Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome

Abstract

The construction of cDNA clones encoding large-size RNA molecules of biological interest, like coronavirus genomes, which are among the largest mature RNA molecules known to biology, has been hampered by the instability of those cDNAs in bacteria. Herein, we show that the application of two strategies, cloning of the cDNAs into a bacterial artificial chromosome and nuclear expression of RNAs that are typically produced within the cytoplasm, is useful for the engineering of large RNA molecules. A cDNA encoding an infectious coronavirus RNA genome has been cloned as a bacterial artificial chromosome. The rescued coronavirus conserved all of the genetic markers introduced throughout the sequence and showed a standard mRNA pattern and the antigenic characteristics expected for the synthetic virus. The cDNA was transcribed within the nucleus, and the RNA translocated to the cytoplasm. Interestingly, the recovered virus had essentially the same sequence as the original one, and no splicing was observed. The cDNA was derived from an attenuated isolate that replicates exclusively in the respiratory tract of swine. During the engineering of the infectious cDNA, the spike gene of the virus was replaced by the spike gene of an enteric isolate. The synthetic virus replicated abundantly in the enteric tract and was fully virulent, demonstrating that the tropism and virulence of the recovered coronavirus can be modified. This demonstration opens up the possibility of employing this infectious cDNA as a vector for vaccine development in human, porcine, canine, and feline species susceptible to group 1 coronaviruses.

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

“…Rapid response and control of exigent emerging pathogens requires an approach to quickly generate full-length cDNAs from which molecularly cloned viruses are rescued, allowing for genetic manipulation of the genome. Full-length cDNAs were isolated for TGEV, HCoV-229E, IBV, and MHV-A59 by using a variety of approaches (17)(18)(19)(20)(21). Our strategy includes a panel of cDNAs spanning the entire CoV genome, which can be systematically and directionally assembled into a genome-length cDNA by in vitro ligation (17,18).…”

Reengineered salivary glands are stable endogenous bioreactors for systemic gene therapeutics

“…The pellet, containing highmolecular-weight genomic DNA, was washed several times with 75% ethanol and finally dissolved in TE buffer (10 mM Tris͞1 mM EDTA, pH 8.0). Low-molecular-weight DNA was either undigested or digested with SmaI, BssH II, or Plasmid Safe DNase (Epicentre Technologies, Madison, WI), an enzymecutting linear DNA forms (19), whereas high-molecular-weight DNA was digested with SmaI. DNA from each sample (30 g) was separated on 1% agarose gels, transferred to nylon membranes, and hybridized with an [␣- …”

“…DNA was obtained from submandibular glands of both AAVhEPO-treated (n ϭ 4; 10 9 particles per animal) and naïve animals (n ϭ 4; 0.9% NaCl) killed in week 8. Plasmid Safe DNase cuts only linear forms of DNA, circular forms remaining intact (19). Undigested low-molecular-weight DNA from the AAVhEPO-treated group showed two hybridization positive bands (Fig.…”

The Missing Link in Coronavirus Assembly

“…We do not know whether the change to a leucine residue at this crucial position for IBV E might suggest a different targeting mechanism. Interestingly, neither the Ϫ3 leucine nor the Ϫ4 lysine residues were found to be conserved in the counterpart E protein sequences of other coronaviruses, including MHV, human coronavirus, and TGEV (23,48,49). Because MHV E was also localized to the ER and IC, a different retention signal may exist in this protein.…”