Mechanism of Nucleic Acid Unwinding by SARS-CoV Helicase

Adedeji, Adeyemi O.; Marchand, Bruno; te Velthuis, Aartjan J. W.; Snijder, Eric J.; Weiss, Susan; Eoff, Robert L.; Singh, Kamalendra; Sarafianos, Stefan G.

Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is the causative agent of SARS, a life-threatening illness characterized by flu-like syndrome such as high fever, pneumonia, dyspnoea, infiltrates on chest radiography and sometimes respiratory failure [1] . The global spread of SARS caused hundreds of deaths and had enormous negative impact to the global economy in [2003] [2004] . SARS-CoV is a positive single-stranded (ss) RNA virus with one of the largest known RNA genomes (,29.7 kb) [2, 3] . SARS-CoV gene expression involves poorly understood transcriptional and translational regulatory mechanisms. Following infection, there is translation of two large replicative polyproteins, the pp1a (the ORF1a polyprotein) and the pp1ab (the polyprotein made from the ORF1a and ORF1b through a 21 ribosomal frameshift during translation). These polyproteins are processed by the virus-encoded papain-like proteinase (PL pro ; residing in nsp3) and nsp5, a 3C-like proteinase [4, 5, 6, 7, 8, 9, 10] . This auto-proteolysis leads to formation of 16 non-structural proteins, including an RNA-dependent RNA polymerase (RdRp) and an NTPase/helicase that are known as non-structural proteins 12 and 13, respectively (nsp12 and nsp13). These are likely to form the core of membrane-bound replication-transcription complexes in double-membrane vesicles at perinuclear regions [11] . Helicases are motor proteins that unwind double-stranded nucleic acids into two single-stranded nucleic acids by utilizing the energy derived from nucleotide hydrolysis [12, 13, 14, 15, 16, 17, 18, 19] . Although, helicases were initially thought as molecular engines that unwind nucleic acids during replication, recombination, and DNA repair [16] , recent reports have shown that they are also involved in other biological processes, including displacement of proteins from nucleic acid, movement of Holliday junctions, chromatin remodeling, catalysis of nucleic acid conformational changes [20, 21, 22, 23, 24, 25] , several aspects of RNA metabolism, including transcription, mRNA splicing, mRNA export, translation, RNA stability and mitochondrial gene expression [11] . Defects in helicase function have been associated with some human diseases, including Bloom's syndrome, Werner's syndrome, and Xeroderma Pigmentosum [26, 27, 28, 29] . The specific function of nsp13 in SARS-CoV replication has yet to be established. It is thought to interact with other SARS-CoV non-structural proteins, such as nsp7, nsp8 and nsp12 [30, 31, 32] and probably contribute to the formation of a replication complex, but the effect of these interactions on nsp13 unwinding activity is unknown.