Cloning and expression of human cyclophilin A and its interaction with human coronavirus NL63 nucleocapsid protein
Coronaviridae family is composed of a number of ribonucleic acid (RNA)-containing viruses currently classified into two genera, the coronavirus and torovirus. The family is classified together with the Arteviridae in the order Nidovirales. Coronaviruses are enveloped single stranded positive sense RNA viruses about 80-160 nm in diameter. The coronavirus is, as in the case of all positive sense RNA virus, a messenger, and the naked RNA is infectious. The 5′-two thirds of the genome encodes for a polyprotein that contains all the enzymes necessary for replication, whereas the 3′-one third encodes for all the structural proteins that mediate viral entry into the host cell. The structural proteins include spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins.Nucleocapsid protein is one of the most crucial structural components of coronaviruses;hence major attention has been focused on characterization of this protein. Some laboratories have demonstrated that this protein interferes with different cellular pathways, thus implying it to be a key regulatory component of the virus (Zakhartchouk, Viswanathan et al. 2005). Furthermore, it has been shown that severe acute respiratory syndrome (SARS)-N protein interacts with cellular proteins, including cyclophilin A (CypA), heterogenous nuclear ribonucleoprotein (hnRNP) A1, human ubiquitin-conjugating enzyme, cyclin dependent kinase (CDK)-cyclin complex protein, Ikappaßalpha (IkBα), cytochrome (Cyt) P450 etc. For the purpose of this study, the focus is based on CypA interaction with human coronavirus (HCoV) NL63-N protein. These interactions might play a role in the pathology of HCoV-NL63. Using glutathione-S-transferase (GST), the interaction of CypA with the nucleocapsid protein can be clearly demonstrated to be direct and specific. Since the N protein is involved in viral RNA packaging to form a helical core, it is suffice to say that both NL63-N and CypA are possibly within the HCoV-NL63 replication/transcription complex and NL63-N/human CypA interaction might function in the regulation of HCoV-NL63 RNA synthesis. In addition, the results will demonstrate that HCoV-NL63-N has only a specific domain for interacting with CypA.