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1 DVP/Office of Vaccines Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, USA
2 Department of Pharmacology, University of Virginia, Charlottesville, VA, USA
Correspondence
Tahir Malik
tahir.malik{at}fda.hhs.gov
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Asp amino acid substitution at position 466 in the haemagglutinin–neuraminidase protein resulted in decreased receptor binding and neuraminidase activity, the Ala/ThrThr selection in the fusion protein resulted in decreased fusion activity, and the IleVal substitution in the polymerase resulted in increased replicative/transcriptional activity. These data suggest a polygenic component (i.e. specific and inter-related roles of these amino acid changes) to MuV neuroattenuation. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Elango et al., 1988). Through an RNA editing mechanism, the P gene also encodes the V and I proteins (Paterson & Lamb, 1990). MuV typically causes a mild respiratory infection; however, evidence of infection of the central nervous system has been reported in approximately half of all clinical cases (Bang & Bang, 1943). Neurological manifestations include aseptic meningitis (Beghi et al., 1984; Rotbart, 2000) and, more rarely, encephalitis (Aygun et al., 2001; Beghi et al., 1984), obstructive hydrocephalus (Ogata et al., 1992; Timmons & Johnson, 1970), transverse myelitis (Caksen & Ustunbas, 2003) and cerebellar ataxia (Cohen et al., 1992). The introduction of MuV vaccination programmes worldwide has resulted in a dramatic reduction in the number of mumps cases and, in some countries, the disease has almost been eliminated (Galazka et al., 1999; Peltola et al., 2000; Slater et al., 1999). However, as disease incidence has declined, concerns about vaccine safety have increased. For example, aseptic meningitis following the use of some vaccines, e.g. Urabe AM9, has resulted in vaccine withdrawal and public resistance to vaccination (Furesz & Contreras, 1990; Miller et al., 1993; Sugiura & Yamada 1991; Ueda et al., 1995). The need for robust immunization programmes and maintenance of public confidence in vaccines has been highlighted by the recent massive mumps epidemics in the USA and UK (Centers for Disease Control and Prevention, 2006; Gupta et al., 2005; Savage et al., 2005).
To support the development and use of safe MuV vaccines, a rat-based test that appears capable of accurately assessing the human neurovirulence potential of MuV strains was developed (Rubin et al., 2000, 2005). Using this prototype neurovirulence safety test, we previously reported the identification of three amino acid changes associated with neuroattenuation of the highly neurovirulent 88-1961 wild-type MuV strain: one each in the F (Ala/Thr-91Thr), HN (Ser-466Asn) and L (Ile-736Val) proteins (Rubin et al., 2003). To understand the basis for virus neuroattenuation in more detail, this study characterized the effect of these amino acid changes on the function of the corresponding proteins. Our results indicated that, relative to the wild-type parental virus strain, the amino acid substitution in the HN protein of the attenuated virus resulted in decreased receptor binding (haemagglutination) and release (neuraminidase) activities, whilst the amino acid selection in the F protein resulted in a dramatic decrease in fusion activity. Lastly, the amino acid substitution in the polymerase protein resulted in increased replicative/transcriptional properties of the protein. Potential ways in which these three amino acid changes, individually or in combination, may directly impact on virus virulence in vivo are discussed, but cannot be firmly ascertained until the development of a reverse-genetics system for this and other wild-type MuV strains.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), kindly provided by K. Conzelmann (Munich, Germany), were maintained in DMEM supplemented with 10 % FBS and 1 mg neomycin (Invitrogen) ml–1. The modified vaccinia virus Ankara strain expressing T7 RNA polymerase (MVA-T7) was kindly provided by B. Moss (Bethesda, MD, USA) and propagated in primary CEF cells (Wyatt et al., 1995). The neurovirulent 88-1961 wild-type MuV strain (88-1961WT) and its CEF-passaged neuroattenuated derivative (88-1961ATT) have been described previously (Amexis et al., 2003; Rubin et al., 2003).
RNA isolation, RT-PCR and sequencing.
MuV RNA was extracted from 88-1961-infected Vero cells using an RNAeasy kit (Qiagen) or from MuV stocks using a QIAamp Mini-elute Virus Spin kit (Qiagen). Reverse transcription (RT) reactions were performed with an appropriate gene-specific primer and Superscript II reverse transcriptase (Invitrogen). PCRs were performed with Pfx polymerase (Invitrogen). Optimal PCR conditions were determined empirically for each primer set. PCR fragments and plasmids were sequenced on an ABI 3100 automated capillary DNA sequencer. Sequence data were analysed using CHROMAS (Technelysium Pty Ltd), JELLYFISH (LabVelocity) and IMAGE-PRO PLUS 4.5 (Media Cybernetics) software packages.
Assessment of heterogeneity within the wild-type 88-1961F ORF.
The consensus sequence of 88-1961WT contains a heterogeneous nucleotide (A/G) population at position 271 of the F ORF, encoding either Ala or Thr at aa 91 (Amexis et al., 2003). In order to assess accurately the impact of this mixed nucleotide population on fusogenicity, the F ORF was amplified by RT-PCR using primers F1 (5'-GGCTTTCTCAGTTATTTGCTTGG-3') and F2 (5'-CCTGATGAGATCATCGACACTACTTG-3'), using viral RNA isolated from a lysate of 88-1961WT, in three independent reactions. The PCR fragment from each of the three reactions was sequenced and measurements of the area under the curve were performed to determine the ratio of A : G using IMAGE-PRO PLUS 4.5 software.
Construction of plasmids.
Total RNA isolated from 88-1961-infected Vero cells was used for RT-PCR amplification of all viral sequences. Restriction sites introduced into the primers used for RT-PCR are highlighted below in bold and all final constructs were sequenced completely to ensure homology with the published sequence (GenBank accession no. AF467767
[GenBank]
).
Expression plasmids for the HN and F genes
The F ORF containing an A at nt 271 was amplified by RT-PCR using the following primers: F-P1-BspHI (5'-TGCTCATGAAGGCTTTCTCAGTTATTTGCTTGG-3') and F-P2-XhoI (5'-CCTCTCGAGTTAGTACCTGATGAGATCATCG-3'). The PCR fragment was digested with BspHI and XhoI and ligated into the pTM1 vector (kindly provided by B. Moss; Moss et al., 1990) between the T7 RNA polymerase promoter and terminator sequences, respectively, at the NcoI/XhoI sites. The resulting plasmid, p-FThr, was modified by site-directed mutagenesis (SDM) to introduce a G at nt 271 using the QuikChange SDM kit (Stratagene). The resultant plasmid was termed p-FAla. To represent the previously reported nucleotide substitution in HN (GA at nt 1397), a wild-type HN expression plasmid containing a G at nt 1397 was constructed by amplifying two PCR fragments using primer pairs HN-P1-NcoI (5'-TGCCCATGGAACCCTCAAAACTCTTCACAATATC-3') and HN-P2-NcoI (5'-CGAGAATCCCCATGGAAACATATTGGTTAGACG-3'), and HN-P3-NcoI (5'-CGTCTAACCAATATGTTTCCATGGGGATTCTCG-3') and HN-P4-XhoI (5'-CCTCTCGAGTCAAGTGATAGTCAATCTGGTTAGC-3'). The PCR fragments were digested with the appropriate restriction enzymes and ligated contiguously into the pTM1 vector at the NcoI/XhoI sites. The resultant plasmid, p-HNWT, was modified by SDM to introduce an A at nt 1397. The modified plasmid was termed p-HNATT.
Expression plasmids for the N, P and L genes
RT-PCR amplification of the N ORF was performed using primers N-P1-BspHI (5'-TGCTCATGAGCTCTGTGCTCAAAGCGTTTG-3') and N-P2-XhoI (5'-CCGCTCGAGTTACTCATCCCAGTCACC-3'). The PCR fragment was digested with BspHI and XhoI and ligated into the pTM1 vector at the NcoI/XhoI sites. The resultant plasmid was termed p-N.
RT-PCR amplification of the P ORF was performed using primers P-P1-NcoI (5'- TGCCCATGGATCAGTTTATAAAACAGGATGAG-3') and P-P2-XhoI (5'- CCGCTCGAGTCATATGGCGCTTCGTATG-3'). The PCR fragment was digested with NcoI and XhoI and ligated into the pTM1 vector at the NcoI/XhoI sites. The P ORF was subsequently modified by SDM to introduce two additional Gs at position 463 (Paterson & Lamb, 1990). The resultant plasmid was termed p-P.
Due to the large size of L, the wild-type L ORF containing an A at nt 2206 was amplified and assembled in three steps. L-P1-BamHI (5'-GGCAATTCTTTTCAACAATAAGGATCCTGCG-3') and L-P2-PstI (5'- CCACTGCAGTTAAATTATGTCTCCGTGG-3') amplified the 3' 857 bp fragment, L-P3-NcoI (5'-GCTAATAACCATGGAATTGGACACCACC-3') and L-P4-BamHI (5'-CGCAGGATCCTTATTGTTGAAAAGAATTGCC3') amplified the middle 3.5 kb fragment, and L-P5-NcoI (5'GGACCATGGATGGGCGTATCTCAGTTTTTATCG-3') and L-P6-NcoI (5'-GGTGGTGTCCAATTCCATGGTTATTAGC-3') amplified the 2.5 kb 5' fragment. The three PCR fragments were digested with the appropriate restriction enzymes and cloned contiguously into pTM1 at the NcoI/PstI sites. The resultant plasmid, p-LWT, was modified by SDM to introduce a G at nt 2206 of the L ORF. This plasmid was termed p-LATT.
Construction of the 88-1961 minireplicon, p88-1961CAT
A minireplicon containing the chloramphenicol acetyl transferase (CAT) reporter gene flanked by the 88-1961 gene start and gene end signals and by the genome termini was the final component constructed for use in the minigenome system. The following primers were used to amplify the 3' and 5' ends of 88-1961WT, respectively: 3' P1-NarI (5'-ATCATTGGCGCCAGCGAGGAGGCTGGGACCATGCCGGCCACCAAGGGGAAAATGGAGATGGG-3') and 3' P2-BsmBI (5'-ATCATTCGTCTCTTTTCAGGGTAGTGTCAAAATGTCTCCTATCAACAATGGAAGGTGAGGATCG-3'), and 5' P1-BsmBI (5'-ATCATTCGTCTCTATCGACTAGGGACTCCTCTGGT-3') and 5' P2-NotI (5'-AAGCTCGGCGGCCGCTTGTAATACGACTCACTATAACCAAGGGGAGAAAGTAAAATC-3'). Primer 3' P1-NarI encompassing the 88-1961WT 3' end also encodes the hepatitis delta virus ribozyme sequence (italicized), whilst primer 3' P2-BsmBI, encompassing the 88-1961WT 5' end, also encodes the sequence for the T7 RNA polymerase promoter (italicized). The following primers with BsmBI overhangs were used to PCR amplify the CAT ORF, using the Jeryl Lynn minireplicon (MUVCAT; Clarke et al., 2000) as template: CAT-P1 (5'-ATCATTCGTCTCGGAAAATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCC-3') and CAT-P2 (5'-ATCATTCGTCTCTCGATTTACGCCCCGCCCTGCCACTC-3'). The three PCR products were digested with the appropriate restriction enzymes and cloned in a four-way ligation into the pBluescript KS+-based MUVCAT minireplicon (Clarke et al., 2000). MUVCAT was pre-digested with NotI/NarI to release all Jeryl Lynn and the previously cloned CAT-related sequences from the vector. The total number of nucleotides (966) in the 88-1961CAT RNA is divisible by six, in agreement with the ‘rule of six’ (reviewed by Conzelmann, 2004).
Assessment of binding activity.
To assess the effect of the single amino acid change in the HN protein on binding activity, Vero cells (4x105 per well of a 12-well plate) were incubated with 88-1961WT or 88-1961ATT at an m.o.i. of 0.05 for 1 h at 37 °C. At 3 days post-infection, the cells were washed twice with ice-cold CO2-independent medium (Invitrogen) and incubated with 5 % (v/v) guinea pig red blood cells (RBCs; Bio-Link) for 1 h at 4 °C. Following the 1 h incubation, non-adherent RBCs were removed by washing three times with ice-cold CO2-independent medium. The binding activity of the HN variants was quantified by lysis of the bound RBCs in 145 mM NH4Cl, 17 mM Tris/HCl (pH 7.4), for 20 min at room temperature, and measuring the absorbance of the released haemoglobin at 540 nm.
The binding activity of each HN variant was normalized for expression of HN at the cell surface. Vero cells infected with 88-1961WT or 88-1961ATT (m.o.i.=0.05) were harvested to quantify expression of HN using a Compartmental Protein Extraction kit (Chemicon). Membrane proteins (8 µg) were resolved by 7.5 % SDS-PAGE and transferred onto a nitrocellulose membrane (Towbin et al., 1979). Polyclonal monospecific antibodies raised in rabbits against a peptide of the mumps HN protein cytoplasmic tail were used for the detection of HN by Western blotting (diluted 1 : 5000). Protein bands were visualized by staining with horseradish peroxidase-conjugated donkey anti-rabbit antibody (diluted 1 : 10000; Chemicon) followed by enhanced chemiluminescence (Amersham Biosciences). Band intensities were determined using QUANTITY ONE software (Bio-Rad).
Assessment of neuraminidase activity.
To assess the role of the single amino acid change in the HN protein on neuraminidase activity, HeLa cells (1x106 cells per well of a six-well plate) were infected with MVA-T7 (m.o.i.=2.0) for 1 h at 37 °C and then transfected with 1 µg p-HNWT plus 1 µg pTM1, 1 µg p-HNATT plus 1 µg pTM1, or 2 µg pTM1 (as assay control) using Lipofectamine (Invitrogen). At 24 h post-transfection (p.t.) the cell monolayers were washed twice with PBS and once with 0.1 M sodium acetate (pH 6) and incubated with 1 ml neuramin-lactose (625 µg µl–1; Sigma-Aldrich) in 0.1 M sodium acetate (pH 6). Following incubation for 20 min at 37 °C, the neuraminidase activity of the HN variants was determined essentially as described by Aminoff (1961). The supernatants were transferred to 15 ml polypropylene centrifuge tubes and incubated with 0.5 ml 25 mM periodic acid (in 62.5 mM H2SO4). Following incubation at 37 °C for 1 h, 0.4 ml 2 % NaAsO4 was added, the mixtures were vortexed and 2 ml of thiobarbituric acid (14.2 mg ml–1, pH 9.0) was added. After boiling each sample for 7.5 min, an equal volume of acid butanol (5 % HCl) was added to extract the free sialic acid. The amount of free sialic acid was determined by reading the absorbance at 549 nm.
Following removal of the neuramin-lactose substrate from the transfected cell monolayers, the cells were washed three times with PBS and then harvested to quantify HN expression as described above. The neuraminidase activity of each HN variant was normalized for expression of HN at the cell surface.
Assessment of fusion activity.
To assess the effects of the single amino acid changes in the F (amino acid selection) and HN (amino acid substitution) proteins on fusion activity, HeLa cells (1x106 cells per well of a six-well plate) infected with MVA-T7 (m.o.i.=2) for 1 h at 37 °C were co-transfected with combinations of p-HNWT, p-HNATT, p-FThr and p-FAla (1 µg each), or 2 µg p-HNWT as control, using Lipofectamine. The fusion promotion activity of the expressed protein combinations was determined as described by Sergel et al. (1993), with some modifications. The differences in size (number of nuclei per syncytium) and number of syncytia were quantified in five independent fields of view (100x magnification) per well every 2 h for a total of 16 h p.t. To eliminate selection bias, fields of view were chosen based on a prescribed uniform pattern.
Assessment of F protein expression and processing.
HeLa cells (1x106 cells per well of a six-well plate) infected with MVA-T7 (m.o.i.=2) for 1 h at 37 °C were transfected with 2 µg p-FAla, p-FThr or pTM1 (assay control) using Lipofectamine. At 36 h p.t., the cells were washed with cold PBS and membrane proteins isolated using a Compartmental Protein Extraction kit. The protein lysates (8 µg) were resolved by 7.5 % SDS-PAGE followed by Western blotting (Towbin et al., 1979). F protein was detected with a rabbit MuV F anti-cytoplasmic tail polyclonal antibody (diluted 1 : 5000) (kindly provided by Dr Cattaneo, Mayo Clinic, Rochester, MN, USA; von Messling et al., 2004). Protein bands were visualized by staining with a donkey anti-rabbit horseradish peroxidase-conjugated IgG (1 : 10 000) followed by enhanced chemiluminescence. Band intensities were determined using IMAGE-PRO PLUS 4.5 software. F protein expression and processing were evaluated based on the relative amounts and sizes of the unprocessed F0 and processed F1 subunits, respectively.
Assessment of polymerase activity.
To assess the effect of the single amino acid change in the L protein on polymerase activity, BsrT7 cells grown on six-well plates to 70 % confluence were co-transfected using Lipofectamine with the minireplicon p88-1961CAT (200 ng) and the three support expression plasmids p-N (300 ng), p-P (50 ng) and either p-LWT or p-LATT (100–600 ng). At 48 h p.t, the cells were lysed and CAT expression, used as an indicator of polymerase activity, was measured by CAT ELISA (Roche Biochemicals). The protein content of each sample was normalized using a BCA protein assay kit (Pierce) prior to determination of CAT expression.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Asp substitution at position 466 of the neuroattenuated 88-1961 HN protein affects protein expression, virus-to-cell binding and neuraminidase activityAsp substitution on HN protein function, we assessed the ability of the 88-1961WT and 88-1961ATT viruses to bind RBCs, a standard method for evaluating relative differences in binding activities for paramyxoviruses. It should be noted that viruses were used here in lieu of HN-transfected cells due to the inability of transfected cells to express the high levels of HN protein required for this assay. In all other assays described in this paper, which are much more sensitive than the binding assay, transfected cells were used. As summarized in Table 1, in cultures infected with 88-1961ATT, RBC binding was reduced by approximately 48 % compared with 88-1961WT-infected cultures. Normalizing for cell-surface HN expression decreased the magnitude of this difference to approximately 29 %, but the difference remained statistically significant (P=0.029, Student's t-test). That the Ser-466Asp change per se was responsible for decreased cell-surface HN expression was confirmed in HeLa cells transfected with p-HNWT or p-HNATT (Table 2). Differences were also measured in neuraminidase activity associated with the Ser-466Asp substitution in HN (Table 2). In p-HNATT-transfected cultures, neuraminidase activity was reduced by approximately 34 % compared with p-HNWT transfected cultures. The magnitude of this difference was decreased following normalization for cell-surface HN expression to approximately 23 % and statistical significance was marginally lost (P=0.056, Student's t-test). Thus, despite the association of the HN Ser-466Asp substitution with a decrease in cell-surface HN expression, the reduction in protein level did not account for decreased binding and neuraminidase activities on a per-HN molecule basis.
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Asp substitution in the HN protein, affects fusogenicity; Tsurudome et al., 1986). We have reported previously that neuroattenuation of 88-1961WT was associated with the selection of an A residue from a previously heterogeneous A/G mixture at nt 271 of the F ORF (Rubin et al., 2003). Electropherogram data indicated the A : G ratio in the wild-type virus to be approximately 1 : 1 (data not shown). To assess the effect of the loss of amino acid heterogeneity (Ala/Thr-91Thr) in F and that of the SerAsp substitution in HN on fusion, an in vitro fusion assay was performed. In this assay HeLa cells were co-transfected with plasmids expressing the wild-type and attenuated F and HN proteins, and the number and size of resulting syncytia were quantified over time. The 88-1961WT virus was represented by FWT (p-FThr and p-FAla, 1 : 1) and HNWT (p-HNWT), whilst 88-1961ATT was represented by FThr (p-FThr) and HNATT (p-HNATT). HeLa cells co-transfected with plasmids representing 88-1961WT displayed syncytia as early as 6 h p.t., with maximum syncytium formation occurring at 12 h p.t. (Fig. 1a, b). In contrast, HeLa cells co-transfected with plasmids representing 88-1961ATT not only displayed a delay in syncytium formation, but the number and size of syncytia was also significantly reduced over the same 16 h period (Fig. 1a, b). Of note, the reduction in the number of syncytia in cultures co-transfected with wild-type F and HN following the 12 h time point was due to the merging of multiple syncytia to form single, large syncytia. To determine whether the decreased fusogenicity was due to FThr or HNATT, or both, HeLa cells were co-transfected with plasmids containing p-FAla plus p-HNATT, p-FThr plus p-HNATT or p-FThr plus p-HNWT, respectively. Our results indicated that the amino acid selection in the F protein was solely responsible for the decreased fusogenic phenotype of the attenuated virus (Fig. 2a–d). Although HNATT was associated with a small increase in the number of syncytia relative to HNWT between 14 and 16 h (Fig. 2a), this trend did not continue (up to 24 h p.t., data not shown).
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), an approximately 3 kDa increase was observed for the F0 subunit expressed by p-FThr (78 kDa) relative to that of p-FAla (75 kDa) (Fig. 3a). This size increase is consistent with the predicted N-glycosylation at aa 89 (corresponding to nt 265–267) following the selection of a Thr at aa 91 (corresponding to nt 271–273) of the F protein (PROSITE database; Bairoch et al., 1997). N-Glycosylation at aa 89 was confirmed by peptide : N-glycosidase F treatment of protein extracts isolated from cells transfected with FThr or FAla. Deglycosylation yielded F0 of an equivalent size (58.5 kDa) for the two F variants (data not shown). There was no difference observed in the size of either F1 variant (63 kDa) (Fig. 3a). This is consistent with the fact that aa 89 and 91 are present in F0 and the processed F2 (Fig. 3b). As the antibody available to us was specific to the C terminus of F (von Messling et al., 2004), we could detect the size difference in F0, but not F2.
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Val substitution in the neuroattenuated 88-1961 L protein leads to an increase in transcriptional/replicative activity). The transcriptional/replication activity of the neuroattenuated polymerase (p-LATT) was significantly greater than that of the wild-type polymerase (p-LWT) across all concentrations of input L plasmid DNA tested (Fig. 4) (all P<0.001; Student's t-test).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The genomes of the clinical isolate (88-1961WT) and the attenuated variant (88-1961ATT) were sequenced and compared. Amino acid selection/substitutions were identified only within the F (Ala/Thr-91Thr), HN (Ser-466Asp) and L (Ile-736Val) proteins. Here, we have shown that each of these single amino acid changes affects protein function and therefore may play a combined role in the neuroattenuation of 88-1961.
The HN glycoprotein is multifunctional, playing key roles in binding of the virus to its cellular receptor (infectious potential), in virus-to-cell and cell-to-cell fusion (spread of virus in the infected tissue) and in neuraminidase activity (release of nascent virions from infected cells). Amino acid substitutions within the HN glycoprotein therefore have the potential to reduce virulence by limiting virus infectivity and spread in the host. Others have reported amino acid changes within the MuV HN protein associated with virus virulence; however, these studies have not examined the effect of such changes on the HN protein in isolation, i.e. changes elsewhere in the virus genome may be responsible for the observed differences in virulence (Afzal et al., 1998; Amexis et al., 2001; Brown et al., 1996; Kovamees et al., 1990; Love et al., 1985). The Ser-466Asp substitution in the HN protein identified by our group is of particular significance, being located near the active site affiliated with receptor binding and neuraminidase activity (Crennell et al., 2000). This substitution is predicted to result in the loss of a glycosylation site (PROSITE database; Bairoch et al., 1997) and possibly leads to structural changes within the protein (Segawa et al., 2000). Whilst we did not observe evidence for altered glycosylation based on protein size in Western blots, the substitution did result in reduced cell-surface expression of HN that was manifested in cell cultures infected with 88-1961ATT compared with 88-1961WT, and in cells transfected with HN-expressing plasmids that only differed at this one site. Even when normalized for HN protein expression levels, reduced binding and neuraminidase activities were found for HNATT compared with HNWT. Our observations of reduced expression and binding and neuraminidase activities for HNATT compared with HNWT confirm that this single amino acid substitution impacts protein function and may potentially affect virus virulence.
The effect of the single amino acid selection in the F glycoprotein on protein function was also investigated and demonstrated a dramatic inhibitory effect on fusion activity in transfected cells. The possibility that reduced fusion activity was due to differences in the transfection efficiency of the two F constructs was ruled out by detecting equivalent amounts of F protein expression in the transfected cells. Cells infected with 88-1961WT also displayed earlier and more extensive syncytium formation compared with cells infected with 88-1961ATT using an identical m.o.i. (S. A. Rubin, unpublished observations). The possibility that FThr may be less fusogenic due to poor cleavage of the inactive precursor protein F0 into the two disulfide-linked active subunits, F1 and F2 (Merz et al., 1983; Rima et al., 1980), was also ruled out. All functional domains identified to date in the MuV F protein have been located in the F1 subunit (Liu et al., 2004; Server et al., 1985; Waxham et al., 1987). Thus, this is the first report suggesting the location of a critical functional domain within the MuV F2 subunit. Furthermore, computer analyses predict that this amino acid selection in the F protein of 88-1961ATT results in glycosylation of the nearby aa 89 (PROSITE database; Bairoch et al., 1997). Indeed, we observed a 3 kDa increase in size of FThr relative to FAla. This difference in size was not observed following deglycosylation of FThr- and FAla-transfected cell extracts (T. Malik, unpublished observations). Whilst others have reported that loss of glycosylation sites in paramyxovirus F proteins can lead to decreased fusogenicity (Bagai & Lamb 1995; McGinnes et al., 2001; Moll et al., 2004; Segawa et al., 2000; von Messling & Cattaneo, 2003, Zimmer et al., 2001), there are few studies indicating that the addition of a glycosylation site can result in decreased fusogenicity (Aguilar et al., 2006). Additionally, based on homology data from related paramyxoviruses, aa 91 is probably located in a third heptad repeat in which amino acid changes have been suggested by others to affect fusogenicity (Plemper & Compans, 2003).
Lastly, our analyses of the Ile-466Val substitution in the polymerase protein also suggest a role for this mutation in neurovirulence (Rubin et al., 2003). The MuV polymerase contains six highly conserved domains separated by regions of high variability (Okazaki et al., 1992; Poch et al., 1990). Domain II contains a highly charged putative RNA-binding motif, whilst domain III is believed to represent an important element of the active site for template recognition and/or phosphodiester bond formation (Okazaki et al., 1992; Poch et al., 1989, 1990). Domains II and III together constitute the polymerase module and are essential for viral RNA synthesis (Jablonski et al., 1991, Muller et al., 1994; Smallwood et al., 2002). The Ile-466Val substitution lies within domain III (Poch et al., 1990). In an attempt to evaluate the impact of the Ile-466Val substitution on viral transcription/replication, we developed a minigenome system representing the 88-1961 MuV strain. Substitution of the 88-1961ATT-derived polymerase into the minigenome system resulted in a significant increase in reporter gene activity relative to the 88-1961WT-derived polymerase. Notably, elevated polymerase activity has also been associated with measles virus attenuation (Bankamp et al., 2002). Elevated 88-1961ATT polymerase activity could result in increased viral transcription, triggering a more-effective RIG I-mediated beta interferon (IFN-
) response and hence rapid viral clearance. This hypothesis is based on the recent publication by Plumet et al. (2007), which proposes that RIG I recognizes mononegavirales transcription and induces a type I interferon (IFN-
/) response after encountering a free cytosolic 5'-triphosphate-ended viral leader transcript. Additional studies are needed to determine whether the increased activity of 88-1961ATT-derived L is a reflection of increased genome transcription or replication, or both.
In summary, we have identified functional differences in vitro arising from a single amino acid change in each of the 88-1961 HN, F and L proteins, and these changes were associated with neuroattenuation. These studies underline the probable complex and polygenic nature of the common cell-passage method of attenuation of virulent wild-type virus strains and the need to determine in this case whether the three amino acid changes act individually or in concert, in vivo, to neuroattenuate 88-1961WT. Such a determination will require the development of a molecular clone for this viral strain in order to shed much-needed light on the pathogenesis of MuV neurotoxicity and mechanisms of neuroattenuation. These studies may have implications for future vaccine design, not only for MuV, but also for other paramyxoviruses.
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ACKNOWLEDGEMENTS |
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Received 20 February 2007;
accepted 20 May 2007.
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