Extensive Positive Selection Drives the Evolution of Nonstructural Proteins in Lineage C Betacoronaviruses

Abstract

Middle East respiratory syndrome-related coronavirus (MERS-CoV) spreads to humans via zoonotic transmission from camels. MERS-CoV belongs to lineage C of betacoronaviruses (betaCoVs), which also includes viruses isolated from bats and hedgehogs.A large portion of the betaCoV genome consists of two open reading frames (ORF1a and ORF1b) that are translated into polyproteins. These are cleaved by viral proteases to generate 16 nonstructural proteins (nsp1 to nsp16) which compose the viral replication-transcription complex. We investigated the evolution of ORF1a and ORF1b in lineage C betaCoVs. Results indicated widespread positive selection, acting mostly on ORF1a. The proportion of positively selected sites in ORF1a was much higher than that previously reported for the surface-exposed spike protein. Selected sites were unevenly distributed, with nsp3 representing the preferential target. Several pairs of coevolving sites were also detected, possibly indicating epistatic interactions; most of these were located in nsp3. Adaptive evolution at nsp3 is ongoing in MERS-CoV strains, and two selected sites (G720 and R911) were detected in the protease domain. While position 720 is variable in camel-derived viruses, suggesting that the selective event does not represent a specific adaptation to humans, the R911C substitution was observed only in human-derived MERSCoV isolates, including the viral strain responsible for the recent South Korean outbreak. It will be extremely important to assess whether these changes affect host range or other viral phenotypes. More generally, data herein indicate that CoV nsp3 represents a major selection target and that nsp3 sequencing should be envisaged in monitoring programs and field surveys. IMPORTANCEBoth severe acute respiratory syndrome coronavirus (SARS-CoV) and MERS-CoV originated in bats and spread to humans via an intermediate host. This clearly highlights the potential for coronavirus host shifting and the relevance of understanding the molecular events underlying the adaptation to new host species. We investigated the evolution of ORF1a and ORF1b in lineage C betaCoVs and in 87 sequenced MERS-CoV isolates. Results indicated widespread positive selection, stronger in ORF1a than in ORF1b. Several selected sites were found to be located in functionally relevant protein regions, and some of them corresponded to functional mutations in other coronaviruses. The proportion of selected sites we identified in ORF1a is much higher than that for the surface-exposed spike protein. This observation suggests that adaptive evolution in ORF1a might contribute to host shifts or immune evasion. Data herein also indicate that genetic diversity at nonstructural proteins should be taken into account when antiviral compounds are developed.

Nsp3 of coronaviruses: Structures and functions of a large multi-domain protein

“…As the largest non-structural protein of CoVs, Nsp3 has also been identified as the major selective target for driving evolution in lineage C betaCoVs on the basis of a high rate of positively selected mutation sites (Forni et al, 2016). Furthermore, the adaptive evolution of Nsp3 of MERS-CoV is still ongoing (Forni et al, 2016).…”

“…As the largest non-structural protein of CoVs, Nsp3 has also been identified as the major selective target for driving evolution in lineage C betaCoVs on the basis of a high rate of positively selected mutation sites (Forni et al, 2016). Furthermore, the adaptive evolution of Nsp3 of MERS-CoV is still ongoing (Forni et al, 2016). For example, the Arg911Cys mutation (located in the palm subdomain of the PL2 pro , corresponding to Arg283 in Lei et al, 2014) of Nsp3 exists in the viral strain KOR/KNIH responsible for the 2015 South Korean outbreak but not in the ancestral strain EMC/2012 (Forni et al, 2016).…”

“…Furthermore, the adaptive evolution of Nsp3 of MERS-CoV is still ongoing (Forni et al, 2016). For example, the Arg911Cys mutation (located in the palm subdomain of the PL2 pro , corresponding to Arg283 in Lei et al, 2014) of Nsp3 exists in the viral strain KOR/KNIH responsible for the 2015 South Korean outbreak but not in the ancestral strain EMC/2012 (Forni et al, 2016). It is interesting to speculate why coronaviruses keep many essential functions in one protein, while this protein shows high-rate genetic diversity during CoV evolution.…”

Genetic characterization and phylogenetic analysis of porcine deltacoronavirus (PDCoV) in Shandong Province, China

“…In MHV, the Nsp3 protein ubiquitin-like domains could interfere with pathways involving ubiquitinylated or ISGylated host targets, thereby leading to the disruption of host anti-viral signal transduction or protein degradation (Chen and Makino, 2004). Nsp3 protein was also recommended as a marker for monitoring coronavirus evolution and for surveying the molecular epidemiology in lineage C betaCoVs (Forni et al, 2016). Additionally, the MERS-CoV Nsp3 Arg911Cys mutation is an example of adaptive evolution (Shokri et al, 2019).…”

MERS coronavirus outbreak: Implications for emerging viral infections

“…The MERS-CoV has a genome of 30,119 nucleotides comprising 7 predicted open reading frames (ORFs) (1a, 1b, 3, 4a, 4b, 5, 8b) and 4 structural genes encoding the spike (S), nucleocapsid (N), membrane (M) and envelope (E) proteins ( Fig. 2) (Forni et al, 2016;Mackay and Arden, 2015;Zhang et al, 2016a). The overlapping ORF1a and 1b are located at the 5′ end of the single stranded positive RNA alongside a 278 nucleotide un-translated region (UTR) (Fig.…”