot 1 ml of cells per transfection and pellet them at 1,200 X g for 2 min. Remove the media and wash the cells in 500 μl of PBS (without Mg²⁺/Ca²⁺). Spin down the cells again at 1,200 X g for 2 min. Note it is advisable to keep cells in DMEM for as long as possible as storage in PBS for long periods of time may compromise the cell viability and transfection rate.
6. Remove PBS from tubes and add 130 μl of resuspension solution (Neon transfection system kit, Invitrogen) to a final concentration of 6 x 10⁷ cells/ml. Care should be taken to resuspend the cells avoiding the formation of bubbles which will cause sparking during transfection and compromise cell survival.
7. Add the appropriate amount of capped MNV transcript to the cells (Figure 2), generally 1.3 μg of capped RNA is added to 130 μl of resuspended cells and mixed gently. Then, collect 100 μl of the mixture in the 100 μl Neon transfection tip. Special care should be taken to ensure that no bubbles are formed in the electroporation cuvette (Neon transfection system kit 100 μl tip) as this will cause failure of the experiment.
8. Electroporate the cells using a single pulse at 1,700V for 25 msec, ensuring the absence of sparks during pulsing which will indicate the presence of bubbles in the sample. In the case sparking should occur, discard the sample and repeat the transfection. Release the cells into an Eppendorf tube containing 1 ml of antibiotic free DMEM containing 10% FCS. Note each tip can be reused up to three times with the same RNA sample if larger numbers of transfected cells are required.
9. Thereafter, distribute the cells from the tube into independent wells containing an appropriate amount of prewarmed antibiotic free DMEM containing 10% FCS. As a general guidance, 150 μl of the cell suspension generated during step 1.2.8 are sufficient for a single well of a 24-dish plate containing 0.5 ml of prewarmed DMEM, whereas 300 μl are appropriate for a well of a 12-dish plate containing 1 ml of prewarmed DMEM.
10. Incubate the cells at 37 °C and 10% CO₂ for 24 to 72 hours. Then, release infectious virions from cells by one (or more) freeze and thaw cycles and determine virus titre in the sample using either plaque assay or TCID50. Note that lysates should be clarified by centrifugation for 1-2 minutes at maximum speed or by their filtering through a 0.22 μm pore filter prior to titration. Typically, MNV reaches titres of around 1 x 10⁶ TCID50/ml at 24 hours post-transfection and up to 1 x 10⁹ at 72 hours post-transfection.
11. The presence and stability of mutations introduced in pT7:MNV 3’Rz are typically determined by sequencing the rescued viruses after 2 to 5 additional passages in Raw264.7 cells.

1.3 Recovery by lipofection into BHK-21 cells:

A more direct and often more cost effective method for the recovery of infectious MNV from capped transcripts is via lipofection (Lipofectamine 2000, Invitrogen). Given that Raw264.7 cells are difficult to transfect using lipid based approaches we normally make use of other easy to transfect cell lines such as BHK-21 which is an immortalised line derived from baby hamster kidney fibroblasts. As a standard approach in our laboratory we use BSR-T7 cells, a derivative of the BHK-21 cell line, as whilst these cells are easy to transfect and support MNV replication, they lack a suitable receptor to allow several rounds of re-infection. As a result, the virus yield generated from this system is an indication of a single cycle of virus replication. This approach is of particular use when examining the effect of mutation on virus recovery as it allows multiple transfections to be performed at substantially reduced costs compared to Neon-mediated transfection and also does not require specialist equipment. It is worth noting that other readily available cell lines such as human embryonic kidney 293T cells also support the efficient recovery of MNV however transfection conditions should first be optimised to ensure efficient RNA delivery.

1. Trypsinise a monolayer of BHK-21 cells (or BSR-T7 cells), seed 7.5 x 10⁵ cells into a 35 mm diameter dish in antibiotic-free growth media and incubate the cells at 37 °C with 10% CO₂ overnight. Double the amount of cells in each plate if the transfections are planned for the same day as the seeding, and allow cells to adhere to the plate for 2-3 hours at 37 °C with 10% CO₂. Note that other cells that are suitable to this approach include human 293T cells, human hepatocellular carcinoma Huh7 cells and African green monkey Cos7 cells.
2. Remove the media from the cells and replace with 3 ml of fresh media without antibiotics to ensure the maximum efficiency of transfection.
3. Prepare a mixture of 1-2 μg of capped MNV transcript into 100 μl of Opti-MEM (Invitrogen) and mix it with 4 μl of Lipofectamine 2000 previously mixed in 100 μl of Opti-MEM. Mix the sample thoroughly by pipetting it up and down 15 times. Leave the mixture at room temperature for 20 minutes.
4. Add the transfection complexes containing capped MNV transcripts in a drop-wise fashion to the cell monolayer and gently shake the plate in perpendicular directions.
5. Incubate the cells at 37 °C and 10% CO₂ for 24 to 72 hours. Afterwards, release infectious virions from cells by freeze and thawing and determine virus titre by plaque assay or TCID50. Typical yields of around 1 x 10⁶ TCID50/ml are reached.

2. Direct Recovery of Infectious MNV from cDNA in Cells Expressing T7 RNA Polymerase

This protocol is designed to allow the recovery of MNV in cells by the transcription of an infectious plasmid harbouring the full genomic cDNA sequence by a T7 polymerase expressed in the cells. Different cell lines can be used to recover infectious MNV by this approach although we typically obtain the highest yields with BHK-21 and BSR-T7 cells ¹⁵. We typically use BSR-T7 cells since they grow faster than the parental BHK clone line. Cells are infected with fowlpox (FPV) encoding for T7 RNA polymerase (FPV-T7) ¹⁸ which functions as a helper virus to drive expression of the viral RNA and subsequent recovery of infectious virus (Figure 4). Although BSR-T7 cells constitutively express T7 RNA polymerase, this expression is not sufficient to rescue infectious MNV after transfection of pT7:MNV 3’Rz in the absence of helper FPV-T7. Whilst the typical yields from this system are at least 10-fold lower than those described above, this approach does provide a rapid method of screening mutants to allow the identification of debilitating mutations. Typically this method is used first to assess the viability of a cDNA construct. Should a construct either fail to produce infectious virus or appear to yield virus at lower levels than that of the wild type infectious clone, then the RNA based approach described above is undertaken.

1. Trypsinise a monolayer of BHK-21 cells (or BSR-T7 cells) and seed 7.5 x 10⁵ cells into a 35 mm dish in antibiotic free growth media and incubate the cells at 37 °C and 10% CO₂ overnight. Add double the amount of cells in each plate if the transfections are planned for the same day as the seeding, and allow cells to adhere to the plate for 2-3 hours at 37 °C and 10% CO₂.
2. Remove cell culture media and add 700 μl of FPV-T7 to each well (Figure 4). A multiplicity of infection (MOI) of ~0.5 PFU per cell, based on titrations on primary chicken embryo fibroblasts, is generally used. However it is worth noting that new preparations of helper virus grown in primary fibroblasts are functionally titrated to determine the dose required for efficient virus recovery. Protocols for the propagation and titration of FPV-T7 have been previously described ¹⁸.
3. Incubate at 37 °C and 10% CO₂ for 1 hour to allow FPV-T7 to infect the cells. Then, add 2 ml of antibiotic free DMEM containing 10% FCS and incubate the cells for an additional hour at 37 °C and 10% CO₂ to allow T7 RNA polymerase expression.
4. To proceed with the transfection of the infectious plasmid, firstly remove the media from the infected cells, wash with 2 ml of media (10% FCS in antibiotic free DMEM), and finally cover the cell monolayer with 3 ml of media. Antibiotics should not be added to the media since they may interfere with the efficiency of Lipofectamine 2000 (Invitrogen).
5. Prepare a mix of 1 μg of wild type MNV cDNA infectious plasmid (e.g. pT7:MNV 3’Rz) in 100 μl of Opti-MEM (Invitrogen) and mix it with 4 μl of Lipofectamine 2000 (Invitrogen) previously mixed with 100 μl of Opti-MEM (Invitrogen). Mix the reaction thoroughly by pipetting it up and down 15 times and keep the mix at room temperature for 20 minutes.
6. The resulting transfection mix should be then added drop-wise to the cell monolayer and the plate should be gently shaken in perpendicular directions.
7. Incubate the FPV-T7 infected, MNV plasmid-transfected cells at 37 °C and 10% CO₂ for 24 to 72 hours. Cells transfected with infectious plasmid pT7:MNV 3’Rz normally render titres from 1 x 10⁴ to 5 x 10⁴ TCID50/ml.

3. Representative Results

Both reverse genetics approaches are highly efficient for the recovery of infectious MNV in cell culture as shown in Figure 5. Infectious MNV with titres exceeding 10⁵ TCID50/ml are recovered at 24 hours after transfection of capped MNV RNA into Raw264.7 cells. Similarly, the transfection of infectious plasmid pT7:MNV 3’Rz into BSR-T7 cells previously infected with helper FPV expressing T7 (FPV-T7) led to viral titres largely exceeding 10⁴ TCID50/ml (Figure 5). These viral titre values obtained with synthetic RNA and DNA molecules are similar to those obtained in transfections involving natural VPg-linked RNA isolated from infectious virions into the same cells (Figure 5). These results highlight the high efficiency of the reverse genetics approaches described here to recover genetically defined MNV variants in cell culture.

Figure 1. Illustration of MNV genome and plasmid for the recovery of infectious virus. A, Schematic representation of MNV genome organisation. Each protein coding region is illustrated as a single white box. ORF1 is translated into 7 different non-structural proteins (NS1/2 to NS7) that are released from precursor polyprotein after self-proteolytic processing. ORF 2 encodes the major capsid protein VP1, ORF 3 encodes the minor capsid protein VP2, and ORF4 overlapping with ORF2 coding region encodes virulence factor VF1. Genomic and subgenomic RNAs contain a polyA tail at their 3′ ends of variable length. B, Plasmid containing MNV cDNA used in our reverse genetic approaches (pT7:MNV 3’Rz). MNV cDNA is fused to a polyA tail of 26 residues in its 3′ end. The MNV cDNA sequence is located immediately downstream of a truncated T7 promoter sequence, to allow T7-driven transcription, and upstream of a unique NheI site and a DNA sequence coding for a self-cleaving ribozyme after it. These sequences are instrumental for ensuring RNA transcription termination right after the genomic polyA tail present at the 3′ end.

Figure 2. Overview of the protocol for the recovery of infectious MNV from RNA transcribed and capped in vitro. The plasmid pT7:MNV 3’Rz is linearised immediately downstream of the MNV genomic sequence using NheI restriction enzyme (step 1). After DNA purification, MNV RNA transcripts are generated in vitro by using T7 RNA polymerase (step 2). Transcription products usually run with an apparent mobility of 2.5-3Kb on a non-denaturing 1% agarose gel (step 3, Figure 3). The template DNA is eliminated using a commercial RNAse-free DNAse. RNA is then purified from free nucleotides by LiCl precipitation (step 4). The purified RNA product may then be in vitro capped after being previously heated at 65 °C to unfold secondary RNA structures (steps 5-6). After purification by LiCl precipitation, RNA is transfected into either Raw264.7 cells (Neon transfection system, Invitrogen) or BSR-T7 cells (Lipofectamine 2000, Invitrogen) (steps 7-8). Once inside the cell, capped RNA transcripts will be translated into viral proteins which would catalyse viral transcripts replication into new MNV RNA molecules containing a proper VPg molecule at their 5′ end. Successive cycles of replication accompanied of viral translation would generate large numbers of viral genomes which will be encapsidated to generate infectious virions. To facilitate virus release from cells, one or several cycles of freeze and thaw are performed (step 9). Viral yields can be then determined by TCID50 or plaque assay procedures.

Figure 3. Analysis of MNV RNA transcripts integrity along the protocol. A, Integrity of MNV RNA synthesised in vitro. The plasmid pT7:MNV 3’Rz is firstly linearised using NheI restriction enzyme. After DNA purification, MNV RNA transcripts are generated in vitro by using T7 RNA polymerase (lane 2). RNA is then purified from free nucleotides by LiCl precipitation (lane 3). Transcription products are run on a non-denaturing 1% agarose gel in parallel to 1-Kb DNA ladder (New England Biolabs, lane 1). Relative mobility of viral transcripts under non-denaturing conditions is similar to a dsDNA product of 2.5-3 Kb. B, Integrity of MNV RNA transcripts after capping. MNV transcripts purified previously by LiCl precipitation (lane 2) are subjected to enzymatic capping (lane 3) and purification by LiCl precipitation (lane 4). C, , Analysis in an Agilent RNA 6000 Nano chip of MNV transcripts (second lane) and capped MNV transcripts (third lane) which have been previously precipitated in LiCl. A ssRNA ladder is run in parallel.

Figure 4. Overview of the protocol for the recovery of infectious MNV from cDNA. Initially, BSR-T7 (or BHK) cells are infected with a recombinant fowlpox virus (FPV) expressing the bacteriophage T7 RNA polymerase (FPV-T7) (step 1). The infected cells are incubated for 2 hours before further treatment to allow the expression of FPV proteins which includes the recombinant T7 RNA polymerase (step 2). Afterwards, pT7:MNV 3’Rz is transfected into the cells by using Lipofectamine 2000 (Invitrogen) (step 3). Once inside the cell, pT7:MNV 3’Rz is recognised by T7 RNA polymerase which synthesises MNV RNA transcripts (step 4). The presence of a self-cleaving δ-Ribozyme sequence at the 3’end of the genome guarantees the transcript 3′ terminus is located just after the polyA tail (step 5). Some viral transcripts are intracellularly capped by an FPV capping enzyme (step 6). The resulting MNV capped transcripts will be translated to generate MNV proteins which would catalyse MNV transcripts replication. Newly synthesised MNV RNA molecules containing a proper VPg molecule at their 5′ end would undergo successive cycles of replication accompanied by viral translation which may finally result in the generation of infectious encapsidated virus. To facilitate virus release from cells, one or several cycles of freeze and thaw are performed (step 7). Viral yields can be then determined by TCID50 or plaque assay procedures.

Figure 5. Representative results of virus titres obtained from different reverse genetics approaches described in the text. Grey bars represent the virus titres obtained at 24 hours after Neon-transfection of 2 x 10⁶ Raw264.7 cells, or after lipo-transfection of 2 x 10⁶ BSR-T7 cells with in vitro transcribed and capped MNV RNA. White bars represent the virus titre typically obtained after lipofection of pT7:MNV 3’Rz (MNV cDNA) into 2 x 10⁶ BSR-T7 cells previously infected for 2 hours with fowlpox virus expressing recombinant T7 polymerase (FPV-T7). As a positive control for the transfection into Raw and BSR-T7cells, we typically use 2 μg of RNA extracted from cells infected with MNV which contain high levels of VPg-linked MNV RNA. Negative controls have been carried out with either MNV RNA or pT7:MNV 3’Rz encoding a frameshift mutation (F/S) which abrogates replication, resulting in no detectable virus (data not shown).