Reverse genetics systems for SARS-CoV-2: Development and applications

Cai, Hou-Li; Huang, Yao-Wei

doi:10.1016/j.virs.2023.10.001

Cited by 7 publications

(7 citation statements)

References 123 publications

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“…Although the term 'virus rescue' has systematically been employed as a synonym of reverse genetics, the la er more properly refers to the a empts to recover virions from mutated or genetically altered viral genomes, provided that the rescue of wild-type virions from unaltered genomes has previously been achieved in a reproducible manner so that it could be set up as a parallel control assay in every reverse genetics experiment. 4) and further autoproteolytic processing that yields the non-structural mature polypeptides (5), synthesis of the genome-length antisense RNA intermediate (6) that serves as the template for both the sgRNA synthesis (7) that is subsequently translated into structural proteins (VP1 and VP2) (8) and the generation of multiple copies of the gRNA (9). The newly synthesized viral components (e.g., capsid proteins, gRNA, and sgRNA) are put together into the progeny viral particles (10), which are ultimately released from the infected cell (11) during late events, concomitantly associated with cell lysis and death.…”

Section: The Challenges Of Reverse Geneticsmentioning

confidence: 99%

Highs and Lows in Calicivirus Reverse Genetics

Álvarez,

Arboleya,

Abade dos Santos

et al. 2024

Viruses

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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.

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Section: The Challenges Of Reverse Geneticsmentioning

confidence: 99%

Highs and Lows in Calicivirus Reverse Genetics

Álvarez,

Arboleya,

Abade dos Santos

et al. 2024

Viruses

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“…Further details of the replication cycle of a model calicivirus are comprehensively reviewed in [3,43]. 4) and further autoproteolytic processing that yields the non-structural mature polypeptides (5), synthesis of the genome length antisense RNA intermediate (6) that serves as the template for both: the sgRNA synthesis (7) that is subsequently translated into structural protein (VP1 and VP2) (8), and for the generation of multiple copies of the gRNA (9). The newly synthesized viral components (e.g., capsid proteins gRNA and sgRNA) are put together into the progeny viral particles (10), which are ultimately released from the infected cell (11) during late events, concomitantly associated to cell lysis and death.…”

Section: Calicivirus Replication Cyclementioning

confidence: 99%

“…Depending on the viral genome size, the construction of a genome-length cDNA vector supporting the synthesis of infectious transcripts can be long and tedious [6,52,53], but once a reproducible workflow is established from genotype (viral genome) to the phenotype (rescued virions), it represents a major leap for virus research. An established and reproducible infectious clone provides a powerful tool for reverse genetics experiments, in which researchers directly manipulate the viral genome (for example introducing point mutations, deletions, insertions, inversions or translocations) and further assess the effects of such manipulations on the phenotype in terms of fitness, replication competence (virus yield, attenuation), tropism, host range, virulence, pathogenicity, immunogenicity, etc.…”

Section: The Challenges Of Reverse Geneticsmentioning

confidence: 99%

“…Regardless of the strategy followed, obtaining an infectious clone provides a powerful tool for the use of reverse genetics techniques. Thus allowing the manipulation of the viral genome and the study of the effects of certain gene changes (point mutations, deletions, insertions, inversions or translocations) on the biology of viruses, their replication cycle, the role of viral proteins in pathogenicity or in the interactions between viruses and the immune response components [3,5,6].…”

Section: Introductionmentioning

confidence: 99%

“…Except for retroviruses, replicons derived from positive-sense RNA viruses do not integrate exogenous genetic information into the host cell genome. Several systems have been described for heterologous gene expression based on infectious cDNA clones of picornavirus [7], flavivirus [8], alphavirus [9] or coronavirus [6,10,11].…”

Section: Introductionmentioning

confidence: 99%

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Highs and Lows in the Calicivirus Reverse Genetics

Álvarez,

Arboleya,

Abade dos Santos

et al. 2024

Preprint

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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 RNA 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 -such as restriction enzyme digestion, gene knock-out or knock-in, site directed mutagenesis, ligation, etc.- can be done. 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 cultures. Despite the successful rescue of Feline calicivirus (FCV) from cDNA containing plasmid more than 20 years ago, reverse genetics methods are not routine procedures than can be easily extrapolated to other caliciviruses. Reports of calicivirus reverse genetics systems have been few and far between, in this review, we discuss the main pitfalls, failures, and delays behind several successful calicivirus infectious clones.

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