Downloaded from www.microbiologyresearch.org by IP: 54.196.252.114 On: Mon, 24 Oct 2016 10:33:32 The rat cytomegalovirus homologue of parvoviral rep genes, r127, encodes a nuclear protein with single- and double-stranded DNA-binding activity that is dispensable for virus replication Koen W. R. van Cleef, Wendy M. A. Scaf, Karen Maes, Suzanne J. F. Kaptein, Erik Beuken, Patrick S. Beisser, Frank R. M. Stassen, Gert E. L. M. Grauls, Cathrien A. Bruggeman and Cornelis Vink Correspondence Cornelis Vink kvi@lmib.azm.nl Department of Medical Microbiology, Cardiovascular Research Institute Maastricht (CARIM), University of Maastricht, PO Box 5800, 6202 AZ Maastricht, The Netherlands Received 1 December 2003 Accepted 25 February 2004 An intriguing feature of the rat cytomegalovirus (RCMV) genome is open reading frame (ORF) r127, which shows similarity to the rep genes of parvoviruses as well as the U94 genes of human herpesvirus type 6A (HHV-6A) and 6B (HHV-6B). Counterparts of these genes have not been found in other herpesviruses. Here, it is shown that the r127 gene is transcribed during the early and late phases of virus replication in vitro as an unspliced 1?1 kb transcript containing the complete r127 ORF. Transcripts of r127 were also detected in various organs of RCMV-infected rats at 1 week post-infection (p.i.), but only in the salivary gland at 4 months p.i. Using rabbit polyclonal antibodies raised against the r127-encoded protein (pr127), pr127 was found to be expressed as early as 12 h p.i. within the nuclei of RCMV-infected cells in vitro. Expression of pr127 was also observed within the nuclei of cells in various organs of RCMV-infected rats at 3 weeks p.i. Moreover, pr127 was demonstrated to bind single- as well as double-stranded DNA. Finally, an RCMV r127 deletion mutant (RCMVDr127) was generated, in which the r127 ORF was disrupted. This deletion mutant, however, was shown to replicate with a similar efficiency as wild-type RCMV (wt RCMV), both in vitro and in vivo. Taken together, it is concluded that the RCMV r127 gene encodes a nuclear protein with single- and double-stranded DNA-binding activity that is dispensable for virus replication, not only in vitro, but also during the acute phase of infection in vivo. INTRODUCTION Cytomegaloviruses (CMVs) are widespread species-specific betaherpesviruses that cause acute, persisting and latent infections in both humans and animals. Infections with human CMV (HCMV) are mainly asymptomatic in immunocompetent individuals, but can be life-threatening in immunocompromised individuals, such as AIDS patients and organ transplant recipients. In our laboratory, we are studying the interaction between rat CMV (RCMV) and its host as a model for HCMV infection and disease (Bruggeman et al., 1982). RCMV contains a linear, double-stranded DNA genome of 230?1 kb. The complete DNA sequence of the RCMV genome has been determined recently and was found to contain at least 167 open reading frames (ORFs) (Vink et al., 2000). Most of these ORFs have counterparts in the genomes of both HCMV and murine CMV (MCMV) (Chee et al., 1990; Rawlinson et al., 1996; Vink et al., 2000). However, an exception is RCMV ORF r127, which is unique among the CMVs. This ORF has the capacity to encode a 337 amino acid protein (pr127) which shows similarity to the non- structural proteins (NS or Rep proteins) that are encoded by the rep genes of parvoviruses (Vink et al., 2000). The predicted amino acid sequence of the r127-encoded protein is most closely related to the sequences of the Rep1/2 proteins of three avian parvoviruses, namely barbary duck parvovirus (BDPV), muscovy duck parvovirus (MDPV) and goose parvovirus (GPV) (Vink et al., 2000; Zadori et al., 1995). Although the RCMV rep gene homologue is unique among the CMVs, it is not unique among the betaherpes- viruses: the U94 genes of human herpesvirus type 6A (HHV- 6A) and 6B (HHV-6B) also show similarity to the parvoviral rep genes (Dominguez et al., 1999; Gompels et al., 1995; Isegawa et al., 1999; Thomson et al., 1991). The U94 ORF was first discovered in HHV-6A and was found to encode a The nucleotide and amino acid sequences discussed in this paper have been deposited in the GenBank database under accession number AF232689. 0007-9864 G 2004 SGM Printed in Great Britain 2001 Journal of General Virology (2004), 85, 2001–2013 DOI 10.1099/vir.0.79864-0