Reverse genetics with a full-length infectious cDNA of severe acute respiratory syndrome coronavirus

Rewiring the severe acute respiratory syndrome coronavirus (SARS-CoV) transcription circuit: Engineering a recombination-resistant genome

“…DNA fragments were isolated from 1.0% agarose gels with the Qiaquick gel extraction kit (Qiagen) and visualized by using Dark Reader technology (Clare Chemical Research, Denver, CO). The six subgenomic cDNA clones (A-F) span the SARS-CoV genome (9). ORF 7a͞b, located within SARS F (nt 27273-27772), was replaced with the Renilla luciferase gene as described with GFP in ref.…”

“…The SARS full-length cDNA was assembled, and full-length transcripts were synthesized and mixed with polyadenylated N gene transcripts and then electroporated into cells (9,40). Viruses were plaque-purified in Vero E6 cells, and stock was grown in 75-cm 2 flasks.…”

“…ORFs 2-8 are encoded in subgenomic mRNAs (mRNAs 2-9) synthesized as a nested set of 3Ј coterminal molecules. Leader RNA sequences, encoded at the 5Ј end of the genome, are joined to body sequences at distinct transcription regulatory sequences (TRSs) that contain the core sequence ACGAAC (7,9). SARS-CoV likely uses transcription attenuation to synthesize both full-and subgenomic-length negative strand RNAs containing antileader sequences, which then function as the templates for the synthesis of like-sized mRNAs (10,11).…”

Construction of a Severe Acute Respiratory Syndrome Coronavirus Infectious cDNA Clone and a Replicon To Study Coronavirus RNA Synthesis

“…Besides containing RNA-dependent RNA polymerase, RNA helicase, and proteases (4,12,15,23,37), which are all common to positive-strand RNA viruses, the CoV replicase was recently predicted to contain a variety of RNA-processing enzymes that are extremely rare or absent in other RNA viruses, including endoribonuclease (NendoU), 3Ј-to-5Ј exoribonuclease (ExoN), 2Ј-O-ribose methyltransferase (2Ј-O-MT), ADP ribose 1ЈЈ-phosphatase, and, in a subset of group 2 coronaviruses, cyclic phosphodiesterase (25, 36). These enzymatic activities might be involved in the replication of the largest known RNA virus genome and in the production of an extensive set of 5Ј-and 3Ј-coterminal subgenomic RNAs (11,14,25,36).The study of CoV molecular biology has been profoundly advanced by the recent construction of full-length cDNA clones (3,6,26,27,(32)(33)(34) and self-replicating RNAs, or replicons (2,28,30). Due to the large size of the CoV RNA genome and the instability of some CoV replicase gene sequences in bacteria, cDNA clones and replicons have been engineered using bacterial artificial chromosomes (BACs) (3), in vitro ligation of CoV cDNA fragments (32), and vaccinia virus as a vector for the propagation of CoV full-length cDNAs (27).…”

Humanized mice develop coronavirus respiratory disease

“…Genetic obstacles were obliterated by Thiel et al (11), who developed the first molecular clone for a HCoV, HCoV-229E, after inserting a full-length genome copy into a poxvirus vector to drive full-length infectious transcripts. Shortly thereafter, Yount et al (12) isolated a molecular clone of the SARSCoV by cloning the genome as six fragments that could be systematically assembled into a full-length cDNA in vitro and serve as the genomic template for the recovery of infectious transcripts (12). Using genetic approaches, the role of the accessory genes in coronavirus pathogenesis has been studied, foreign genes have been expressed, and coronavirus replicon particles have been developed as delivery vehicles for heterologous vaccines and therapeutics (13).…”