Downloaded from www.microbiologyresearch.org by IP: 54.157.140.51 On: Sun, 28 Feb 2016 09:39:38 Short Communication The P gene of Newcastle disease virus does not encode an accessory X protein Ben Peeters, Paul Verbruggen, Frank Nelissen and Olav de Leeuw Correspondence Ben Peeters ben.peeters@wur.nl Division of Infectious Diseases, Animal Sciences Group, Wageningen University and Research Centre, PO Box 65, NL-8200 AB Lelystad, The Netherlands Received 2 April 2004 Accepted 28 April 2004 Many paramyxoviruses encode non-essential accessory proteins that are involved in the regulation of virus replication and inhibition of cellular antiviral responses. It has been suggested that the P gene mRNA of Newcastle disease virus (NDV) encodes an accessory protein – the so-called X protein – by translation initiation at a conserved in-frame AUG codon at position 120. Using a monoclonal antibody that specifically detected the P and X proteins, it was shown that an accessory X protein was not expressed in NDV-infected cells. Recombinant NDV strains in which the AUG was changed into a GCC (Ala) or GUC (Val) codon were viable but showed a reduction in virulence, probably because the amino acid change affected the function of the P and/or V protein. The family Paramyxoviridae includes important pathogens that can cause severe disease in humans as well as animals. Well-known examples of human pathogens are measles virus, mumps virus, Hendra virus and Nipah virus. Animal pathogens include rinderpest virus, bovine respiratory syncytial virus and Newcastle disease virus (NDV). The subfamily Paramyxovirinae consists of five genera, Respiro- virus, Morbillivirus, Rubulavirus, Henipavirus and Avulavirus (Mayo, 2002a, b). Paramyxoviruses have a non-segmented negative-sense single-stranded RNA genome that encodes six to ten genes (Lamb & Kolakofsky, 2001). Characteristic of most, if not all, paramyxoviruses is the ability to gener- ate multiple proteins from the P gene. These so-called accessory proteins are generated by an RNA-editing event and in some instances by the use of alternative open read- ing frames (ORFs) present within the P gene. For respiro- viruses, morbilliviruses and avulaviruses, the P protein is encoded by an unedited transcript of the P gene, whereas the V and W proteins are the result of an mRNA-editing event in which one (V) or two (W) G residues are inserted at a specific position within the P gene mRNA. For rubulaviruses, the unedited mRNA generates the V pro- tein while the P and W proteins are the result of mRNA- editing. Additional proteins derived from the P gene mRNA may be generated by translation initiation at different start points in the +1 reading frame (C proteins) or in the same reading frame (X protein) (Curran et al., 1998). Morbilliviruses express at least one C protein, whereas some respiroviruses express two or more. Apart from the P protein, the Sendai virus P gene seems to encode a total of at least seven accessory proteins, i.e. V and W by RNA-editing, C, C9, Y1 and Y2 from the +1 reading frame, and X from an in-frame reading frame (Curran et al., 1998). Expression of an accessory C protein, or X protein, has not yet been reported for rubulaviruses or avulaviruses. However, McGinnes et al. (1988) reported the existence of 38 and 29 kDa non-structural proteins derived from the P gene ORF of NDV (genus Avulavirus) and suggested that these proteins could have been generated by in-frame translation initiation at amino acid positions 82 and 120, respectively. Analysis of the P gene sequences of 23 dif- ferent NDV strains showed that the AUG codon at position 82 was not conserved, whereas the one at position 120 was completely conserved in all strains (Locke et al., 2000). These results led Locke and co-workers to suggest that – in addition to the P and V/W proteins – an additional protein, termed the X protein, could potentially be expressed by the P gene of NDV (Fig. 1). To determine whether NDV encodes an accessory X pro- tein, we tried to detect the X protein in NDV-infected cells using two different monoclonal antibodies (mAbs) against the P protein. To determine the specificity of these mAbs, we first expressed the individual P, V, W and X proteins in eukaryotic cells by means of an expression vector. The different ORFs were amplified by PCR using the Z-Taq system (Takara) with full-length NDV cDNA as a template (Peeters et al., 1999; GenBank accession no. AF077761). The P ORF was amplified using primers pRT1 (59-CAA- AGAATTCAGAAAAAAGTACGGGTAGAAG-39) and p2 (59-GCAGTCTAGATTAGCCATTCACTGCAAGGCGC-39). The X ORF was amplified using primers XpF (59-GACG- AATTCCGTCGACACACAGTTCAGG-39) and p2. The X ORF provided with an optimized translation initiation site (Kozak, 1987) was amplified using primers XpFkz (59- AACGAATTCGCCGCC ATGCTTGACAAGCTAGCAATA- AATCG-39) and p2. The V ORF was amplified by means of fusion PCR using primer pRT1 as forward primer and 0008-0160 G 2004 SGM Printed in Great Britain 2375 Journal of General Virology (2004), 85, 2375–2378 DOI 10.1099/vir.0.80160-0