Highs and Lows in Calicivirus Reverse Genetics.

In virology, the term reverse genetics refers to a set of methodologies in which changes are introduced into the viral genome and their effects on the generation of infectious viral progeny and their phenotypic features are assessed. Reverse genetics emerged thanks to advances in recombinant DNA technology, which made the isolation, cloning, and modification of genes through mutagenesis possible. Most virus reverse genetics studies depend on our capacity to rescue an infectious wild-type virus progeny from cell cultures transfected with an "infectious clone". This infectious clone generally consists of a circular DNA plasmid containing a functional copy of the full-length viral genome, under the control of an appropriate polymerase promoter. For most DNA viruses, reverse genetics systems are very straightforward since DNA virus genomes are relatively easy to handle and modify and are also (with few notable exceptions) infectious per se. This is not true for RNA viruses, whose genomes need to be reverse-transcribed into cDNA before any modification can be performed. Establishing reverse genetics systems for members of the Caliciviridae has proven exceptionally challenging due to the low number of members of this family that propagate in cell culture. Despite the early successful rescue of calicivirus from a genome-length cDNA more than two decades ago, reverse genetics methods are not routine procedures that can be easily extrapolated to other members of the family. Reports of calicivirus reverse genetics systems have been few and far between. In this review, we discuss the main pitfalls, failures, and delays behind the generation of several successful calicivirus infectious clones.

Reverse Genetics System for Rabbit vesivirus.

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.

Role of annexin A2 in cellular entry of rabbit vesivirus.

The mechanisms of calicivirus attachment and internalization are not well understood, mainly due to the lack of a reliable cell-culture system for most of its members. In this study, rabbit vesivirus (RaV) virions were shown to bind annexin A2 (ANXA2) in a membrane protein fraction from HEK293T cells, using a virus overlay protein-binding assay and matrix-assisted laser desorption/ionization time-of-flight analysis. A monoclonal anti-ANXA2 antibody and small interfering RNA-mediated knockdown of ANXA2 expression in HEK293T cells reduced virus infection significantly, further supporting the role of ANXA2 in RaV attachment and/or internalization.