Reverse Genetics System for Rabbit vesivirus

Abstract

Most caliciviruses are refractory to replication in cell culture and only a few members of the family propagate in vitro. Rabbit vesivirus (RaV) is unique due to its ability to grow to high titers in several animal and human cell lines. This outstanding feature makes RaV an ideal candidate for reverse genetics studies, an invaluable tool to understand the molecular basis of virus replication, the biological functions of viral genes and their roles in pathogenesis. The recovery of viruses from a cDNA clone is a prerequisite for reverse genetics studies. In this work, we constructed a RaV infectious cDNA clone using a plasmid expression vector, under the control of bacteriophage T7 RNA-polymerase promoter. The transfection of permissive cells with this plasmid DNA in the presence of T7 RNA-polymerase, provided in trans by a helper recombinant poxvirus, led to de novo synthesis of RNA transcripts that emulated the viral genome. The RaV progeny virus produced the typical virus-induced cytopathic effect after several passages of cell culture supernatants. Similarly, infectious RaV was recovered when the transcription step was performed in vitro, prior to transfection, provided that a 5'-cap structure was added to the 5' end of synthetic genome-length RNAs. In this work, we report an efficient and consistent RaV rescue system based on a cDNA transcription vector, as a tool to investigate calicivirus biology through reverse genetics.

Keywords: Vesivirus; calicivirus; infectious cDNA clone; reverse genetics; virus rescue.

FIGURE 1

(A) Genomeorganization of Rabbit vesivirus. The proteolytic cleavage of ORF1-encoded polyprotein, as well as the capsid leader (LC) cleavage site, are indicated with arrowheads, along with the names of mature peptides generated. Numbers refer to nucleotide positions within the genome. (B) Schematic representation of the genetic elements in the infectious clone design, including the nucleotide sequence neighboring the +1 transcription site. Scissors represent the HDV antigenome ribozyme cleavage site. (C) Detail of the site-directed mutagenesis of nucleotide residues 7,862–7,865 for the generation of tagged RaV, involving the loss of a NheI restriction site and the appearance of a novel XhoI site. Numbers refer to nucleotide positions within the genome. (D) Formaldehyde-agarose denaturing electrophoresis gels. Analysis of capped (+) and uncapped (–) genome-length RaV RNA products, obtained using in vitro transcription of NotI-linearized pT7-RaV and pT7-RaV/Xh infectious clones. Virion RNA extracted from a concentrated purified RaV stock was run in the first lane, for comparative purposes.

FIGURE 2

Microscope images (100 X) of 293T cells either (A) infected with rFPV-T7 and further transfected with DNA infectious clones or (B) transfected with capped or uncapped synthetic genome-length viral RNA. GFP expression from pT7-GFP plasmid visualized using a fluorescence filter served as a transfection efficiency control. (C) Bright field microscope images (40 X) of Vero cells infected with either wtRaV, rRaV, or rRaV/Xh (MOI = 0.1, passage 3). The photos were taken at 6, 12, and 24 hpi. (D) Morphology of wild-type and rescued RaV lysis plaques in Vero cells under 1% agarose overlay.

FIGURE 3

(A) Formaldehyde-agarose denaturing gel analysis of virion RNA extracted from concentrated stocks of wtRaV, rRaV, and rRaV/Xh. gRNA: genomic RNA; sgRNA: subgenomic RNA; MWM, molecular weight marker High Range (HR) RiboRuler (Fermentas); Kb, kilobases. (B) Sequences of RNA 5′ ends found after 5′-RACE assays. RNA was extracted from cells infected with rFPV-T7 and further transfected with pT7-RaV or pT7-RaV/Xh (passage 0). The first 430 nt of the 5′ ends of pT7-RaV and pT7-RaV/Xh-derived transcripts were identical to those of wtRaV RNA, though only the first 67 nt are shown.

FIGURE 4

Agarose gel electrophoresis of RT-PCR products derived from de novo synthesized viral RNA in cells infected with rescued viruses rRaV and rRaV/Xh after 2 blind virus passages. (A) Amplicons from cells infected with RaV (passage 3) recovered after transfection using synthetic capped or uncapped genome-length RNA transfections (capped or uncapped refers to RNA used in passage 0). The absence or presence of RT enzyme during the first strand synthesis step is indicated by –/+, respectively. (B) Cells infected with RaV recovered after rFPV-T7 infection followed by DNA transfection. The absence or presence of RT enzyme during the first strand synthesis step is represented by –/+, respectively. (C,D) Digestion products obtained after treating the PCR amplicons from A and B with XhoI restriction enzyme. The –/+ signs indicate the absence or presence of XhoI enzyme when applicable. MWM, molecular weight marker O’Generuler 1 Kb Plus DNA ladder (Fermentas); bp, base pairs.

FIGURE 5

Western blot analyses of RaV NS3 and VP1 proteins during passages 0, 1, and 3 of rescued rRaV and rRaV/Xh using either (A) synthetic capped or uncapped RNA transfections, or (B) rFPV-T7 infection followed by DNA transfections. MWM, molecular weight marker Broad Range (BR) Spectra (Fermentas).

FIGURE 6

(A) One-step growth curves of wtRaV and rRaV/Xh in Vero cells infected at MOI = 10 and incubated for 14 h. No significant statistical differences were found between both viral titers within each time point (Student’s t-tests, p > 0.05). (B) Effect of the DNA amount used in transfection (passage 0) over the titer of viruses recovered in passage 1 supernatants. Transfections were performed in 6-well plates with either 1, 3, or 5 μg of the infectious cDNA clone. For each DNA quantity used in passage 0, plaque assay wells inoculated with the corresponding passage 1 supernatant dilutions 10^–3 and 10^–4 are shown.