The matrix protein of Human respiratory syncytial virus localises to the nucleus of infected cells...

Arch Virol (2003) 148: 1419–1429DOI 10.1007/s00705-003-0112-y

The matrix protein of Human respiratory syncytial viruslocalises to the nucleus of infected cells

and inhibits transcription

Brief Report

R. Ghildyal, C. Baulch-Brown, J. Mills, and J. Meanger

Children’s Virology Research Unit, Macfarlane Burnet Institute for MedicalResearch and Public Health, Prahran, Vic., Australia

Received November 11, 2002; accepted February 26, 2003Published online June 2, 2003 c© Springer-Verlag 2003

Summary. We studied the kinetics of localisation of matrix (M) protein of Humanrespiratory syncytial virus (RSV) in infected cells. M protein was detected in thenucleus early in infection, by confocal microscopy and by immunoblotting ofnuclear fractions. We next tested the possibility that M protein may be involved ininhibition of host cell transcription. Nuclear extracts from RSV infected cells hadless transcriptional activity in vitro when compared to nuclear extracts from mockinfected cells. In addition, nuclear extracts from RSV infected cells inhibited thetranscriptional activity of nuclear extracts from mock infected cells, suggestingthat an inhibitory activity was transferred with nuclear extracts from RSV infectedcells. Our data suggest that M protein may play a role early in the infection byinhibiting host cell transcription.

∗The matrix protein of some enveloped RNA viruses is a major structural proteinwith several roles in the virus life cycle [6, 19]. Matrix proteins play a centralrole in assembly of the virus, bringing together the nucleocapsid and the envelopecomplex; they inhibit transcription activity from the viral genome in the nucleo-capsid prior to packaging and budding and may inhibit host cell transcription inthe initial stages of infection [19].

Although most RNA viruses replicate predominantly in the cytoplasm, someare known to inhibit host cell transcription [16]. Current knowledge of this phe-nomenon is based on studies on Poliovirus and Vesicular stomatitis virus (VSV)[1, 3, 11, 15, 23, 24, 27]. The poliovirus and VSV proteins responsible for host

1420 R. Ghildyal et al.

transcription inhibition (3C protease and M protein respectively) are located incytoplasm as well as the nucleus of infected cells [5, 14].

Little is known of the functions of the Human respiratory syncytial virus (RSV)matrix (M) protein in infected cells. It is assumed that the RSV M protein has anequivalent role to that of other paramyxovirus matrix proteins [19]. Routledgeet al. showed that the RSV M protein is distributed in perinuclear patches similarto the F and G glycoproteins, suggesting Golgi localisation and interaction withenvelope glycoproteins [22]. Recently, we have shown that the RSV M protein as-sociates with the nucleocapsid late in infection, where it inhibits viral transcriptaseactivity [8].

In this report we describe the localisation of M protein of RSV in the earlystages of infection. We show that M protein is present in the nucleus of the infectedcell, where it may inhibit host cell transcription.

HEp-2 cells (Victorian Infectious Disease Reference Laboratory, Melbourne,Australia) and RSV strain A2 (a gift from Dr Paul Young, Department of Micro-biology, University of Queensland, Australia) were used throughout this study.HEp-2 cells were infected with RSV at multiplicity of infection of 5 as previouslydescribed [7]. Mock infected HEp-2 cells were used as negative control in allexperiments. Monoclonal antibody C781, specific for M protein, was a gift fromProf Erling Norrby, Karolinska Instituten, Sweden [18].

The localisation of M protein in RSV infected cells was studied by immuno-fluorescence as described earlier for the matrix proteins of VSV and Newcastledisease virus (NDV) [14, 20]. RSV infected HEp-2 cells were fixed at various timespost infection (p.i.) with 4% formaldehyde in PBS (phosphate buffered saline, pH7.2) for 10 min, followed by permeabilisation with 0.1% Triton X-100. The cellswere probed with C781 for 30 min, washed, and binding of C781 detected withTRITC conjugated antibody to mouse immunoglobulins (DAKO corporation).Cells were analysed by confocal microscopy using a Leica TCS-NT with ArKrLaser confocal microscope as described before [8, 17]. The red channel was usedto analyse fluorescence. At 12 h p.i. M protein was seen predominantly in thenucleus; at 14 h and 16 h p.i. M protein was seen both in the nucleus and cytoplasm(Fig. 1). At 16 h p.i. M protein was also seen at the cytoplasmic membranes. At18 h p.i. M protein was seen mainly within cytoplasmic inclusions and diffused,at a low level throughout the cytoplasm and nucleus. We have shown earlier thatat 20 h, M protein is almost completely absent from the nucleus and is presentpredominantly in cytoplasmic inclusions [8]. At the multiplicity of infection usedhere (5) we consistently observe viral proteins (G, M, F, N, P, M2-1) at 12 h p.i.,

�Fig. 1. RSV infected cells were fixed with formaldehyde and permeabilised with 0.1% TritonX-100 at 12 h, 14 h, 16 h and 18 h p.i. M protein was detected by incubating with C781followed by TRITC conjugated goat antibodies to mouse Ig. Images were analysed using thered channel output of a laser scanning confocal microscope. Uninfected cells were similarlyanalysed, at 18 h. The hours p.i. are indicated above the respective images. White arrows inthe 18 h p.i. image show cytoplasmic inclusions. The scale bar indicates 10 µm

RSV M protein in the nucleus of infected cells 1421


with the expression reaching a peak at 18 h, consistent with reports from othergroups [9, 12]. The observation of M protein in the nucleus is not due to itspresence in the cytoplasm above the nucleus as sequential optical slices throughthe nucleus also showed nuclear localisation. However, to address this concern,we carried out the immunofluorescence assay in conditions of varying membranepermeabilisation.

RSV infected cells were fixed at 16 h p.i. with formaldehyde and perme-abilised with various concentrations of Triton X-100 or left unpermeablilised.The cells were probed with C781 followed by FITC conjugated antibody tomouse immunoglobulins (DAKO corporation) and counterstained with propidiumiodide. Propidium iodide stains nucleic acids and can be observed in the confocalmicroscope using the red channel. The images in Figure 2a are computer generatedmerges produced by the superposition of the red and green channel outputs ofthe same cell at the same optical plane and yellow areas denote colocalisation.Figure 2a shows that in the absence of permeabilisation and when 0.01% Tritonwas used M protein was observed only in the cytoplasm, indicating that in theabsence of nuclear membrane permeabilisation, M protein in the nucleus was notaccessible to antibody. The nucleus can be clearly observed in these cells, stainedred with propidium iodide. When cells were permeabilised with ≥0.1% TritonX-100, the antibody was able to enter the nucleus and M protein was seen in thenucleus (0.1% and 1%) as well as the cytoplasm.Yellow patches are observed in thenucleus in these images denoting colocalisation of M protein with the red stainingfor propidium iodide. As previously shown by Lyles et al., Westaway et al., andPeeples [14, 20, 25], permeabilisation of the nuclear membrane with Triton X-100 is required to stain proteins in the nucleus. This differential staining observedwhen using different concentrations of Triton X-100 reflects the differences in theability of the antibody to cross the nuclear membrane, not a change in the locationof the protein.

�Fig. 2. a. RSV infected cells were fixed either with formaldehyde alone or fixed andpermeabilised with 0.01, 0.1 or 1.0% Triton X-100 as indicated above the respective images.Cells were probed for M protein using C781, followed by FITC conjugated goat antibodiesto mouse Ig. Cells were counterstained with propidium iodide. Stained cells were analysedin a laser scanning confocal microscope using the green and red channel outputs. The imagesshown represent computer generated merge produced by the superposition of the red andgreen channel outputs of the same cell at the same optical plane. The yellow areas indicateco-localisation. The scale bar indicates 10 µm. b. Nuclei were purified from RSV infectedHEp-2 cells at 16 h p.i. and separated into nuclear membrane and nucleoplasm fractions. Theproteins were separated on SDS-PAGE, transferred to nitrocellulose membranes and probedfor presence of M protein using C781 followed by horseradish peroxidase conjugated speciesspecific secondary antibodies, and ECL. 1,3,5 – RSV infected cells; 2,4 – mock infectedcells; 1 – nuclei; 2,3 – nuclear membrane; 4,5 – nucleoplasm. N – nuclei, N.M. – nuclearmembrane, Np – nucleoplasm; M – protein specific band is indicated on the left. Molecularweight standards are indicated on the right, in kilodaltons


To confirm the presence of M protein within the host cell nucleus, we extractednuclei from RSV infected cells at 16 h p.i. and separated them into nuclear mem-brane and nucleoplasm fractions [14]. Briefly, RSV infected cells were disruptedat 16 h p.i. in reticulocyte standard buffer (RSB) by passing 25 times through a26G needle; the lysate layered over a two step gradient of 45 and 61% sucrosein RSB containing 1 mg/ml of BSA; centrifuged at low speed and purified nucleicollected from the interface. Nuclei were washed in RSB and resuspended in0.1 mM MgCl2 to give 2 × 107 cells/ml. DNA was removed by enzyme digestionand nuclear membrane and nucleoplasm fractions obtained. Mock infected cellswere also treated as above and nuclear membrane and nucleoplasm fractions


obtained. Purified nuclei, nuclear membranes and nucleoplasm fractions wereanalysed by SDS PAGE followed by immunoblotting with C781. The amountof protein loaded per well corresponded to 4 × 105 cells for nuclei and nu-clear membranes, and 4 × 104 cells for nucleoplasm. Separated proteins weretransferred to nitrocellulose membranes (HybondC, Pharmacia Ltd., Transblot,Bio-Rad). Membranes were blocked with BSA (10 mg/ml BSA in Tris bufferedsaline, pH 7.5) and probed with C781 diluted 1:1000 in BSA (5 mg/ml BSA inTris buffered saline containing 0.1% Tween 20). Membranes were washed andbound antibody detected with secondary antibodies to mouse immunoglobulinsconjugated to horseradish peroxidase (DAKO corporation) followed by ECL(Amersham). Figure 2b shows the presence of M protein in RSV infected cellsbut not in mock infected cells (lanes 1,3,5 compared to lanes 2,4). M proteinis present in whole nuclei, the nuclear membrane, and the nucleoplasm. Weobserved two major protein bands; the identity of the higher band is unknownat this time, but its size raises the possibility that it may be a dimer of M protein.The apparent difference in M protein content between the nuclear membrane andthe nucleoplasm fractions reflects the difference in number of cells representedby the fractions. Our data show that RSV M protein is present within the nucleusas well as the nuclear membrane.

Our results show that the M protein of RSV, like the matrix proteins of VSV,Sendai virus and NDV, is localised to the host cell nucleus early in infection. Themechanism of transport of M across the nuclear membrane is not clear as RSVM protein lacks canonical nuclear localisation signals. The nuclear localisation isnot due to leakiness of nuclear membrane induced as an affect of cytopathology,as M protein was present in the nucleus early in infection when cytopathologywas not evident. These observations, when considered together with our previousdata that M protein is not observed in the nucleus later in infection [8], led

�Fig. 3. Dot blot analysis of the in vitro transcription reaction. Nuclear extracts were preparedfrom mock and RSV infected cells at 16 h p.i. and transcription reactions were performedusing an exogenous template as described in the text. a. The reaction products were spottedonto nitrocellulose membrane in duplicate (samples 1–5). 1 µg of the vector fragment ofBamHI/SacI digest was spotted as negative control (sample 6). The membrane was probedwith 32P-dCTP labelled BamHI/SacI insert fragment. 1–15 µg of nuclear extract from mockinfected cells, with 15 µg of nuclear extract from RSV infected cells; 2–15 µg of nuclearextract from RSV infected cells with 15 µg of BSA; 3–15 µg of nuclear extract from mockinfected cells with 15 µg of BSA; 4–15 µg of nuclear extract from RSV infected cellswith 15 µg of BSA, without template; 5–15 µg of nuclear extract from mock infected cellswith 15 µg of BSA, without template; 6 – BamHI-SacI vector fragment. N.E. – nuclearextract. Template – pEGFP-N1 containing 320 bp ORF3 gene of feline calicivirus (FCV) waslinearised with BamHI to generate the template. The presence or absence (+/−) of the variousreaction constituents in every sample is indicated above the dot blot. b. The spots on the X-rayfilm of the northern blot were scanned and analysed by ImageGauge software. The densityvalues so obtained were corrected by subtraction of the value obtained from an equal area offilm between the spots. Data are represented as relative scan units


us to hypothesise that M protein may be inhibiting host cell transcription. Totest this hypothesis, we determined the ability of nuclear extracts from RSVinfected cells to support transcription of an exogenous template in an in vitroreaction.

The 320 bp ORF3 gene of Feline calicivirus (FCV) cloned into the pEGFP-N1 vector (Clontech industries) was used as template for in vitro transcriptionreactions. Transcription from pEGFP-N1 is controlled by a cytomegalovirus im-mediate early promoter (CMVIE). The FCV gene was cloned into the SacI andBamHI sites of the multicloning site in front of the EGFP cassette of the vector.This plasmid was linearised with BamHI to generate the template for transcriptionreactions. The FCV gene was extracted from the plasmid by digestion with BamHIand SacI and used to generate a 32P-dCTP labelled probe by nick translation using acommercial kit (Promega). The large vector fragment was used as negative controlfor the Northern blot.

Nuclear extracts were prepared from mock and RSV infected HEp-2 cells at16 h p.i., as described by Dignam et al. [4] and the protein content estimated by theBio-Rad Coommassie Blue protein assay reagent. A 50 µl transcription reactioncontained 1 µg DNA template and 30 µg of total protein. The reactions contained15 µg of nuclear extract from either mock or RSV infected cells or both. Thetotal protein was made up to 30 µg with BSA where required. Control reactionscontained nuclear extracts without template. The transcription reaction was carriedout for 60 min at 30 ◦C, and stopped by adding 175 µl of stop solution (0.3 MTrisHCl pH 7.4 at 25 ◦C, 0.3 M sodium acetate, 0.5% SDS, 2 mM EDTA) followedby digestion of DNA by 10 U of DNAse 1 for 15 min, phenol chloroform extractionand precipitation of RNA with ethanol. Transcription products were suspendedin nuclease free water and spotted onto nitrocellulose membranes under vacuum,using the Bio-dot apparatus (Bio-Rad).As a control for the specificity of the probe,the vector fragment of the plasmid generated by BamHI and SacI digestion wasalso spotted. Nucleic acids were crosslinked to the membrane by exposure to UV.The blot was probed with the 32P-dCTP labelled probe using ExpressHyb perthe manufacturer’s recommendations (Clontech) and the membrane exposed to Xray film at −70 ◦C. The developed film was scanned and the intensity of the dotsanalysed by ImageGauge software. The density values so obtained were correctedby subtraction of the value obtained from an equal area of film between the spots.Data are represented as relative scan units.

Nuclear extracts from mock infected cells supported transcription more effi-ciently than nuclear extracts from RSV infected cells (Fig. 3, compare sample 3with sample 2). When the two extracts were mixed, lower amounts of transcriptionproduct were detected (sample 1). The transcription activity in sample 1 wascomparable to that of sample 2. The amount of nuclear extract from mock and RSVinfected cells used in sample 1 was the same as in samples 3 and 2 respectively.The amount of transcription product formed in sample 1 should have been thesum of the transcription products formed in samples 2 and 3, i.e., at least asmuch as that formed in sample 3 by nuclear extracts from mock infected cells;unless there was an inhibitory activity associated with the RSV infected nuclear


extract. As M protein is the only RSV protein we have been able to detect in thenucleus, these data suggest that the inhibition is due to the activity of M protein.The radiolabelled probe used in the dot blot was specific as it did not bind to thecontrol DNA which represented the vector fragment of the template plasmid afterdigestion by BamHI and SacI (sample 6). There was no background transcriptionobserved in the absence of template by either extracts from mock or RSV infectedcells (samples 4, 5), indicating that the positive signals observed in samples 1–3are specific.

Our finding that nuclear extracts from RSV infected cells were deficient intranscription activity was interesting, as RSV infection of cells is not known toresult in host cell shut off as is documented for VSV, and to a lesser extent,for NDV [10]. However, an early study showed that DNA synthesis is reducedby 50% and RNA synthesis by 35% in RSV infected cells compared to mockinfected cells [13]. Our data lead us to suggest that M protein may be involvedin selective shut off of some host cell genes early in infection to facilitate viralreplication. Our finding that nuclear extracts from RSV infected cells were ableto inhibit transcription activity of extracts from mock infected cells is in contrastto that seen in VSV [28]. In VSV, the mechanism of inhibition is indirect; matrixprotein inactivates TFIID, leading to transcription inhibition [27]. Based on thedata presented here, we suggest that the excess M protein in nuclear extracts frominfected cells was able to directly inhibit transcription by nuclear extracts frommock infected cells.

The matrix proteins of two other viruses belonging to the family Paramyxoviri-dae, Sendai virus and NDV, have been shown to be present in nuclei of infectedcells, [20, 26]. In both these cases, the matrix protein was observed in the nucleusearly in infection, prior to any cytopathology. The NDV matrix protein contains abipartite nuclear localisation signal and is able to enter the nucleus when expressedalone in a transfection system [2, 21]. There are no reports on the function of eitherthe Sendai or the NDV matrix proteins in the nucleus.

In our present study, we have shown that RSV M protein is present in the nu-cleus early in infection, where it appears to inhibit host cell transcription. Our datasuggest that the M protein is responsible for inhibition of macromolecule synthesisobserved by Levine et al. [13]. We have previously shown that M protein is presentin the cytoplasm later in infection where it inhibits viral transcriptase activity [8].It is tempting to speculate that RSV sequesters M protein within the nucleus earlyin infection, removing it from the cytoplasm where virus transcription is able toproceed without hindrance.

Acknowledgement

We thank Prof Erling Norrby and Dr Mariethe Ehnlund for the monoclonal antibody to Mprotein and Prof Douglas Lyles for discussions throughout this study and critical reading of themanuscript.All confocal microscopy was done at Monash Micro Imaging, Monash University,Melbourne, Australia. This work was supported in part by a grant from Tattersalls, through theestate of the late George Adams and by the Research Fund of the Macfarlane Burnet Institutefor Medical Research and Public Health.


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Author’s address: Dr. Reena Ghildyal, Department of Respiratory and SleepMedicine, Monash Medical Centre, Clayton Road, Clayton 3168, Australia; e-mail:[email protected]

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