Amino acid substitution L232F in non-structural protein 6 identified as a possible human-adaptive mutation in clade B MERS coronaviruses

Middle East respiratory syndrome coronavirus (MERS-CoV) causes zoonotic disease. Dromedary camels are the source of zoonotic infection. We performed sequence analysis of clade B MERS-CoV after excluding potential bias arising multiple sequences from a single zoonotic transmission chain and identified a substitution of amino acid leucine to phenylalanine in the codon 232 position of the non-structural protein 6 (nsp6) (nsp6 L232F) that occurs preferentially in human clade B MERS-CoV, with a rate of 16.9% (20/118) in human sequences vs a 0.6% (1/160) in camel sequences. Using a human clade B MERS-CoV strain GD01 as backbone, we generated a pair of isogenic recombinant MERS-CoV with nsp6 232L and 232F residues, respectively, and showed that the nsp6 L232F substitution confers higher replication competence in ex vivo culture of human nasal and bronchial tissues and in lungs of mice experimentally infected in vivo. Mechanistically, the nsp6 L232F substitution was found to associate with higher exocytic virus egress, while innate immune responses, autophagic restriction, and zippering activity of the endoplasmic reticulum remained unaffected. Our study suggests an adaptive mutation that occurs in clade B MERS-CoV associated with inter-species transmission to humans that may facilitate higher viral replication in the human respiratory tract. This highlights the importance of MERS-CoV as a zoonotic threat and the need for continued virus surveillance in camels and humans.IMPORTANCEViral host adaptation plays an important role in inter-species transmission of coronaviruses and influenza viruses. Multiple human-adaptive mutations have been identified in influenza viruses but not so far in MERS-CoV that circulates widely in dromedary camels in the Arabian Peninsula leading to zoonotic transmission. Here, we analyzed clade B MERS-CoV sequences and identified an amino acid substitution L232F in nsp6 that repeatedly occurs in human MERS-CoV. Using a loss-of-function reverse genetics approach, we found the nsp6 L232F conferred increased viral replication competence in vitro, in cultures of the upper human respiratory tract ex vivo, and in lungs of mice infected in vivo. Our results showed that nsp6 L232F may be an adaptive mutation associated with zoonotic transmission of MERS-CoV. This study highlighted the capacity of MERS-CoV to adapt to transmission to humans and also the need for continued surveillance of MERS-CoV in camels. Viral host adaptation plays an important role in inter-species transmission of coronaviruses and influenza viruses. Multiple human-adaptive mutations have been identified in influenza viruses but not so far in MERS-CoV that circulates widely in dromedary camels in the Arabian Peninsula leading to zoonotic transmission. Here, we analyzed clade B MERS-CoV sequences and identified an amino acid substitution L232F in nsp6 that repeatedly occurs in human MERS-CoV. Using a loss-of-function reverse genetics approach, we found the nsp6 L232F conferred increased viral replication competence in vitro, in cultures of the upper human respiratory tract ex vivo, and in lungs of mice infected in vivo. Our results showed that nsp6 L232F may be an adaptive mutation associated with zoonotic transmission of MERS-CoV. This study highlighted the capacity of MERS-CoV to adapt to transmission to humans and also the need for continued surveillance of MERS-CoV in camels.