Downloaded from www.microbiologyresearch.org by IP: 54.242.161.225 On: Tue, 10 May 2016 17:35:52 Barley stripe mosaic virus-encoded proteins triple-gene block 2 and cb localize to chloroplasts in virus-infected monocot and dicot plants, revealing hitherto-unknown roles in virus replication L. Torrance, G. H. Cowan, T. Gillespie, A. Ziegler and C. Lacomme Correspondence L. Torrance Lesley.Torrance@scri.ac.uk Plant Pathology Programme, Scottish Crop Research Institute (SCRI), Invergowrie, Dundee DD2 5DA, UK Received 23 February 2006 Accepted 2 April 2006 Replication of Barley stripe mosaic virus (BSMV), genus Hordeivirus, is thought to be associated with vesicles in proplastids and chloroplasts, but the molecular details of the process and identity of virus proteins involved in establishing the virus replication complexes are unknown. In addition, BSMV encodes a triple-gene block of movement proteins (TGBs) that putatively share functional roles with their counterparts in other hordei-, pomo- and pecluviruses, but detailed information on the intracellular locations of the individual TGBs is lacking. Here, the subcellular localizations of BSMV-encoded proteins TGB2 and cb fused to green or red fluorescent proteins were examined in epidermal cells of Nicotiana benthamiana and barley (Hordeum vulgare ‘Black Hulless’). The fusion proteins were expressed from a BSMV vector or under the control of the cauliflower mosaic virus 35S promoter. The subcellular localizations were studied by confocal laser-scanning microscopy (CLSM). CLSM studies showed that both proteins were recruited to chloroplasts in the presence of viral RNA and that virus RNA, coat protein and cb protein were detected in plastid preparations from infected leaves. Electron microscope images of thin sections of virus-infected leaves revealed abnormal chloroplasts with cytoplasmic inclusions containing virus-like particles. In addition, cellular localizations of BSMV TGB2 suggest subtle differences in function between the hordei-like TGB2 proteins. The results indicate that TGB2 and cb proteins play a previously unknown functional role at the site of virus replication. INTRODUCTION Positive-sense single-stranded RNA (ssRNA) virus replica- tion occurs in association with intracellular membranes of different origins, including the endoplasmic reticulum (ER) (e.g. Brome mosaic virus; BMV), multivesicular bodies derived from mitochondria, peroxisomes or vacuoles (e.g. Cucumber mosaic virus) and chloroplasts (e.g. Turnip yellow mosaic virus) (reviewed by Ahlquist et al., 2003; Salonen et al., 2005). Viral proteins associated with replication co-localize with viral genomic RNA and host factors to form a replication complex that is assembled on intracel- lular membranes. Often the membranes are invaginated to create compartments that bring together the components of replication (Ahlquist et al., 2003); such compartmentaliza- tion helps to isolate them from host-cytoplasmic compon- ents and, arguably, may also help to avoid RNA-mediated defence responses (Ahlquist et al., 2003). Replication of Barley stripe mosaic virus (BSMV) is associated with plastids (Carroll, 1970; Lin & Langenberg, 1985), but the identity of the viral proteins involved and how the complex is recruited to the plastid are unknown. The BSMV genome contains a triple-gene block of move- ment proteins (TGBs) (Fig. 1). TGB-containing viruses have been classified into two groups: the hordei-like group, including BSMV and Potato mop-top virus (PMTV), and the Fig. 1. Diagram of BSMV genome organization; the mass of the encoded proteins is given within the boxes. Triple gene- block proteins 1, 2 and 3 are indicated on RNA b. Solid rec- tangles indicate tRNA-like structures. 0008-1975 G 2006 SGM Printed in Great Britain 2403 Journal of General Virology (2006), 87, 2403–2411 DOI 10.1099/vir.0.81975-0