The interaction of the SARS coronavirus non-structural protein 10 with the cellular oxido-reductase system causes an extensive cytopathic effect

Abstract

The pathological mechanism of SARS-CoV infection was investigated. The gene for the SARS-CoV non-structural protein 10, which is located in the open reading frame of pp1a/pp1ab gene, was synthesized and used to screen for the specific cellular gene coding for the protein interacting with this nsp10 protein in a human embryo lung cDNA library using a yeast trap method. The results indicated that apart from the two subunits of cellular RNA polymerase complex, BTF3 and ATF5, this nsp10 protein was also able to interact specifically with the NADH 4L subunit and cytochrome oxidase II. Further study revealed that the activity of the NADH-cytochrome was altered and the inner mitochondrial membrane was depolarized in the transfected human embryo lung fibroblast by the nsp10 protein gene. The cytopathic effect of the Coronavirus 229E strain appeared more extensive in these cells than in the control cells.

Fig. 1

Specific binding activities of the proteins identified using a yeast two-hybrid system with the SARS-CoV non-structural protein 10 in mating assays. The identified gene clone in the yeast trap was transfected into Y187 strain together with pGBK-7-nsp10. This transfectant was grown in the selection media of SD/-Leu/-Trp/-His/+25 mM 3-AT followed by growth in a 1.5 ml SD medium. The supernatants were collected after being centrifuged at 10,000 rpm in an Eppendorf tube. The OD₆₀₀ of the culture was recorded. One hundred microliters of the supernatant was reacted with O-nitropheng/β-d-galacto-pyranoside (ONPG) in Z-buffer with β-mercaptoethanol. The final OD₄₂₀ nm value was recorded after at least 30 min. The negative sample is supernatant of yeast Y187 transfected with pGBKT7 and pGADT7. The positive control is from the yeast Y187 transfected with pGBKT7-53 and pGADT7-T as provided by the manufacturer. The β-galactosidase units were calculated using the formula: units = 1000 × OD₄₂₀/(t × v × OD₆₀₀); T, elapsed time of incubation; v, 0.1 ml × concentration factor.

Fig. 2

The interaction of SARS-CoV nsp10 protein and cytochrome oxidase complex. (a) Pull-down test of SARS-CoV nsp10 and the extract of KMB-17 cells labeled with ³⁵S-methioine. Approximately 4 ug expressed and purified GST-nsp10 fusion protein and GST protein were respectively mixed with the 500 ul labeled extract of KMB-17 cells (5 × 10⁶ cells), which were lysed in lysis buffer at 4 °C overnight. The GST and GST-nsp10 complex were conjugated with glutathione-Sepharose 4B beads in a total volume of 500 μl of buffer in 25 °C for 4 h. After centrifugation, the beads were washed three times with cold PBS and boiled at 100 °C with 20 ul sample buffer for 2 min. After centrifugation, the supernatants were loaded in a 12% SDS-PAGE gel for electrophoresis. Lane a-1, GST protein interacting with the extract of KMB-17 cells; Lane a-2, GST-nsp10 fusion protein interacting with the extract of KMB-17 cells. (b) Western blot of antibody against cytochrome oxidase complex to detect the proteins collected from pull-down test. The protein samples collected from pull-down test were separated in SDS-PAGE gel and transferred to NC membrane. This membrane was interacted with the polyclonal antibody against cytochrome oxidase complex and visualized. Lane b-1, proteins from the sample of GST-nesp10 fusion protein interacting with the extract of KMB-17 cells; Lane b-2, proteins from the sample of GST protein interacting with the extract of KMB-17 cells. (c) The distribution of nsp10-GFP fusion protein in KMB-17 cells. The nsp10 gene was inserted into pGFP plasmid and expressed as a fusion protein of nsp10-GFP after the pGFP-nsp10 was transfected into KMB-17 cells. The distribution of nsp10-GFP fusion protein in cells was observed under a fluorescence microscope with the amplification of 400×. Lane c-1, observation of nsp10-GFP fusion protein in KMB-17 cells; Lane c-2, observation of mitochondria stained with Rhodamine-123 fluorescence in KMB-17 cells.

Fig. 3

The functional impact of mitochondria by the nsp10 protein expressed in vivo. (a) Activity of NADH-cytochrome c oxidase in the cells transfected by the pcDNA-nsp10. The human embryo fibroblasts transfected by the pcDNA-nsp10 for 18 h were grown in DMEM-5% FBS for 12, 24 and 36 h and collected by scraping followed by rinsing with 0.9% NaCl buffer. The cells were treated with sonication and centrifugation. The supernatant was used for detecting the activity of cytochrome c oxidase with its substrate. The results of O₂⁻ production were read at OD₅₅₀. The cellular protein in supernatant was quantified using the Lowry method. (b) Distribution of the cells transfected by the pcDNA-nsp10 according to their rhodamine-123 fluorescence intensities. The human embryo lung fibroblast transfected by the pcDNA-nsp10 or pcDNA-Vp3 plasmid and the control cells, were stained using rhodamine-123 at 24 and 48 h after transfection, and analyzed using flow cytometry. Lane b-1, rhodamine-123 fluorescence profiles of the cells transfected by the pcDNA-nsp10 or pcDNA-Vp3 at 24 h after transfection and the control cells treated using only Lipotamine™ ²⁰⁰⁰ under the same conditions; Lane b-2, Rhodamine-123 fluorescence profiles of the cells transfected by the pcDNA-nsp10 or pcDNA-Vp3 at 48 h after transfection and the control cells treated using only Lipotamine™ ²⁰⁰⁰ under the same conditions (1, transfected cells with pcDNA-nsp10; 2, transfected cells with pcDNA-Vp3).

Fig. 4

Cytopathic effect induced by Coronavirus 229E infection in cells transfected with pcDNA-nsp10. (a) Cytopathic effect induced by Coronavirus 229E in cells transfected by the nsp10 gene as observed under a light microscope (200×). Lane a-1, cells transfected with the pcDNA-nsp10 and then infected with Coronavirus 229E, moi 0.5, 24 h post-infection; Lane a-2, control cells transfected with the pcDNA-Vp3 and then infected with Coronavirus 229E, moi 0.5, 24 h post-infection. (b) The pathological structural change in the cytopathic effects induced by Coronavirus 229E in cells transfected with the nsp10 gene as observed under an electron microscope. Lane b-1, cells transfected with the pcDNA-nsp10 and then infected by Coronavirus 229E, moi 0.5 24 h post-infection (the arrows indicate vacuolar degeneration, karyolysis and lysis of plasma membrane); Lane b-2, control cells transfected with the pcDNA-Vp3 and then infected by Coronavirus 229E, moi 0.5, 24 h post-infection (the arrows indicate the complete membrane and the nucleus).

Fig. 5

Analysis of the viral replication in the cells transfected by the pcDNA-nsp10. Titration of the harvested Coronavirus 229E grown in the cells transfected by the pcDNA-nsp10 or pcDNA-Vp3.