Childrens Virology Research Unit, Macfarlane Burnet Institute for Medical Research and Public Health, PO Box 254, Yarra Bend Road, Fairfield, Victoria 3078, Australia1
Department of Medical Laboratory Science, School of Medical Sciences, RMIT University, Bundoora, Victoria 3083, Australia2
Author for correspondence: J. Meanger. Fax +613 9282 2100. e-mail jayesh{at}burnet.edu.au
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Abstract |
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Main text |
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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 permeabilized with 0·1% Triton X-100 for 5 min (Lyles et al., 1988
). We chose formaldehyde as it is a gentle fixative compared to acetone or methanol. Fixed cells were incubated with an anti-M monoclonal antibody (mAb C781) (Orvell et al., 1987
) diluted 1:100 in PBS (pH 7·2) for 30 min, followed by goat anti-mouse immunoglobulin conjugated to TRITC (Sigma) (diluted 1:40 in PBS). Data for three time-points, 16, 18 and 20 h p.i., are presented in Fig. 1
. The M protein was observed throughout the cell at 16 h p.i. (Fig. 1a
), whereas at 18 h p.i., the M protein was present in cytoplasmic inclusions as well as diffused throughout the cell (Fig. 1b
). At 20 h p.i., the M protein was present predominantly within cytoplasmic inclusions and very little was observed in the rest of the cell (Fig. 1c
). The specificity of mAb C781 was demonstrated by the low level of staining in mock-infected cells (Fig. 1d
).
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To determine if the M protein was indeed associated with RNPs, we purified RNPs from RSV-infected cells and performed immunoblotting assays. RNPs were purified from infected and mock-infected cells, as described previously (Barik, 1992 ), with some modifications. At 16 h p.i., cells were exposed to actinomycin D (2 µg/ml) for 2 h. At 18 h p.i., cells were washed and lysed (Barik, 1992
). The cell lysate was then cleared of nuclei and cell debris and centrifuged at 150000 g for 4 h through 40% glycerol in 50 mM Trisacetate (pH 8) and 1 mM DTT onto a 100% glycerol cushion. The RNPs were collected and diluted in a small volume of 20 mM Trisacetate (pH 8) containing 1 mM EDTA, re-centrifuged through 40% glycerol and used immediately for immunoblotting or transcription assays (Fig. 3
). Mock-infected cells were treated in a similar manner. Soluble proteins were collected from the top of the glycerol gradient as well as the RNP fraction.
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The antibodies used in this experiment (Fig. 3a) were specific for RSV proteins, as no bands were observed in the mock lanes (Fig. 3a
, lanes 1, 3, 5 and 7). The purified RNP preparation contained the M protein (Fig. 3a
, lane 2). When the blot was re-probed, RNPs were found, as expected, to contain the N protein (Fig. 3a
, lane 4). The smaller band seen in lane 4 is residual M protein, remaining due to the incomplete stripping of primary antibodies from the previous experiment. To confirm that the M protein co-purified with the RNPs and was not a contaminant from the membrane or cytosol fractions, we looked for the presence of the RSV G protein in the same samples (Fig. 3a
, lanes 5 and 6). The G protein is found in the cytoplasm and membrane of RSV-infected cells and is not associated with RNPs. There was no G protein in the RNP preparation (Fig. 3a
, lane 6), indicating that the M protein co-purified with the RNPs. The antibody to G protein recognized a band of 8590 kDa in RSV-infected cell extracts (Fig. 3a
, lane 8). The final probe for the N protein was positive (data not shown).
Next, we studied the role of M protein in association with the RNPs by carrying out transcription reactions with purified RNPs in the presence of increasing concentrations of mAb C781 (Fig. 3b). Standard transcription assays were performed as described previously (Barik, 1992
). Briefly, each reaction contained 50 mM Trisacetate (pH 8), 120 mM Kacetate, 5 mM MgCl2, 5% glycerol, 1 mM DTT, actinomycin D (2 µg/ml), 20 µCi [
-32P]CTP, UTP and GTP, each at 400 µM, 1 mM ATP, 10 µg RNPs and 8 µg soluble protein from mock-infected cells. The indicated amounts of mAb C781 or a non-specific antibody (mAb 1420) (BioDesign) were used in a 20 µl total volume. Reactions containing mAb C781 but without soluble proteins or without RNPs were also included as controls. Soluble proteins from mock-infected cells were added to render the RNPs transcriptionally active, as previous reports have shown that purified RNPs require cellular factors for transcriptase activity (Burke et al., 2000
). Reactions were carried out at 30 °C for 3 h. Synthesis of labelled RNA was quantified by applying 5 µl of the reaction mixture to DEAE-embedded glass fibre filtermats. These were processed for scintillation counting in a Wallac Microbeta counter. Incorporation of 32P over a 3 h period was used to measure transcription. Data from one experiment, representative of three separate experiments, are shown in Fig. 3(b)
.
Results from these assays show that soluble proteins from mock-infected cells were essential for virus transcription, which was enhanced specifically in a dose-dependent manner in the presence of mAb C781, suggesting that the presence of the M protein inhibited virus transcription.
Our studies show that, during RSV infection, the M protein is present in characteristic cytoplasmic inclusions and is associated with RNPs containing the N, P and M2-1 proteins (Collins et al., 1996 ). This study also provides evidence that the M protein may function as a virus transcription inhibition factor.
An earlier report by Routledge et al. (1987) on the immunofluorescent localization of the RSV M protein using mAb C781 did not show the M protein in cytoplasmic inclusions. This discrepancy is attributable to the fixative used (Lyles et al., 1988
; Westaway et al., 1997
). We found that acetone fixation led to patchy staining around the nucleus (Routledge et al., 1987
), while fixation with formaldehyde led to diffuse cytoplasmic staining with intense staining in cytoplasmic inclusions. Another report on RSV M protein localization used formaldehydeglutaraldehyde fixing followed by immunoelectron microscopy and this report also failed to observe the M protein in cytoplasmic inclusions (Garcia et al., 1993
). These results differ from those reported here: this may be due to the different antibodies used in the two studies.
We have also shown that, like many other negative-strand RNA viruses, RSV nucleocapsids and the M protein interact in cytoplasmic inclusions within infected cells. Interactions between the matrix and the nucleocapsid proteins of Sendai virus (Markwell & Fox, 1980 ; Ryan & Kingsbury, 1988
), Newcastle disease virus (Portner & Murti, 1986
) and parainfluenza virus type 3 (De et al., 1991
) have been documented previously.
The nucleocapsid of negative-strand RNA viruses has to be rendered inactive before it can be packaged into the virus particle; this function has been attributed to the matrix proteins (Collins et al., 1996 ). Vesicular stomatitis virus, measles virus and influenza virus matrix proteins interact with, and inhibit transcription by, viral nucleocapsids in infected cells (Carroll & Wagner, 1979
; Perez & Donis, 1998
; Suryanarayana et al., 1994
). The data presented here suggest that the RSV M protein is involved in such a function. Depleting the M protein from RNPs in the absence of soluble proteins from mock-infected cells did not lead to the activation of RNPs, confirming that cellular proteins are necessary for transcription by RNPs. Barik (1992)
stated that RNPs isolated at 16 h p.i. were activated by the addition of soluble proteins but a later purification (2430 h p.i.) resulted in RNPs incapable of being activated by the addition of soluble proteins. One explanation for this phenomenon could be that the M protein is associated with RNPs at later times p.i. We observed that, at 16 h p.i., the M protein is not present in cytoplasmic inclusions, whereas at 20 h p.i., it is found mainly within cytoplasmic inclusions.
Our data, taken together with earlier reports of RSV transcription, imply that the M protein associates with RNPs late in the virus life cycle in order to inhibit virus transcription activity, thus facilitating virus assembly. This makes the M protein the third RSV protein to be shown to regulate virus transcription. Previously, the NS1 protein was shown to inhibit virus RNA replication and transcription in a minigenome system and the M2-2 protein was shown to act as a switch from transcription to replication (Atreya et al., 1998 ; Bermingham & Collins, 1999
). In light of our current data, it is obvious that RSV has a very complex mechanism for the modulation of transcription and replication during virion assembly.
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Acknowledgments |
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References |
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Received 28 August 2001;
accepted 15 November 2001.