The protein X4 of severe acute respiratory syndrome-associated coronavirus is expressed on both virus-infected cells and lung tissue of severe acute respiratory syndrome patients and inhibits growth of Balb/c 3T3 cell line.
BACKGROUND
The genome of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) includes sequences encoding the putative protein X4 (ORF8, ORF7a), consisting of 122 amino acids. The deduced sequence contains a probable cleaved signal peptide sequence and a C-terminal transmembrane helix, indicating that protein X4 is likely to be a type I membrane protein. This study was conducted to demonstrate whether the protein X4 was expressed and its essential function in the process of SARS-CoV infection.


METHODS
The prokaryotic and eukaryotic protein X4-expressing plasmids were constructed. Recombinant soluble protein X4 was purified from E. coli using ion exchange chromatography, and the preparation was injected into chicken for rising specific polyclonal antibodies. The expression of protein X4 in SARS-CoV-infected Vero E6 cells and lung tissues from patients with SARS was performed using immunofluorescence assay and immunohistochemistry technique. The preliminary function of protein X4 was evaluated by treatment with and over-expression of protein X4 in cell lines. Western blot was employed to evaluate the expression of protein X4 in SARS-CoV particles.


RESULTS
We expressed and purified soluble recombinant protein X4 from E.coli, and generated specific antibodies against protein X4. Western blot proved that the protein X4 was not assembled in the SARS-CoV particles. Indirect immunofluorescence assays revealed that the expression of protein X4 was detected at 8 hours after infection in SARS-CoV-infected Vero E6 cells. It was also detected in the lung tissues from patients with SARS. Treatment with and overexpression of protein X4 inhibited the growth of Balb/c 3T3 cells as determined by cell counting and MTT assays.


CONCLUSIONS
The results provide the evidence of protein X4 expression following SARS-CoV infection, and may facilitate further investigation of the immunopathological mechanism of SARS.
Chen
Ying-yu
YY
Center for Human Disease Genomics, Peking University, Beijing 100083, China.
Shuang
Bao
B
Tan
Ya-xia
YX
Meng
Min-jie
MJ
Han
Pu
P
Mo
Xiao-ning
XN
Song
Quan-sheng
QS
Qiu
Xiao-yan
XY
Luo
Xin
X
Gan
Qi-ni
QN
Zhang
Xin
X
Zheng
Ying
Y
Liu
Shun-ai
SA
Wang
Xiao-ning
XN
Zhong
Nan-shan
NS
Ma
Da-long
DL
D000595:Amino Acid Sequence; D000818:Animals; D041702:BALB 3T3 Cells; D002522:Chlorocebus aethiops; D006131:Growth Inhibitors / Q000032:analysis* / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D008168:Lung / Q000737:chemistry*; D051379:Mice; D008969:Molecular Sequence Data; D045473:Severe acute respiratory syndrome-related coronavirus / Q000737:chemistry*; D045169:Severe Acute Respiratory Syndrome / Q000378:metabolism*; D014709:Vero Cells; D015678:Viral Structural Proteins / Q000032:analysis* / Q000502:physiology
D006131:Growth Inhibitors; D015678:Viral Structural Proteins