siRNA silencing of angiotensin-converting enzyme 2 reduced severe acute respiratory syndrome-associated coronavirus replications in Vero E6 cells

Abstract

The outbreak of severe acute respiratory syndrome (SARS) in 2002-2003 has had a significant impact worldwide. No effective prophylaxis or treatment for SARS is available up to now. Angiotensin-converting enzyme 2 (ACE2) is the cellular receptor for SARS-associated coronavirus (SARS-CoV). By expressing a U6 promoter-driven small interfering RNA containing sequences homologous to part of ACE2 mRNA, we successfully silenced ACE2 expression in Vero E6 cells. By detecting negative strand SARS-CoV RNA and measuring RNA copy numbers of SARS-CoV by real-time reverse transcription polymerase chain reaction (RT-PCR), we demonstrated that SARS-CoV infection was reduced in the ACE2-silenced cell lines. These findings support the involvement of ACE2 in SARS-CoV infections and provide a basis for further studies on potential use of siRNA targeting ACE2 as a preventive or therapeutic strategy for SARS.

Fig. 1

ACE2 was silenced by siRNA in Vero E6. a Western blotting of ACE2 in various clones of Vero E6 stably transfected with pSilencer-ACE2, which expressed siRNA targeting ACE2 (lower panel). Relative levels of ACE2 expression determined by quantitative analysis of chemiluminescence and normalized by β-actin expressions are shown in the upper panel. Clones A4 and C4 showed the lowest levels of ACE2 expression. b Quantitative RT-PCR of ACE2 mRNA. The numbers over the bars denote relative copy numbers of ACE2 mRNA normalized by GAPDH copy numbers. Clones A4 and C4 showed less than 4% of ACE2 mRNA left in comparison to the original Vero E6 cells. c Expressions of ACE2 in Vero E6 and clone A4 were determined by flow cytometry. The thin dotted line represents unstained control. ACE2 expression was hardly detectable in the majority of A4 cells

Fig. 2

Inoculation of SARS-CoV resulted in typical cytopathic effects in Vero E6 but not A4 cells. Microscopic pictures of Vero E6 cells and its ACE2-silenced clone (A4) were taken before (a, b) and 16 h after (c, d, e) inoculation (h.p.i.) with SARS-CoV. Vero E6 cells inoculated with medium alone remained healthy and grew into a high density (c). At 16 h post-inoculation of SARS-CoV, almost all Vero E6 cells rounded up and died (d), while only a few foci of typical cytopathic effects (arrow in e) were shown in ACE2-silenced A4 cells (e)

Fig. 3

Negative strand of SARS-CoV RNA was detected in Vero E6 cells but not in ACE2-silenced A4 or C4 cells. RT-PCR using negative strand SARS-CoV RNA-specific primer for was done at 0 and 24 h after inoculation for Vero E6, A4, and C4 cells. A positive result for negative strand RNA of SARS-CoV was shown for Vero E6 cells at 24 h after inoculation. A4 and C4 cells whose ACE2 had been silenced showed no negative strand of SARS-CoV. GAPDH was amplified as an internal control

Fig. 4

Replication of SARS-CoV was blocked in ACE2-silenced A4 cells at low inoculation doses. Viral loads of SARS-CoV were determined by real-time RT-PCR in Vero E6 and A4 cells with different inoculation doses at different time points. a At the inoculation dose of 1 MOI, no difference in viral loads was noted between Vero E6 and A4 cells at various time points. The velocities of SARS-CoV replication were significantly lower in A4 cells than in Vero E6 with the inoculation doses of 10^–1 MOI (b) and 10^–2 MOI (c). When the inoculation dose was decreased to 10^–3 MOI, no increase in viral load was noted in A4 cells, while viral load increased to normal levels at 72 h in Vero E6 (d)