Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Wed, 12 Dec 2018 12:05:13 Expression profile and subcellular localization of Torque teno sus virus proteins Laura Martı ´nez-Guino ´, 1 Maria Ballester, 1 Joaquim Segale ´s 1,2 and Tuija Kekarainen 1 Correspondence Tuija Kekarainen tuija.kekarainen@cresa.uab.cat Received 8 April 2011 Accepted 28 June 2011 1 Centre de Recerca en Sanitat Animal (CReSA), UAB-IRTA, Campus de la Universitat Auto ` noma de Barcelona, 08193 Bellaterra, Barcelona, Spain 2 Departament de Sanitat i Anatomia Animals, Universitat Auto ` noma de Barcelona, 08193 Bellaterra, Barcelona, Spain In the present study, the expression, generation and subcellular localization of Torque teno sus virus (TTSuV) proteins were characterized into two genetically distinct TTSuV species (TTSuV1 and TTSuV2). Following transfection of three TTSuV1 and TTSuV2 full-length ORF (ORF1, ORF2 and ORF3) expression constructs into porcine kidney cells, alternative splice variants encoding new TTSuV protein isoforms were identified for the first time. Proteins encoded from ORF1 and ORF3 were localized in the nucleoli of porcine kidney cells and that of ORF2 in the cytoplasm and nucleus excluding the nucleoli. The subcellular localization of the different protein isoforms was not only similar between distinct TTSuV species but also to the ones described in human Torque teno virus (TTV). Results of the present in vitro study were not based on full-length viral clones but suggested that alternative splicing strategy to generate TTSuV protein isoforms probably occurs in vivo. Obtained data provide new information on molecular biology of TTSuV and anelloviruses, which until now has been solely based on results obtained from human TTV. INTRODUCTION The family Anelloviridae includes Torque teno viruses (TTVs), which are vertebrate infecting, small, non- enveloped, circular, ssDNA viruses. TTV was first dis- covered in human and later detected in domestic and wild species including swine (Martı´nez et al., 2006; Nishizawa et al., 1997). Domestic pig and wild boar-infecting Torque teno sus virus 1 (TTSuV1) and Torque teno sus virus 2 (TTSuV2) are classified into the genus Iotatorquevirus. It is believed that TTVs might influence the development of some diseases or even modulate the outcome of disease by being present in blood or tissues (Okamoto, 2009). Even a clear-cut pathogenic role for TTSuVs has not been demonstrated to date, its role during co-infection with other pathogens is under debate, especially with regards to porcine circovirus diseases (PCVDs) (Ellis et al., 2008; Kekarainen et al., 2006; Taira et al., 2009). TTVs share conserved genomic regions with economically important circular ssDNA viruses of swine and poultry [Porcine circovirus-2 (PCV2) and Chicken anemia virus (CAV), respectively] (Biagini, 2009) both members of the family Circoviridae. TTSuVs have similar genomic organ- ization with human-infecting TTVs but share less than 45 % nt sequence identity (Niel et al., 2005; Okamoto et al., 2002). Recent studies also demonstrated a high degree of genetic variability between TTSuV1 and TTSuV2 (Cortey et al., 2011). The genome of TTSuV is approximately 2.8 kbp in length and two major potential protein-coding genes, ORF1 and ORF2, can be deduced from the nucleotide sequence. By analogy with related ssDNA viruses, ORF1 is believed to encode the viral capsid protein. ORF2 encodes a non-structural protein, assumed to be involved in viral replication (Hijikata et al., 1999; Huang et al., 2010). TTV ORF2 has also been associated with the NF-kB pathway suppression (Zheng et al., 2007). Analysis of TTSuV nucleotide sequence reveals the existence of an additional ORF, ORF3, generated by splicing and sharing its 59 end with ORF2. ORF3 is believed to encode a non-structural protein with unknown function (Biagini et al., 2001; Okamoto et al., 2000). Research on anelloviruses has been based almost solely on PCR techniques. Recently, tissue culture systems support- ing human TTV replication, with an inefficient propaga- tion, have been reported (Kakkola et al., 2007; Leppik et al., 2007). To date, few studies have focused on molecular virology, transcription and expression strategies of differ- ent human TTV genotypes and results are fairly contra- dictory. Three mRNAs were produced after transfection with a plasmid containing a TTV genotype 1 genome driven by a putative promoter in COS-1 cells (Kamahora et al., 2000). Moreover, after alternative splicing and alternative translation process, six different proteins from Supplementary material is available with the online version of this paper. Journal of General Virology (2011), 92, 2446–2457 DOI 10.1099/vir.0.033134-0 2446 033134 G 2011 SGM Printed in Great Britain