Visualization of feline calicivirus replication in real-time with recombinant viruses engineered to express fluorescent reporter proteins

Abstract

Caliciviruses are non-enveloped, icosahedral viruses with a single-stranded, positive sense RNA genome. Transposon-mediated insertional mutagenesis was used to insert a transprimer sequence into random sites of an infectious full-length cDNA clone of the feline calicivirus (FCV) genome. A site in the LC gene (encoding the capsid leader protein) of the FCV genome was identified that could tolerate foreign insertions, and two viable recombinant FCV variants expressing LC fused either to AcGFP, or DsRedFP were recovered. The effects of the insertions on LC processing, RNA replication, and stability of the viral genome were analyzed, and the progression of a calicivirus single infection and co-infection were captured by real-time imaging fluorescent microscopy. The ability to engineer viable recombinant caliciviruses expressing foreign markers enables new approaches to investigate virus and host cell interactions, as well as studies of viral recombination, one of the driving forces of calicivirus evolution.

Figure 1. Recombinant FCV constructs

(A) Schematic diagram showing the recombinant FCV that was engineered to have a multiple cloning site encoded within the LC. The location of the multiple cloning site in the LC is indicated by an arrow. E/A corresponds to the cleavage site of the ORF2 precursor. Nucleotide numbers correspond to the Urbana strain of FCV (Genbank accession no. L40021). (B) Recombinant FCV constructs. cDNA infectious clone constructs showing the site of insertion of GFP (pR6-LC-GFP) and DsRed (pR6-LC-DsRed). (C) pCI expression vectors containing the subgenomic region of pR6, pR6-LC-GFP or pR6-LC-DsRed. rLC, recombinant LC.

Figure 2. Recombinant FCV expressing GFP and DsRed fused to the LC are viable and show growth kinetics similar to wt FCV

(A) Virus serial dilutions were added to CRFK monolayers of a 6-well plate and incubated for 60 min, at which point an agarose overlay was added. Approximately 48 h later, fluorescent and phase images were captured of vR6 (panels 5-6, 9-10), vR6-LC-GFP (panels 7, 11) and vR6-LC-DsRed (panels 8, 12) plaques using a Leica DMI4000 B microscope. Fluorescent images correspond to signals detected when using filters specific to GFP and DsRed (panels 5-8). The agarose overlay was removed, and the cells were stained with crystal violet and images were taken of plaques (panels 1-4). (B) CRFK monolayers were infected at a multiplicity of infection (MOI) of 0.01 and virus titer was determined by plaque assay. The mean log₁₀ titer is shown with a standard deviation of 1. Time-points were taken in duplicate, and each time-point duplicate was plaqued in duplicate. T=0 represents the virus titer of washed monolayers after a 1 hr incubation period to allow for virus adsorption.

Figure 3. Evidence for efficient cleavage of the LC precursor of pR6-LC-GFP and pR6-LC-DsRed constructs in vitro and in vivo

(A) The subgenomic region of vR6, vR6-LC-GFP and vR6-LC-DsRed were cloned into the pCI expression vector. The cloned pCI expression vectors were used in a coupled in vitro transcription and translation reaction, and labeled with [³⁵S] methionine. The samples were subjected to SDS-PAGE in a 4-12% Tris-glycine gel (Lonza). The proteins were stained with GelCode Blue Stain (Pierce), dried and exposed to film. (B) TNT samples were treated with either unlabeled mock (lanes 1-3) or vR6-infected (lanes 4-6) lysates for 3 h. The treated samples were immunoprecipitated using rabbit hyperimmune anti-LC sera, and subsequently subjected to SDS-PAGE in a 4-12% Tris-glycine gel (Lonza). The proteins were stained with GelCode Blue Stain (Pierce), dried and exposed to film. (C) CRFK monolayers of a 6-well plate were infected with vR6, vR6-LC-GFP and vR6-LC-DsRed at an MOI of 1, and lysates of a single well were collected at 2, 4, and 6 hours p.i. Lysates were loaded on a 4-20% Tris-glycine gel and subjected to SDS-PAGE. The gel was transferred to a nitrocellulose membrane and incubated with FCV-virion specific serum to detect mature VP1 production. (D) Resuspended pellets from CRFK cells infected with the recombinant FCV variants, as well as vR6 as a positive control, were analyzed by SDS-PAGE (4-20% Tris-glycine gel), and subsequently transferred to a nitrocellulose membrane. Western blotting was performed with antibodies specific to LC (rabbit polyclonal raised against bacterially expressed LC), GFP (affinity purified mouse monoclonal; Clontech) or DsRed (rabbit polyclonal; Clontech). After detecting the viral protein of interest, blots were stripped (Thermo Scientific) and the membrane reacted with anti-β-actin to serve as a loading control (anti-β-actin HRP conjugated antibody; Sigma). Molecular mass standards in kilodaltons (kDa) are indicated on the left and were calculated using SeeBlue Plus2 Pre-Stained Standard (Invitrogen).

Figure 4. Northern blot analysis of genomic and subgenomic RNA from recombinant FCV-infected cells

CRFK monolayers of a 6-well plate were infected with vR6, vR6-LC-GFP and vR6-LC-DsRed at an MOI of 1, and approximately 8 h later total RNA was extracted using Trizol reagent (Invitrogen). The RNA was analyzed on a 1% agarose gel that was dried and treated with a biotinylated RNA sense probe that was then exposed to film. G, genomic RNA; SG, subgenomic RNA.

Figure 5. Real-time imaging of a co-infection

CRFK cells were co-infected with vR6-LC-GFP (MOI of 2) and vR6-LC-DsRed (MOI of 1). 2.5 h p.i. the cells were transferred to a closed chamber (37°C/5% CO₂) and monitored over time. Images were captured every 12 min for 14 h. Time (h) represents time p.i. in hours. GFP and DsRed channels correspond to signals detected when using filters specific to GFP and DsRed. Scale bars = 10 μm. For a movie derived from this experiment of LC-GFP/LC-DsRed expression during an infection, see Supplementary Video 1.

Figure 6. RT-PCR diagnostics and sequence analysis of vR6-LC-PmeIDsRed and revertants

(A) Schematic of the ORF2 organization and the diagnostic PCR primers with the expected amplified fragment lengths for vR6 and vR6-LC-PmeIDsRed. The grey box corresponds to the DsRed insertion. E/A corresponds to the amino acid cleavage site. (B) RT-PCR analysis of a fluorescent plaque (lane 2), six non-fluorescent plaques (lanes 3-6), and vR6 (lane 9) virus with primers flanking the site of insertion. M, Invitrogen's 100 bp DNA molecular weight marker (lanes 1 and 11). Products were analyzed in a 1.5% agarose gel. Sizes are indicated in nucleotides. (C) Schematic representation of the sequence analysis corresponding to the six non-fluorescent plaques. Dotted lines correspond to deleted portions of the insertion. Numbers identify the positions with regard to the parental plasmid (pR6-LC-PmeIDsRed).