Abstract
The SARS coronavirus (SARS-CoV) open reading frame 7a (ORF 7a) encodes a 122 amino acid accessory protein. It has no significant sequence homology with any other known proteins. The 7a protein is present in the virus particle and has been shown to interact with several host proteins; thereby implicating it as being involved in several pathogenic processes including apoptosis, inhibition of cellular protein synthesis, and activation of p38 mitogen activated protein kinase. In this study we present data demonstrating that the SARS-CoV 7a protein interacts with human Ap4A-hydrolase (asymmetrical diadenosine tetraphosphate hydrolase, EC 3.6.1.17). Ap4A-hydrolase is responsible for metabolizing the "allarmone" nucleotide Ap4A and therefore likely involved in regulation of cell proliferation, DNA replication, RNA processing, apoptosis and DNA repair. The interaction between 7a and Ap4A-hydrolase was identified using yeast two-hybrid screening. The interaction was confirmed by co-immunoprecipitation from cultured human cells transiently expressing V5-His tagged 7a and HA tagged Ap4A-hydrolase. Human tissue culture cells transiently expressing 7a and Ap4A-hydrolase tagged with EGFP and Ds-Red2 respectively show these proteins co-localize in the cytoplasm.
Highlights
Severe acute respiratory syndrome coronavirus (SARSCoV) has been shown to be the etiological agent for the global SARS outbreak in the winter 2002/2003 that affected about 30 countries [1].SARS-CoV is an enveloped, positive-sense RNA virus with ~30 kb genome
Identification of human cellular proteins interacting with SARS-CoV protein 7a In order to identify cellular proteins that interact with the 7a protein, we performed a yeast two-hybrid screening of the Hybrid HunterTM Premade cDNA library constructed in pYESTrp2 (Invitrogen)
We have shown an interaction between the 7a protein and Ap4A-hydrolase, an enzyme involved in a number of biological processes [18,19,20,21]
Summary
SARS-CoV is an enveloped, positive-sense RNA virus with ~30 kb genome. It contains 14 potential ORFs. It contains 14 potential ORFs Some of these ORFs encode proteins that are homologues to the structural proteins founded in other coronaviruses, namely the replicase (ORF 1a and 1b), membrane, nucleocapsid, envelope and spike proteins [2,3]. Other ORFs encode group-specific or accessory proteins which are unique to SARS-CoV. Accessory proteins are not necessary for viral replication in cell culture systems and in mice, but may be important for virus-host interactions and may contribute to viral strength and/or pathogenesis in vivo [4,5,6]
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