Human coronavirus 229E (HCoV-229E) infection, persistence and spike glycoprotein.

摘要

Human coronavirus 229E (HCoV-229E) is a large enveloped, plus strand RNA virus that causes 15 to 30% of common colds. Epidemiological studies have also implicated HCoVs in reactive airway disease, lower respiratory tract infection, and possibly in enteric and heart disease. Recently, reports have demonstrated the finding of HCoV-229E RNA in human brains. HCoV-229E uses human aminopeptidase N as its receptor.; To explore the possible mechanisms of HCoV-229E persistence, persistent infections with HCoV-229E were established in five parallel cultures of human cells. Infection yielded persistently infected carrier cultures in which 10–30% of the cells expressed the virus S protein. Mutant viruses with small plaque phenotypes replaced large plaque wild type virus in all persistently infected cultures. The mutant viruses were shown to have mutations in the gene encoding the spike (S) protein.; Persistent infection with HCoV-229E involved changes in the virus and also changes in the host cells. Cells persistently infected with HCoV-229E were found to express markedly reduced levels of the virus receptor hAPN on the cell surface compared to the level of expression of hAPN in wild type L-132 cells.; The virus spike glycoprotein is believed to bind the receptor. Viruses with mutations in the spike glycoprotein that were selected in persistently infected cultures could alter the specificity of receptor binding. To identify the region on the HCoV-229E S glycoprotein that binds to hAPN, three soluble six-histidine-tagged carboxy-terminal truncated proteins were expressed in a baculovirus expression vector. Proteins partially purified by nickel affinity chromatography were shown to be glycosylated by PNGaseF treatment and reacted with polyclonal and monoclonal anti-HCoV-229E antibodies. A protein containing the first 547 amino acids of S bound to live mouse 3T3 cells expressing hAPN but not to mouse 3T3 cells with empty vector. Binding of S547 to hAPN-3T3 cells was blocked by an anti-hAPN MAb and by anti-HCoV-229E polyclonal antibody. Truncated S proteins containing the first 417 and 268 amino acids did not bind to hAPN-3T3 cells. Treatment of S547 with polyclonal anti-S antibody pre-adsorbed with S417 or S268 blocked binding to hAPN-3T3 cells but treatment with pre-adsorbed serum with S547 did not block binding. Thus, the region of S between amino acids 417 and 547 is critical for binding to the hAPN receptor.; Characterization of the molecular interactions between the HCoV-229E spike glycoprotein and its hAPN receptor will elucidate the mechanism by which HCoV-229E enters cells, the species specificity of HCoV-229E infection and may lead to the discovery of a drug to block HCoV-229E infection.