Engineering a Replication-Competent, Propagation-Defective Middle East Respiratory Syndrome Coronavirus as a Vaccine Candidate

Almazán, Fernando; DeDiego, Marta L.; Sola, Isabel; Zuñiga, Sonia; Nieto-Torres, Jose L.; Marquez-Jurado, Silvia; Andrés, German; Enjuanes, Luis

Previous reports from our laboratory showed that deletion of the transmissible gastroenteritis coronavirus (TGEV) E gene leads to a propagation-defective virus that can only spread from cell to cell by expression of the E protein in trans (24, 32) . To analyze whether rMERS-CoV-⌬E could also be complemented in cells transiently expressing E protein, the rescue of rMERS-CoV-⌬E and of rMERS-CoV as a control was analyzed in Huh-7 cells that did not express E protein (E Ϫ ) and in cells transiently expressing the E protein (E ϩ ). The transfection efficiencies in E ϩ cells varied between 40 and 50% in each independent experiment. Infectious rMERS-CoV was rescued from both E ϩ and E Ϫ cells with virus titers of around 4 ϫ 10 5 TCID 50 /ml and 1 ϫ 10 6 TCID 50 /ml, respectively (Fig. 5A ). In contrast, rMERS-CoV-⌬E was rescued in E ϩ cells with titers of around 1 ϫ 10 3 TCID 50 /ml but not in control E Ϫ cells, in which the virus was not detectable from passage 1 (limit of detection, 50 TCID 50 /ml) (Fig. 5A) . These data indicated that the E protein was necessary for either viral RNA synthesis or virus propagation. To evaluate the role of the E protein in viral RNA synthesis, the level of genomic RNA (gRNA) was evaluated by quantitative reverse transcription-PCR (RT-qPCR) at each passage. Viral gRNA was detected for rMERS-CoV in both E Ϫ -and E ϩ -expressing cells, as expected. However, MERS-CoV-⌬E viral RNA was detected at high levels in E ϩ cells at passages 0, 1, 2, and 3, whereas it was only detected at similar levels in E Ϫ cells at passage 0, suggesting that MERS-CoV-⌬E was a replication-competent virus (Fig. 5B) . To further confirm these data, viral RNA synthesis was analyzed in a single-cycle infection. E Ϫ cells were infected with either rMERS-CoV or rMERS-CoV-⌬E grown in E ϩ cells. At 5 h.p.i., the levels of gRNA and subgenomic mRNA 8 (sgmRNA N) were evaluated by RT-qPCR (Fig. 6 ). Similar levels of gRNA and sgmRNA N were detected in cells infected with both viruses, indicating that E protein was not required for efficient viral replication and transcription. Overall, these data indicated that rMERS-CoV-⌬E was a replicationcompetent, propagation-defective virus.