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.

Highs and Lows in Calicivirus Reverse Genetics

“…In general, it must be considered that most of the transfection reagents available may cause a cytotoxic effect to some extent and that such an effect could sometimes be morphologically indistinguishable from virus-induced CPE observed in positive controls. Reportedly, the use of helper viruses, such as rFPV-T7 or rMVA-T7, to deliver RNA polymerase in trans may further increase the chance of observing misleading CPE, easily confounded with inexistent calicivirus recovery [43,73,74,79,80].…”

Highs and Lows in the Calicivirus Reverse Genetics

“…The roles of all non-structural proteins have not yet been fully elucidated, however, it is clear that NS3 possesses NTPase/helicase activity; NS5 (also referred to as VPg) is a paradigm-breaking polypeptide, since it acts as a protein primer for RNA synthesis initiation [25]. VPg also serves for the recruitment of translation initiation factors (such as eIF4A, eIF4E, eIF4G) onto ribosomes during viral RNA translation [26][27][28][29], a function which when absent is reportedly recoverable by simply adding a regular eukaryotic 7-methylguanosine cap structure to the 5′ end of viral RNA [2,30]. NS6 is a protease very similar to the picornavirus 3C cysteine protease, which is why the former is referred to as 3C-like protease in the literature.…”

“…Uncontrolled sequence rearrangements and mutations that render the derived RNA transcripts non-functional, have been systematically reported [54][55][56]. Cloning the genomic cDNA of interest into an expression vector, trying a different bacterial strain, lowering the culture temperature (for example, using 30-32°C instead of 37°C), or even increasing the bacterial culture volume to compensate for the reduced plasmid yield during maxi-preps, could eventually help [30].…”

“…In general, the larger the genome size, the lower the probability of obtaining an error-free, genome-length cDNA copy in a row. Therefore, sometimes this goal is achieved by assembling several smaller cDNA pieces into the whole genomic cDNA, by means of ligation following small PCRs and enzymatic cut within unique endonucleases restriction sites [30,56] or through Gibson assembly [57].…”

“…Schematic representation of a calicivirus, genome-length cDNA-expressing vector, showing the main genetic regulatory elements that should be included in an ideal infectious clone to generate functional "genome-emulating" RNA transcripts of a defined length with a precise 5′ terminus and a sufficiently long, polyadenylated free 3′ end. See further details in the text (Adapted from [30]).…”