Downloaded from www.microbiologyresearch.org by IP: 54.162.190.106 On: Tue, 09 Feb 2016 01:08:04 Journal of General Virology (2002), 83, 753–757. Printed in Great Britain .......................................................................................................................................................................................................... SHORT COMMUNICATION Respiratory syncytial virus matrix protein associates with nucleocapsids in infected cells R. Ghildyal, 1 J. Mills, 1 M. Murray, 1 N. Vardaxis 2 and J. Meanger 1 1 Children’s Virology Research Unit, Macfarlane Burnet Institute for Medical Research and Public Health, PO Box 254, Yarra Bend Road, Fairfield, Victoria 3078, Australia 2 Department of Medical Laboratory Science, School of Medical Sciences, RMIT University, Bundoora, Victoria 3083, Australia Little is known about the functions of the matrix (M) protein of respiratory syncytial virus (RSV). By analogy with other negative-strand RNA viruses, the M protein should inhibit the viral polymerase prior to packaging and facilitate virion assembly. In this study, localization of the RSV M protein in infected cells and its association with the RSV nucleocapsid complex was investigated. RSV-in- fected cells were shown to contain characteristic cytoplasmic inclusions. Further analysis showed that these inclusions were localization sites of the M protein as well as the N, P, L and M2-1 proteins described previously. The M protein co-purified with viral ribonucleoproteins (RNPs) from RSV- infected cells. The transcriptase activity of purified RNPs was enhanced by treatment with antibodies to the M protein in a dose-dependent manner. These data suggest that the M protein is associated with RSV nucleocapsids and, like the matrix proteins of other negative-strand RNA viruses, can inhibit virus transcription. Human respiratory syncytial virus (RSV) is a non-seg- mented negative-strand RNA virus belonging to the family Paramyxoviridae, genus Pneumovirus (Collins et al., 1996). RSV- infected cells contain characteristic cytoplasmic inclusions. Previous studies have shown that these inclusions contain the nucleocapsid (N) protein, the viral polymerase (P and L proteins) (Collins et al., 1996) and the transcription elongation factor protein M2-1 (Garcia et al., 1993). It has been proposed that, as with Sendai virus, the P protein interacts with the newly synthesized N protein to prevent incorrect assembly of the nucleocapsid and to deliver it to the nascent RNA during genome replication (Curran et al., 1995 ; Garcia-Barreno et al., 1996), whereas the M2-1 protein is required for the synthesis of full-length viral RNA (Collins et al., 1996). These obser- Author for correspondence : J. Meanger. Fax 613 9282 2100. e-mail jayeshburnet.edu.au vations suggest that the inclusions are ribonucleoprotein (RNP) aggregates (Garcia-Barreno et al., 1996). Matrix proteins of negative-strand RNA viruses have been shown to associate with viral nucleocapsids in infected cells and virions. This association serves two functions : (1) to facilitate assembly and (2) to inhibit the transcriptase activity of the nucleocapsid prior to encapsidation (Coronel et al., 2001 ; Kaptur et al., 1991 ; Lenard, 1996). The two functions are genetically unrelated (Kaptur et al., 1991). In this paper, we provide evidence that the M protein of RSV is associated with RNPs and also show that it has transcriptase inhibition activity. M protein localization in RSV-infected HEp2 cells (Ghildyal et al., 1999) was investigated by immunofluorescence assays at time-points ranging from 12 to 20 h post-infection (p.i.). Cells were fixed with 4 % formaldehyde for 10 min and per- meabilized with 01% Triton X-100 for 5 min (Lyles et al., 1988). We chose formaldehyde as it is a gentle fixative (a) (b) (c) (d) Fig. 1. Localization of the M protein in infected cells. RSV-infected cells were fixed with formaldehyde and Triton X-100 at (a) 16, (b) 18 and (c) 20 h p.i. Mock-infected cells were fixed at 20 h p.i. (d). the M protein was detected by incubating with mAb C781 and TRITC-conjugated goat antibodies to mouse immunoglobulin. Stained cells were observed by confocal microscopy. Cytoplasmic inclusions are indicated by arrows. Bar, 10 μm. 0001-8066  2002 SGM HFD