Open reading frame 8a of the human severe acute respiratory syndrome coronavirus not only promotes viral replication but also induces apoptosis.
BACKGROUND
A unique genomic difference between human and civet severe acute respiratory syndrome coronaviruses (SARS-CoVs) is that the former has a deletion of 29 nucleotides from open reading frame (orf) 8a' that results in the generation of orf8a and orf8b. The objectives of the present study were to analyze antibody reactivity to ORF8a in patients with SARS and to elucidate the function of ORF8a.


METHODS
Western-blot and immunofluorescent antibody assays were used to detect anti-ORF8a antibody. SARS-CoV HKU39849 was used to infect stable clones expressing ORF8a and cells transfected with small interfering RNA (siRNA). The virus loads (VLs) and cytopathic effects (CPEs) were recorded. Confocal microscopy and several mitochondria-related tests were used to study the function of ORF8a.


RESULTS
Two (5.4%) of 37 patients with SARS had anti-ORF8a antibodies. The VLs in the stable clones expressing ORF8a were significantly higher than those in control subjects 5 days after infection. siRNA against orf8a significantly reduced VLs and interrupted the CPE. ORF8a was found to be localized in mitochondria, and overexpression resulted in increases in mitochondrial transmembrane potential, reactive oxygen species production, caspase 3 activity, and cellular apoptosis.


CONCLUSIONS
ORF8a not only enhances viral replication but also induces apoptosis through a mitochondria-dependent pathway.
Chen
Chia-Yen
CY
Institute of Public Health, National Yang-Ming University, Taipei, Taiwan.
Ping
Yueh-Hsin
YH
Lee
Hsin-Chen
HC
Chen
Kuan-Hsuan
KH
Lee
Yuan-Ming
YM
Chan
Yu-Juin
YJ
Lien
Te-Cheng
TC
Jap
Tjin-Shing
TS
Lin
Chi-Hung
CH
Kao
Lung-Sen
LS
Chen
Yi-Ming Arthur
YM
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D053148:Caspase 3 / Q000378:metabolism; D002460:Cell Line; D002522:Chlorocebus aethiops; D006801:Humans; D016366:Open Reading Frames; D010101:Oxygen Consumption / Q000502:physiology; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species / Q000378:metabolism; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics*; D045169:Severe Acute Respiratory Syndrome / Q000821:virology; D013997:Time Factors; D014779:Virus Replication / Q000502:physiology*
D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D053148:Caspase 3