Non structural proteins 8 and 9 of human coronavirus 229E

The genome of human coronavirus 229E (HCoV-229E) consists of 27, 277 nucleotides. The viral replicase gene comprises two large, overlapping open reading frames (ORFs), ORF1a and ORF1b.1 ORF1a encodes the polyprotein pp1a with a calculated molecular mass of 454 kDa. The downstream ORF1b is expressed as a fusion protein with pp1a by a mechanism involving a ( 1) ribosomal frame shift during translation.1,2 The ORF1a/1b gene product has a calculated molecular mass of 754 kDa and is referred to as polyprotein 1ab (pp1ab). pp1a and pp1ab are processed by two virusencoded, papain-like proteases and the main protease Mpro, resulting in at least 16 non structural proteins (Nsps).3 Several or all of these nonstructural proteins build the replicase complex, probably mediating all the functions necessary for polyprotein processing, viral transcription, and replication.4 The 3´ region of the coronaviral ORF1a encodes a set of relatively small polypeptides (Nsp6 to Nsp11), of which only SARSCoV Nsp9 has had a function assigned, i.e., as ssDNA/RNA-binding protein.5,6 In mouse hepatitis CoV, several of these polypeptides colocalize with other components of the viral replication complex in the perinuclear region of the infected cell.7 Thus, the HCoV-229E polypeptides Nsp 6, 7, 8, and 10 are probably involved, directly or indirectly, in the viral replication complex. In this communication, we will describe the expression of genes coding for HCoV-229E Nsp8 and Nsp9, as well as the purification and biophysical characterization of the proteins. The two proteins are shown to bind tRNA using zoneinterference electrophoresis and fluorescence spectroscopy.