Study Of Relationship Among ACE2 Variants, SARS-CoV Entry, And Severity Of Pulmonary Lesions

Severe acute respiratory syndrome (SARS) is an acute infectious disease caused by a novel coronavirus (CoV) variant, SARS-CoV. SARS-CoV is highly transmissible in humans, with a fatality rate near 10%. After people developed SARS, diffuse alveolar damage (DAD) in patients' lung which leads to the failure of lung function is one of important reasons for high fatality and the precise mechanism is still unclear. Soon after SARS-CoV was isolated, angiotensin-converting enzyme-two (ACE2) was identified as the primary receptor for SARS-CoV. The SARS-CoV S-bearing pseudovirus entry assay showed the entry efficiency of mouse and rat ACE2 gene was lower than that of hu-ACE2 gene. These data suggest that ACE2 gene variants among different species are associated with pesudoviral entry. It also demonstrate the ACE2 gene variants of mouse and rat may be one of important reasons for SARS-CoV susceptibility. ACE2 expression of lung tissue was downregulated when mice was experimentally infected with SARS-CoV. S protein of SARS-CoV also can induce ACE2 down-regulation and enhance acute lung injury. Inhibitor of ACE2 substrate can apparently decrease the degree of acute lung injury. These data show that ACE2 play a important role in SARS pathogenesis. However, these studies mainly focus on mice and are not enough to extrapolate human. Our recent studies indicated that Chinese rhesus macaque, challenged with SARS-CoV, can develop mild or severe interstitial pneumonia. To determine the role of ACE2 in SARS pathogenesis, tissue specimens of mild and severe SARS monkey were used for this study. Based on rh-ACE2 sequence analysis of mild and severe SARS monkey, we wished to find some important amino acid sites that were associated with pseudoviral entry. Furthermore, we introduced the same mutation to hu-ACE2 and observed the efficiency of pseudovirus entry. We hoped to find some key sites of hu-ACE2 gene that could affect pseudoviral entry. Finally, the relationship between rh-ACE2 gene variants and SARS severity was involved.Based on pathologic features of lung tissue, the lung and kidney tissue samples were obtained from 6 mild and 6 severe SARS Chinese rhesus macaque in this study. Briefly, the mild lung damage includes alveolar septa broadening and mild mononuclear macrophages infiltration in alveolar septa. The severe lung damage is characterized by DAD, including extensive alveolar septa broadening, restricted fusion of thick septa by pressure, variably filled with fibrin and protein-rich edema fluid in alveolar cavities. Two uninfected monkeys were included as controls. The full-length rh-ACE2 genes were amplified by nested PCR and cloned to the PVX1 vector. Sequence analysis indicated that there are 55 sporadic non-synonymous (NS) variants in rh-ACE2 genes among 14 Chinese rhesus monkeys. 8-2 clone generated from no. 8 monkey was acted as consensus amino acid sequences, Based on the comparison of consensus amino acid sequences, we found that there are 38 differences between the hu-ACE2 gene and rh-ACE2 gene. After sequence analysis, HEK293T cells were transiently transfected by two representive plasmids of each monkey. The rh-ACE2 expression and pseudovirus entry was detected. The result showed rh-ACE2 expression and pseudovirus entry of 11-7 clone is significantly lower than that of 11-1 clone. Sequence analysis revealed difference of two mutations, including R192G and Y217N, was observed between 11-1 and 11-7 clone. After analysis of site-directed mutagenesis, we found the decrease of protein expression and pseudovirus entry were caused by Y217N mutation of rh-ACE2 gene. We also observed that Y217N mutation of hu-ACE2 could induce the decrease of protein expression and pseudovirus entry. To determine relationship between rh-ACE2 variants and the severity of SARS pathology, sequence alignment of all rh-ACE2 genes was carried out. Sequence analysis showed the A/G heterogeneity was found in one mild SARS monkey (1/6) on the 1677 position of rh-ACE2 but was found in three severe SARS monkeys (3/6). Fisher test showed that no significant difference was found between mild SARS monkey and severe SARS monkey on A/G heterogeneity rate.Our findings indicated that apparent rh-ACE2 gene variants were observed among our Chinese rhesus monkeys. Y217N mutation of rh-ACE2 and hu-ACE2 gene could cause the down-regulation of expression and reduce viral entry efficiency as well. No relationship was observed between rh-ACE2 gene variants and SARS severity. Our study also provide some clues for understating the role of ACE2 in SARS pathogenesis and drug design targeting ACE2 gene.