期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 110, 期 40, 页码 16157-16162出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1311542110
关键词
emerging pathogen; zoonosis; synthetic genome
资金
- National Institutes of Health [U19AI100625, U19AI107810, AI108197, AI085524]
- Defense Threat Reduction Agency (DTRA) [HDTRA1-12-C-0035]
Severe acute respiratory syndrome with high mortality rates (similar to 50%) is associated with a novel group 2c betacoronavirus designated Middle East respiratory syndrome coronavirus (MERS-CoV). We synthesized a panel of contiguous cDNAs that spanned the entire genome. Following contig assembly into genome-length cDNA, transfected full-length transcripts recovered several recombinant viruses (rMERS-CoV) that contained the expected marker mutations inserted into the component clones. Because the wild-type MERS-CoV contains a tissue culture-adapted T1015N mutation in the S glycoprotein, rMERS-CoV replicated similar to 0.5 log less efficiently than wild-type virus. In addition, we ablated expression of the accessory protein ORF5 (rMERS center dot ORF5) and replaced it with tomato red fluorescent protein (rMERS-RFP) or deleted the entire ORF3, 4, and 5 accessory cluster (rMERS-Delta ORF3-5). Recombinant rMERS-CoV, rMERS-CoV center dot ORF5, and MERS-CoV-RFP replicated to high titers, whereas MERS-Delta ORF3-5 showed 1-1.5 logs reduced titer compared with rMERS-CoV. Northern blot analyses confirmed the associated molecular changes in the recombinant viruses, and sequence analysis demonstrated that RFP was expressed from the appropriate consensus sequence AACGAA. We further show dipeptidyl peptidase 4 expression, MERS-CoV replication, and RNA and protein synthesis in human airway epithelial cell cultures, primary lung fibroblasts, primary lung microvascular endothelial cells, and primary alveolar type II pneumocytes, demonstrating a much broader tissue tropism than severe acute respiratory syndrome coronavirus. The availability of a MERS-CoV molecular clone, as well as recombinant viruses expressing indicator proteins, will allow for high-throughput testing of therapeutic compounds and provide a genetic platform for studying gene function and the rational design of live virus vaccines.
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