Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK1
Author for correspondence: Andrew Easton.Fax +44 1203 523701. e-mail ae{at}dna.bio.warwick.ac.uk
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Abstract |
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Introduction |
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Sequence analysis of other pneumovirus genes has shown that some mRNAs contain a second, small, alternative ORF. Of particular note is the presence of a second ORF (ORF2) in the mRNA encoding the M2 proteins of all RS viruses and APV (Collins & Wertz, 1985 ; Elango et al., 1985
; Baybutt & Pringle, 1987
; Collins et al., 1990
; Ling et al., 1992
; Zamora & Samal, 1992
; Alansari & Potgieter, 1994
). Each of these internal ORFs partially overlaps the upstream major ORF. These second ORFs show conservation in location but not in the predicted sequence of the putative polypeptide. This striking degree of conservation suggests that the alternative ORFs may be functional. Studies involving the rescue of infectious RS virus from a full-length cDNA clone have suggested that the product of the second ORF of the M2 gene plays a role in transcription and replication of the virus genome (Collins et al., 1995
, 1996
; Hardy & Wertz, 1998
). To date the polypeptide products of these second ORFs have not been detected directly. We report here the sequence of the M2 gene of PVM and the identification of polypeptides encoded by ORF2 of the pneumovirus M2 genes, together with their localization within the infected cell.
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Methods |
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Nucleic acid manipulations.
mRNA was extracted from infected BSC-1 cells as described previously (Ling et al., 1992 ). The production and preliminary characterization of cDNA clones has been described previously (Chambers et al., 1990
). Clones representing the M2 gene were isolated and the complete nucleotide sequence determined. For PCR amplification, cDNA was prepared by using oligo(dT) as a primer and aliquots were used to amplify the regions of the M2 gene containing ORF2. The entire RS virus M2 gene was amplified by using primers RS22K1A (5' GGGAATTCGGGGCAAATATGTCACG17) and RS22K7 (5' GGGTCGACGGGATCCATTTTGTCCC ACAAC1000) and ORF2 alone was amplified by using primers RS22K6 (5' GGGAATTCGTGATACAAATGACC568) and RS22K7. The entire PVM M2 gene was amplified by using primers PVM22K1A (5' GGGAATTCAGGATGAGTGTGAGAC16) and PVM22K6 (5' CGAGGGTACCGGAATACAGTCCACA765) and ORF2 alone was amplified with primers PVM22K5 (5' TACGGAGCTCAGGAGTACTATGC471) and PVM22K6. The entire APV M2 gene was amplified with primers APV22K1A (5' GGGAATTCGGGACAAGTGAAGATGTCT19) and APV22K4 (5' GACGTCGACAATTAACTAATT ATATG742) and ORF2 alone was amplified by using primers APV22K3 (5' CCCGGGATCCACAATGCCAATGG540) and APV22K4. The numbers refer to the 3' nucleotide in the gene to which the oligonucleotides anneal and restriction enzyme cleavage sites included for cloning purposes are underlined. PCR products were purified and inserted into appropriate plasmid vectors by using the restriction endonucleases for which recognition sites had been included in the PCR primer oligonucleotides and the nucleotide sequence of each insert was confirmed. The DNA fragments representing ORF2 of the M2 genes were inserted into the multiple cloning site of the expression vector pGEX5X-1 (Pharmacia) between the EcoRI and SalI recognition sites. In this way, the putative M2 ORF2 protein was expressed as a carboxy-terminal extension of GST.
Generation of polyclonal antisera and indirect immunofluorescence.
The purified GST fusion proteins were used to raise polyclonal antisera after injection into rats (for PVM and APV) and rabbits (for RS virus). The sera were shown to detect the bacterially expressed proteins by Western blot analysis (not shown). For immunofluorescence, BSC-1 cells grown on coverslips were infected with RSV at an m.o.i. of 1 p.f.u. per cell or with PVM and APV at an m.o.i. of 0·1 p.f.u. per cell and incubated at 37 °C until a cytopathic effect was visible. The cells were washed three times with PBS and fixed with a 1:1 (v/v) mixture of acetone and methanol (precooled at -20 °C) for 20 min at room temperature. After three washes with PBS, the cells were incubated with the primary antibody for 1 h at 37 °C. The antibody was removed and the cells were washed three times with PBS, after which they were incubated with the appropriate biotinylated anti-species antibody, diluted 1:400 in PBS, for 1 h at 37 °C. The cells were washed as before and incubated with streptavidinFITC conjugate for 15 min at 37 °C in the dark. The cells were washed three times with PBS and then mounted in 80% glycerol in PBS and fluorescence was visualized.
Preparation of antigen for Western blot analysis.
Twenty-four hours after infection, BSC-1 cells were washed twice with PBS, scraped into PBS, pelleted by centrifugation and lysed in lysis buffer (0·6% NP40, 150 mM NaCl, 1·5 mM MgCl2, 10 mM TrisHCl). The proteins were separated by PAGE and transferred to filters for Western blot analysis.
In vitro transcription and translation.
The M2 genes were transferred into pBluescribe plasmids such that they were under the control of the bacteriophage T7 promoter. RNA was used to direct the synthesis of proteins in a rabbit reticulocyte translation system (Amersham) as described previously (Chambers et al., 1990 ). The synthesized polypeptides were labelled by the addition of [35S]methionine in the translation reaction. The presence of globin in the reticulocyte lysates affected the apparent molecular mass of the M2 ORF2 proteins. In order to avoid this, the products were immunoprecipitated as described by Ling & Pringle (1989a
, b
) and were subsequently analysed by PAGE followed by detection by autoradiography.
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Results and Discussion |
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Sera raised against a fusion protein containing sequences encoded by the APV M2 ORF2 failed to detect a polypeptide in infected cells, though it precipitated a polypeptide of approximately 8·6 kDa from an in vitro translation (Fig. 3). It is possible that this is due either to lack of expression of this ORF in vivo or to the protein product being expressed at levels below those that are detectable using this assay.
The monospecific antisera raised against the three fusion proteins were also used to detect the possible expression of the products of ORF2 in virus-infected cells by immunofluorescence. As seen with the Western blot analysis, the APV ORF2-specific antibody failed to provide any evidence for expression of this protein in vivo. As before, this may be due to either the absence or very low levels of expression of the APV M2 ORF2 protein. In contrast, the antisera directed against the ORF2 products of the M2 genes of PVM and RS virus gave positive fluorescence signals in the infected cells (Fig. 4). No fluorescence was seen with mock-infected cells or with infected cells treated with pre-immune serum. In RS virus-infected cells, fluorescence was visible in the cytoplasm, with some evidence of localized cytoplasmic inclusions. There was frequently a higher degree of fluorescence in the vicinity of the nucleus. With the PVM-specific antiserum, as with the anti-RS virus serum, no fluorescence was seen with mock-infected cells. In PVM-infected cells, immunofluorescence was mostly distributed diffusely in the cytoplasm. However, some particulate forms of fluorescence could be seen, suggesting the increased localization of the PVM M2 ORF2 protein in specific regions of the cytoplasm. The small numbers of cytoplasmic inclusions containing the PVM and RSV proteins may indicate the presence of aggregates of these proteins or the presence of an interaction(s) with other virus or infected-cell proteins. Most of the inclusion bodies in RSV-infected cells are thought to be aggregates of virus nucleocapsids (Garcia et al., 1993
).
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The kinetics of synthesis of the M2 ORF2 protein in RSV-infected cells was analysed by Western blot analysis of samples prepared at 12, 18, 24 and 48 h post-infection (p.i.) (Fig. 5). The RSV M2 ORF2 protein could not be detected at 12 or 18 h p.i., was initially detected at 24 h p.i. and continued to accumulate throughout the infectious cycle to 48 h. This pattern of accumulation as the infection proceeds has been described for the RSV structural polypeptides (Lambert et al., 1988
) and the non-structural NS1 protein but contrasts with the NS2 protein, which has been shown to turn over rapidly during infection (Evans et al., 1996
). The low level of expression at early times in infection may reflect the relative use of the M2 gene ORF2, not only because of the position of the M2 gene on the virus genome relative to the promoter at the 3' end, but also because the location of this ORF in the M2 gene mRNA transcript may mean that it is only rarely accessed by ribosomes. The increasing amount of this protein over a long time during infection suggests that it is stably accumulated in the infected cells and may indicate that this protein is needed in the late stages of the virus life-cycle. Together with the observation that the M2 ORF2 protein inhibits transcription from the RS virus genome in a concentration-dependent manner (Collins et al., 1995
, 1996
), these data suggest that the protein may possibly act as the trigger, when an appropriate intracellular concentration is achieved, to precipitate the virus from a transcriptional mode into assembly of progeny nucleocapsids suitable for maturation into infectious virus particles, and is consistent with the observation that transfection of a plasmid expressing the M2 ORF2 protein enhanced packaging of a synthetic minigenome (Teng & Collins, 1998
). Elucidation of the role of these proteins in the life-cycle of the pneumoviruses may provide further insights into the mechanisms by which these viruses control their gene expression.
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Acknowledgments |
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Footnotes |
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References |
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Received 17 February 1999;
accepted 17 May 1999.