A versatile reverse genetics platform for SARS-CoV-2 and other positive-strand RNA viruses

Nat Commun. 2021 Jun 8;12(1):3431. doi: 10.1038/s41467-021-23779-5.

Abstract

The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We demonstrate that despite the large size of the viral RNA genome (~30 kb), infectious full-length cDNA is readily assembled in vitro by a circular polymerase extension reaction (CPER) methodology without the need for technically demanding intermediate steps. Overlapping cDNA fragments are generated from viral RNA and assembled together with a linker fragment containing CMV promoter into a circular full-length viral cDNA in a single reaction. Transfection of the circular cDNA into mammalian cells results in the recovery of infectious SARS-CoV-2 virus that exhibits properties comparable to the parental virus in vitro and in vivo. CPER is also used to generate insect-specific Casuarina virus with ~20 kb genome and the human pathogens Ross River virus (Alphavirus) and Norovirus (Calicivirus), with the latter from a clinical sample. Additionally, reporter and mutant viruses are generated and employed to study virus replication and virus-receptor interactions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Chlorocebus aethiops
  • Culicidae / virology
  • Furin / metabolism
  • Genome, Viral
  • HEK293 Cells
  • Humans
  • Mice
  • Mutation / genetics
  • NIH 3T3 Cells
  • Polymerase Chain Reaction
  • RAW 264.7 Cells
  • Receptors, Virus / metabolism
  • Reverse Genetics*
  • SARS-CoV-2 / genetics*
  • Vero Cells
  • Viral Proteins / chemistry
  • Virus Replication

Substances

  • Receptors, Virus
  • Viral Proteins
  • Furin