Generation of Bi-Reporter-Expressing Tri-Segmented Arenavirus

Abstract

Reverse genetics systems provide a powerful tool to generate recombinant arenavirus expressing reporters to facilitate the investigation of the arenavirus life cycle and also for the discovery of antiviral counter-measures. The plasmid-encoded viral ribonucleoprotein components initiate the transcription and replication of a plasmid-driven full-length viral genome, resulting in infectious virus. Thereby, this approach is ideal for the generation of recombinant arenaviruses expressing reporter genes that can be used as valid surrogates for virus replication. By splitting the small viral segment (S) into two viral segments (S1 and S2), each of them encoding a reporter gene, recombinant tri-segmented arenavirus can be rescued. Bi-reporter-expressing recombinant tri-segmented arenaviruses represent an excellent tool to study the biology of arenaviruses, including the identification and characterization of both prophylactic and therapeutic countermeasures for the treatment of arenaviral infections. In this chapter, we describe a detailed protocol on the generation and in vitro characterization of recombinant arenaviruses containing a tri-segment genome expressing two reporter genes based on the prototype member in the family, lymphocytic choriomeningitis virus (LCMV). Similar experimental approaches can be used for the generation of bi-reporter-expressing tri-segment recombinant viruses for other members in the arenavirus family.

1. Introduction

The family Arenaviridae consists of four genera: Mammarenavirus, Reptarenavirus, Hartmanivirus, and Antennavirus [1, 2]. Based on serological cross-reactivity and sequence-based phylogenetic studies, the genus Mammarenavirus, encompassing viruses that infect mammals, is further divided into two groups, the Old World and the New World mammarenaviruses [3]. Several mammarenaviruses can cause severe hemorrhagic fever disease with a very high case fatality rate, representing important threats to human health within the viruses’ endemic regions.

Mammarenaviruses are enveloped RNA viruses with a bi-segmented, negative sense, single-stranded genome, which is composed of the small (S, ~3.5 kb) and the large (L, ~7.2 kb) segments [4] (see Fig. 1). Mammarenavirus L and S viral segments have highly conserved 3′ and 5′ terminal noncoding regions (NCR) that are predicted to form panhandle structures, and serve as cis-acting elements or promoters required for viral genome replication and gene transcription (see Fig. 1a) [5]. Each viral (v)RNA segment encodes, using an ambisense coding strategy, two polypeptides in opposite orientation, separated by a noncoding intergenic region (IGR) [6, 7] (see Fig. 1a). The viral nucleoprotein (NP) and the envelope glycoprotein precursor (GPC) are encoded by the S segment, whereas the viral polymerase (L) protein and a small multi-functional matrix-like (Z) protein are encoded by the L segment (see Fig. 1a) [8]. GPC is co-translationally cleaved by cellular signal peptidases to generate a 58 amino acid-long stable signal peptide (SSP) and the immature GP1/2 precursor (see Fig. 1a). The immature GP1/2 precursor is post-translationally processed by the cellular protease subtilisin kexin isozyme-1 (SKI-1)/site 1 protease (S1P) to generate the mature virion surface glycoproteins GP1 and GP2 (see Fig. 1a) [9, 10]. GP1, GP2, and SSP form a tripartite heterotrimeric GP complex that mediates virion receptor recognition and cell entry [11]. NP and L are the minimum viral factors responsible for viral genome replication and gene transcription (see Fig. 1a) [12, 13]. The arenavirus particles are surrounded by a lipid bilayer containing the post-translationally processed viral glycoprotein subunits involved in receptor recognition (GP1) and cellular entry (GP2), a layer of the matrix-like Z protein, which is involved in viral assembly and budding, located underneath the lipid bilayer, and the core of arenavirus is formed by viral ribonucleoprotein (vRNP) complexes, made of the viral segments encapsidated with the viral NP and attached by the viral RNA-dependent RNA polymerase (L) protein (see Fig. 1b).

Fig. 1.

Arenavirus genome organization, viral replication and transcription, and virion structure. (a) Genomic organization, viral replication and translation: Arenavirus replication takes place in the cytoplasm of infected cells. The viral L polymerase and NP are the are the minimum components required for viral genome replication and gene transcription. The association of the L polymerase with the vRNPs initiates transcription from the untranslated regions, UTRs (black boxes). Primary transcription results in the synthesis of NP (S segment) and L polymerase (L segment) mRNAs. Transcription is terminated in the intergenic region (IGR). Subsequently, L polymerase moves across the IGR to generate a copy of the full-length S and L antigenome vRNAs (replication). The antigenomic vRNAs serve as templates for the synthesis of GPC (S segment) and Z (L segment) mRNAs. The antigenomic vRNAs also serve as templates for the amplification of the corresponding S or L vRNA genomes, a process mediated by the L polymerase and the vRNPs. (b) Virion structure: Arenavirus particles are surrounded by a lipid bilayer containing the post-translationally processed viral glycoprotein subunits GP1 and GP2. Underneath the lipid bilayer is a layer composed of the matrix-like Z protein, which is involved in viral assembly and budding. The core of the virus is formed by vRNP complexes, made of the viral S and L segments encapsidated with the viral NP. The viral L polymerase is also attached to each of the viral genomic RNAs. The incorporation of newly synthesized vRNPs into nascent virions is mediated by the interaction between NP and Z

Reverse genetics represent an excellent approach to investigate the phenotype of recombinant viruses containing changes in their noncoding and/or coding sequences. In virus research, reverse genetics can be used to answer basic virology question and generate useful recombinant viruses, particularly those expressing reporter genes. Rescue plasmids consist of a full-length cDNA copy of the viral S and L segments under the control of a promoter which allows the expression of the corresponding viral genomic RNA in mammalian cells (see Fig. 2a). By providing the ribonucleoprotein complex proteins NP and L required for viral replication and transcription (see Fig. 2a), the viral genomic RNA segments can be replicated into complementary antigenomic RNAs and transcribed into mRNAs, respectively, to produce, thereby initiating the virus life cycle (see Fig. 2b).

Fig. 2.

Schematic representation of the reverse genetics to generate r3LCMV. (a) Plasmids used for r3LCMV rescue: Three plasmids are used for the expression of the r3LCMV vRNA genomes. The L, S1, and S2 vRNA segments are placed downstream of a polymerase I (Pol-I) promoter (gray arrow) and Pol-I terminator (gray box) cassette. The EGFP and GLuc reporter genes are used to substitute the viral NP and GPC in the modified S1 and S2 viral segments, respectively. LCMV L polymerase and NP are cloned between the chicken β-actin promoter (black arrow) and poly(A) signal (black box) in the pCAGGS plasmid, to generate the minimal components to initiate viral genome replication and gene transcription. The CMV-IE enhancer and intron regions (white boxes) are used to increase the expression level of the mRNA transcribed from the chicken β-actin promoter. (b) r3LCMV rescue: To generate r3LCMV, BHK-21 cells are transiently transfected, using LPF2000 with the indicated mPol-I viral RNA and the pCAGGS protein expression plasmids. After changing the media (day 2), transfected cells are scaled up in a T75 flask (day 4). Tissue culture supernatants from BHK-21 cells in the T75 flask are collected at 7 days post-transfection for viral detection (day 7)

In this chapter, we describe the experimental approaches to generate a replication-competent recombinant tri-segmented (r3) lymphocytic choriomeningitis virus (LCMV) expressing two reporter genes. This r3LCMV expressing both an enhanced green fluorescent protein (EGFP) and Gaussia luciferase (GLuc) represents an excellent option to study the biology of the virus and for the identification and characterization of therapeutic approaches for the treatment of LCMV infection. Similar experimental approaches can also be used for the generation of tri-segmented, bi-reporter-expressing virus for other members in the Arenaviridae family.

2. Materials

2.1. Reagents

1. 10× phosphate-buffered saline (PBS): The stock solution is diluted with cell culture grade water to generate 1 × PBS.

2. 100× penicillin-streptomycin L-glutamine (PSG): The stock solution is diluted with cell culture grade water to generate 1 × PSG before use.

3. Dulbecco’s Modified Eagle Medium (DMEM).

4. Fetal bovine serum (FBS).

5. Growth medium: DMEM containing 10% (v/v) FBS and 1 × PSG.

6. Lipofectamine 2000 (LPF2000).

7. Opti-MEM.

8. Post-infection medium: DMEM containing 2.0% (v/v) FBS and 1 × PSG.

9. Trypsin/EDTA solution.

10. Coelenterazine (CTZ) solution: 10 μM native CTZ dissolved in 1 × PBS.

2.2. Plasmids

1. vRNA-expressing plasmids: Full-length plasmids encoding the viral L (mPol-I L) and split S (mPol-I S1/EGFP-GP and mPol-I S2/NP-GLuc) segments have been previously described [13-15] (see Fig. 2a) (see Note 1). The mPol-I L encodes the full-length LCMV viral L segment. The artificial mPol-I S1 segment encodes the viral GPC and EGFP instead of the viral NP (see Fig. 2a). The artificial mPol-I S2 segment encodes the viral NP and GLuc instead of the viral GPC (see Fig. 2a). The mPol-I L and mPol-I S1 and mPol-I S2 plasmids encode the viral RNAs under a mouse Pol-I promoter and mouse Pol-I terminator cassette (see Fig. 2a).

2. Protein expression plasmids: pCAGGS plasmids encoding the viral L (pCAGGS-L) and NP (pCAGGS-NP) proteins required for viral genome replication and gene transcription have been previously described [16, 17] (see Fig. 2a) (see Note 2). LCMV L and NP coding sequences are cloned into pCAGGS using conventional molecular biology methods.

2.3. Cells

1. Baby hamster kidney (BHK-21) cells (ATCC: CCL-10): The cells for transfection and initial virus rescue (see Note 3). They are maintained in growth media.

2.4. Labware

1. 6-well plate for cell culture.

2. 96-well microplate for cell culture.

3. T75 flasks for tissue culture.

4. 20-μL, 200-μL, and 1,000-μL universal pipette tips and their corresponding pipttes.

5. 1.5-mL cryotubes.

6. 15-mL, and 50-mL Falcon tube.

2.5. Instrumentation

1. Humidified 5% (v/v) CO₂ incubator.

2. Fluorescent microscope.

3. Bioluminescence plate reader.

4. Centrifuge.

5. Biosafety cabinet.

3. Methods (See Note 4)

3.1. Generation of r3LCMV

1. (Opti-MEM-LPF2000 mix) Prepare a mixture of 250 μL of Opti-MEM with 12 μL of LPF2000 (2.5 μg LPF2000 per μg plasmid DNA) per transfection (see Note 5). Incubate the Opti-MEM-LPF2000 mixture for 5 min at room temperature (RT).

2. (Opti-MEM-plasmid DNA mix) In a separate tube, prepare the DNA plasmid mixture (1.4 μg mPol-I L, 0.8 μg mPol-I S1, 0.8 μg mPol-I S2, 1.0 μg pCAGGS-L, and 0.8 μg pCAGGS-NP) (see Fig. 2a) in a total volume of 250 μL of Opti-MEM per transfection.

3. (Opti-MEM-LPF2000-plasmid DNA mix) Pipette 250 μL of Opti-MEM-LPF2000 (see step 1 in Subheading 3.1) into the Opti-MEM-plasmid DNA mix (see step 2 in Subheading 3.1) and incubate at RT for 20–30 min.

4. (Cell preparation) Before manipulating the cells, warm up 1 × PBS, trypsin/EDTA solution, and growth media to 37 °C and proceed the following steps:

   1. Wash the cells twice with 10 mL 1 × PBS.
   2. Trypsinize the cells using 2 mL trypsin/EDTA solution. Cell detachment is usually accomplished by incubating the cells at 37 °C for ~5 min at 37 °C in the CO₂ incubator.
   3. Carefully resuspend the cells in 8 mL of growth media. Place the cell suspension in a 15-mL Falcon tube and centrifuge the cells for 5 min at 500 × g.
   4. Remove the supernatant and resuspend the cells in 10 mL of fresh growth media and count the cells using a hemocytometer. Adjust the cell concentration to 5 × 10⁵ cells/mL and dispense 2 mL (10⁶ cells) into individual wells in a 6-well plate.

5. After 30 min incubation, pipette 500 μL of the Opti-MEM-LPF2000-plasmid DNA mix (see step 3 in Subheading 3.1) into individual wells in the 6-well plate (see step 4 in Subheading 3.1) (see Fig. 2b) (see Note 6).

6. Incubate the cells at 37 °C in the CO₂ incubator for 24 h, replace the 2 mL transfection media with 2 mL of post-infection media, and return the cells to 37 °C in the CO₂ incubator for another 60 h (see Fig. 2b).

7. After 3-day incubation, proceed the following steps if transfected cells reach 100% confluence:

   1. Remove the tissue culture supernatant and gently wash the cells, twice, with 1 × PBS.
   2. Trypsinize the cells by adding 500 μL of trypsin/EDTA solution per well. Return the cells to the CO₂ incubator at 37 °C and incubate for 5 min.
   3. Carefully resuspend the cells with 5 mL of growth media and transfer to a 15-mL Falcon tube.
   4. Centrifuge the cells for 5 min at 500 × g.
   5. Remove the tissue culture supernatant and resuspend the cells in 5 mL of post-infection media and plate the cells in a T75 flask (see Fig. 2b). Bring up the volume to 15 mL with post-infection media and incubate the cells at 37 °C in the CO₂ incubator for 3–4 days (see Fig. 2b).

8. After 3–4 days of incubation (see Note 7), collect the tissue culture supernatant from the T75 flask (see step 7 in Subheading 3.1) into a 50-mL Falcon tube and centrifuge at 2500 × g, 4 °C for 5 min (see Fig. 2b). Collect the tissue culture supernatant containing the virus and discard the cell pellet (see Note 8).

9. Aliquot the virus into two cryotubes (~1 mL/cryotube), labeled as P0, and store them at −80 °C.

3.2. Confirmation of Viral Rescue and Titration of r3LCMV

1. Prepare 96-well microplates (10⁴ cells/well, triplicates) with BHK-21 cells (see Note 9).

2. Take one vial of the P0 from −80 °C, and serially dilute (1:10) the virus in 180 μL of post-infection media.

3. Infect the BHK-21 cells in the 96-well microplate (triplicate) with 50 μL/well of the serially diluted virus P0 stock for 1 h at 37 °C. After 1 h viral absorption, discard the supernatant and add 100 μL of post-infection media per well.

4. At 16 h post-infection, calculate the titer of r3LCMV P0 by observing EGFP expression under a fluorescent microscope and confirm GLuc activity in the tissue culture supernatant of infected cells (see Note 10). Viral titers are determined by the number of EGFP-positive cells (fluorescent forming units, FFU)/mL.

3.3. Amplification and Generation of a r3LCMV Stock

1. Once the rescue of rLCMV has been confirmed, prepare fresh monolayers of BHK-21 cells in T75 flasks (~8.0 × 10⁴ cells).

2. Infect the monolayer of BHK-21 cells with r3LCMV at a multiplicity of infection (MOI) of 0.01 for 1 h at 37 °C. After virus adsorption, discard the virus inoculum and add 12 mL of post-infection media.

3. Incubate the infected BHK-21 cells and observe them under a fluorescent microscope for EGFP expression every 24 h (see Note 11).

4. Once EGFP expression reaches to 100%, collect the tissue culture supernatant from the T75 flask into a 15-mL Falcon tube and centrifuge at 2500 × g at 4 °C for 5 min to remove cell debris.

5. After centrifugation, collect the tissue culture supernatant and discard the cell pellet.

6. Aliquot the virus into cryotubes, label as P1, and store them at −80 °C (see Note 12).

7. Titrate the stock as described above.

3.4. In Vitro Characterization of r3LCMV

1. Infect (MOI 0.1) monolayers of fresh BHK-21 cells (6-well plate format, 10⁶ cells/well, triplicate) with r3LCMV as described above.

2. Determine EGFP (fluorescent microscopy) and GLuc (bioluminescence plate reader) expression in the tissue culture supernatants from the infected cells at different times post-infection (see Fig. 3a) (see Note 13). The GLuc expression is determined by addition of the CTZ solution.

3. Also, use the tissue culture supernatants collected at the same times post-infection to assess viral titers as described above (see Note 14).

Fig. 3.

Characterization of r3LCMV. (a) EGFP and GLuc expression: Fresh monolayers of BHK-21 cells (6-well plate format, 10⁶ cells/well, triplicates) are infected (MOI 0.01) with r3LCMV, and, at the indicated times post-infection, expression of EGFP (top) and GLuc (bottom) is determined under a fluorescent microscope and a bioluminescence plate reader, respectively. Results show the median and SD of triplicate wells. Scale bar, 100 μm. In these experiments, a rLCMV/WT was used as the internal control. Expression of NP from rLCMV/WT-infected BHK-21 cells was determined by immunofluorescence assay using an antibody against the viral NP. (b) Viral growth kinetics: Tissue culture supernatants from BHK-21 cells infected in panel A were used to determine viral titers by fluorescent expression. Results show the median and SD of triplicate wells. Viral titers of rLCMV/WT were determined by immunofluorescence using an antibody against the viral NP

4. Notes

1. We recommend growing the mPol-I L plasmid in bacteria at 30 °C for 24 h and the mPol-I S1 and mPol-I plasmids at 37 °C for 16–24 h.

2. While we and many others use pCAGGS plasmids for the expression of the viral L and NP proteins, other RNA polymerase II expression plasmids will certainly be suitable for virus rescue. However, expression levels from other expression plasmids will vary, and the amount of each plasmid for protein expression would need to be optimized accordingly.

3. Human HEK293T and Vero cells can also be used to generate recombinant arenaviruses, including r3LCMV. However, because the transcriptional activity of the Pol-I promoters exhibits stringent species specificity, the cells selected for viral rescue should fit the species origin of the Pol-I promoter. To generate r3LCMV from HEK293T or Vero cells, vRNA expression plasmids should contain the human Pol-I promoter.

4. The rescue and characterization of r3LCMV should be performed at biosafety level 2 (BSL2) laboratories under approvement of institutional biosafety committee.

5. To increase the likelihood of successful r3LCMV rescue, we recommend conducting the transfections in triplicate. Therefore, prepare enough Opti-MEM-LPF2000 and Opti-MEM-plasmid DNA based on the number of virus rescues planned.

6. It is important to rock the plates front to back and side to side rather than swirling them in a circular fashion, as the latter results in transfection complexes being pushed to the edge of the well and produces uneven transfection of the monolayer.

7. The percentage of EGFP-positive cells can be easily determined under a fluorescent microscope before collecting the supernatant.

8. It is possible that r3LCMV titers in tissue culture supernatants from initial virus rescue are low and, therefore, the viruses need to be amplified to generate a stock. To that end, we recommend infecting fresh monolayers of BHK-21 cells at low MOI (0.01) and allow virus amplification for 48–72 h before collecting the tissue culture supernatants to increase viral titers.

9. Viral titers are usually evaluated in triplicate wells of 96-well microplates by assessing EGFP expression at 18 h to prevent overestimation of viral titers due to secondary infections.

10. LCMV does not display a cytopathic effect. If r3LCMV does not encode a fluorescent protein, the presence and quantification of the r3LCMV can be evaluated by immunofluorescence using an antibody specific for arenavirus NP or other viral antigens.

11. After 24 h infection, we recommend observing the infected cells for EGFP expression every 12 h.

12. We usually aliquot the virus stock in 500 μL aliquots per tube to avoid decomposition by repeated freezing and thawing.

13. We recommend including either mock-infected or rLCMV/WT-infected cells as controls for measuring EGFP and GLuc expression levels in these experiments.

14. Since rLCMV/WT does not express EGFP or GLuc, the presence of the virus in infected cells needs to be assessed by immunofluorescence assay using an antibody against the viral NP or any other viral antigen.