Contributions of Matrix and Large Protein Genes of
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病菌学杂志 2005年第24期
Department of Virology, Faculty of Medicine, Kyushu University, Fukuoka 812-8582, Japan
ABSTRACT
The Edmonston strain of measles virus (MV) was obtained by sequential passages of the original isolate in various cultured cells. Although attenuated in vivo, it grows efficiently in most primate cell lines. Previous studies have revealed that MV tropism cannot be solely explained by the use of CD150 and/or CD46 as a cellular receptor. In order to evaluate the contributions of individual genes of the Edmonston strain to growth in cultured cells, we generated a series of recombinant viruses in which part of the genome of the clinical isolate IC-B (which uses CD150 as a receptor) was replaced with the corresponding sequences of the Edmonston strain. The recombinant virus possessing the Edmonston hemagglutinin (H) gene (encoding the receptor-binding protein) grew as efficiently in Vero cells as the Edmonston strain. Those viruses having either the matrix (M) or large (L) protein gene from the Edmonston strain could also replicate well in Vero cells, although they entered them at low efficiencies. P64S and E89K substitutions were responsible for the ability of the M protein to make virus grow efficiently in Vero cells, while the first half of the Edmonston L gene was important for better replication. Despite efficient growth in Vero cells, the recombinant viruses with these mutations had growth disadvantage in CD150-positive lymphoid B95a cells. Thus, not only the H gene but also the M and L genes contribute to efficient replication of the Edmonston strain in some cultured cells.
INTRODUCTION
Measles virus (MV), a member of the genus Morbillivirus of the family Paramyxoviridae, is an enveloped virus with a nonsegmented negative-strand RNA genome. The MV genome has six genes that encode the nucleocapsid (N), phospho- (P), matrix (M), fusion (F), hemagglutinin (H), and large (L) proteins. The P gene encodes additional accessory proteins, the V and C proteins, by a process of RNA editing and by an alternative translational initiation in a different reading frame, respectively (11).
MV was first isolated in a primary culture of human kidney cells inoculated with a sample from a child with measles (9). This isolate, the original Edmonston strain, was subsequently adapted to a variety of cultured cells, giving rise to currently used live vaccines (35). The Edmonston strain grows well in continuous cell lines, including Vero cells derived from the African green monkey kidney, and has become the most extensively studied MV strain in laboratories. In 1990, Kobune etal. reported that the marmoset B-lymphoid cell line B95a is highly sensitive to MV, allowing efficient MV isolation from clinical specimens (16). Whereas the Edmonston strain replicates efficiently in almost any primate cell line, B95a cell-isolated strains were found to grow in a restricted number of cell lines (16, 45). This riddle was resolved when it was demonstrated that the signaling lymphocyte activation molecule (SLAM) (also called CD150) acts as a cellular receptor for MV (10, 14, 46, 48). SLAM is expressed on certain types of cells of the immune system (23). On the other hand, besides SLAM, the Edmonston strain can use as a receptor CD46, which is expressed on all nucleated cells (7, 22, 46). Thus, the ubiquitous expression of CD46 appears to explain the ability of the Edmonston strain to replicate in most primate cell lines.
However, there have been reports that the receptor usage cannot readily explain MV cell tropism. The recombinant Edmonston MV expressing the H protein of the B cell-isolated WTF strain spread in Vero cells, although the parental WTF strain did not (15). Since the H protein is responsible for the receptor binding, this finding suggests that cell tropism of therecombinant virus may be determined at a level other than the H protein-receptor interaction. Takeuchi et al. reported a similar finding (44). Furthermore, an MV strain, isolated in peripheral blood mononuclear cells, which uses SLAM but not CD46 as a receptor was successfully adapted to Vero cells without the acquisition of the ability to interact with CD46 (18). Takeuchi et al. also reported that there was no sequence difference in the H gene between MV strains isolated in B95a and in Vero cells from the same measles patient, although they exhibited distinct tropisms (43). We and others have previously demonstrated the presence of SLAM- and CD46-independent cell entry of MV (1, 12). However, as its efficiency is 2 to 3 log units lower than that of SLAM- or CD46-dependent entry, this inefficient entry can only partly account for the above observations. Thus, there must be some mechanism(s) which allows virus to replicate efficiently at a postentry step(s) even when it enters cells inefficiently.
In an attempt to determine the roles of individual genes of the Edmonston strain in growth in cultured cells, we here generated a series of recombinant MVs in which part of the genome of the B95a cell-isolated IC-B strain (16, 17, 42) was replaced with the corresponding sequences of the Edmonston strain (32). Our results showed that the M and L genes, in addition to the H gene, are important for efficient replication of the Edmonston strain in Vero cells. We have also determined the amino acid residues of the Edmonston M protein and the region of the L gene that are responsible for efficient growth in Vero cells.
MATERIALS AND METHODS
Cells and viruses. Vero, 293T (a human embryonic fibroblast cell line), and RK13 (a rabbit kidney cell line) cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (ICN Biomedicals, Aurora, Ohio) supplemented with 7.5% fetal bovine serum (FBS). B95a cells (a marmoset B-cell line transformed with Epstein-Barr virus) (16) were maintained in RPMI 1640 medium (ICN Biomedicals) supplemented with 10% FBS. Vero cells constitutively expressing human SLAM (Vero/hSLAM) (26) were maintained in DMEM supplemented with 7.5% FBS and 500 μg of Geneticin (G418) (Nacalai Tesque, Tokyo, Japan) per ml. Chinese hamster ovary (CHO) cells constitutively expressing human SLAM (CHO/hSLAM) (46) were maintained in RPMI 1640 medium supplemented with 7.5% FBS and 500 μg of G418 per ml. Recombinant MVs were generated from cDNAs by using CHO/hSLAM cells and the vaccinia virus encoding T7 RNA polymerase, vTF7-3 (a gift from B. Moss), as reported previously (40). Generated MVs were propagated in B95a cells, and virus stocks at two or three passages in B95a cells were used for experiments.
Construction of full-length cDNAs of recombinant virus genomes. The plasmid p(+)MV323-EGFP, which encodes the full-length antigenomic cDNA of the B95a-isolated IC-B strain (16, 17, 42) with an additional transcriptional unit of the enhanced green fluorescent protein (EGFP), was described previously (12). The plasmid p(+)MV2A (a gift from M. A. Billeter) encodes the full-length antigenomic cDNA of the Edmonston B vaccine strain with some modifications (GenBank accession number Z66517) (32). The rescued virus from p(+)MV2A is referred to as the Edmonston tag strain. By using appropriate restriction enzymes, a series of the genomic regions of p(+)MV323-EGFP were replaced with the corresponding regions of p(+)MV2A, generating 12 plasmids carrying the full-length genomes of recombinant chimeric viruses. The chimeric construct p(+)MV-EGFP-m1 was generated by replacing the BlpI-BlpI fragment (nucleotides [nt] 127 to 3146) containing most of the N and P genes of p(+)MV323-EGFP with the corresponding fragment of p(+)MV2A (Fig. 1). In this paper, nucleotide position numbers are shown in accordance with the sequence of the parental IC-B strain genome (42, 43) (GenBank accession number NC_001498). The SapI-SapI fragment [the first SapI site exists just upstream of the T7 promoter sequence of the pBluescriptII-KS(+) vector (42) and the second at nt 1813 in the IC-B sequence] was exchanged between p(+)MV323-EGFP and p(+)MV-EGFP-m1, generating two chimeric constructs, p(+)MV-EGFP-m2 and p(+)MV-EGFP-m3. Thus, p(+)MV-EGFP-m2 and p(+)MV-EGFP-m3 contain the BlpI-SapI fragment (nt 127 to 1813) and SapI-BlpI fragment (nt 1813 to 3146) of p(+)MV2A, respectively. Chimeric constructs p(+)MV-EGFP-m4, p(+)MV-EGFP-m5, p(+)MV-EGFP-m6, and p(+)MV-EGFP-m7 were generated by replacing SacII-BsmBI (nt 2040 to 5475), SacII-PacI (nt 2040 to 7238), BlpI-PacI (nt 3146 to 7238), and BstEII-PacI (nt 4807 to 7238) fragments of p(+)MV323-EGFP with the corresponding fragments of p(+)MV2A, respectively. The plasmid p(+)MV323/EdH-EGFP was described previously (12) and contains a PacI-SpeI fragment (nt 7238 to 9175) which covers the entire open reading frame (ORF) of the H protein of p(+)MV2A. p(+)MV323/EdH-EGFP is referred to as p(+)MV-EGFP-m8 in this paper. Chimeric constructs p(+)MV-EGFP-m9 and -m12 were generated by replacing SpeI-NotI and PmlI-NotI fragments, respectively, of the p(+)MV323-EGFP plasmid (the SpeI and PmlI sites exist in the IC-B sequence at nt 9175 and 14795, while the NotI site is just downstream of the ribozyme sequence by which the IC-B genome sequence is flanked [42]). p(+)MV-EGFP-m10 and p(+)MV-EGFP-m11 were generated by replacing SpeI-NheI (nt 9175 to 12216) and SpeI-PmlI (nt 9175 to 14795) fragments of p(+)MV323-EGFP with the corresponding fragments of p(+)MV2A, respectively.
Three amino acid substitutions (P64S, E89K, and A209T) found in the M protein of the Edmonston tag strain were introduced either independently or in combination into p(+)MV323-EGFP. To produce the P64S and E89K substitutions, nucleotide substitutions C to T at nt 3627 and G to A at nt 3702 were introduced into the p(+)MV323-EGFP plasmid by site-directed mutagenesis using the complementary primer pairs M-P64Sf (5'-TTGAGGACAGCGATTCCCTAGGGCCTCC-3') plus M-P64Sr (5'-GGAGGCCCTAGGGAATCGCTGTCCTCAA-3') and M-E89Kf (5'-AGCAAAACCCGAGAAACTCCTCAAAGAGG-3') plus M-E89Kr (5'-CCTCTTTGAGGAGTTTCTCGGGTTTTGCT-3'), respectively (substituted nucleotides are underlined), and KOD Plus DNA polymerase (Toyobo, Osaka, Japan). To introduce the A209T substitution, an EcoRI-EcoRI fragment (nt 3972 to 4383) of the p(+)MV323-EGFP plasmid was replaced with the corresponding fragment of p(+)MV2A. This fragment contains four synonymous substitutions in addition to the A209T substitution. The resulting plasmids having either the P64S, E89K, or A209T substitution were named p(+)MV-IC-EGFP-M/P64S, -M/E89K, and -M/A209T, respectively, and those having two of the three substitutions (P64S and E89K, P64S and A209T, and E89K and A209T) were named p(+)MV-IC-EGFP-M/P64S/E89K, -M/P64S/A209T, and -M/E89K/A209T, respectively. The plasmid having all three substitutions was named p(+)MV-IC-EGFP-M/P64S/E89K/A209T.
Virus titration. Monolayers of Vero/hSLAM cells on six-well cluster plates were infected with serially diluted virus samples and incubated for 1 h at 37°C. After the inoculum was removed, cells were washed with phosphate-buffered saline and overlaid with DMEM containing 5% FBS and 1% agarose. At 3 days postinfection (p.i.), PFU was determined by counting the number of plaques under a fluorescence microscope. Alternatively, monolayers of Vero/hSLAM cells on 24-well cluster plates were infected with 50 μl of serially diluted virus samples and incubated for 1 h at 37°C. After 1 h of incubation, 150 μl of DMEM supplemented with 7.5% FBS and 100 μg of the fusion block peptide Z-D-Phe-Phe-Gly (Peptide Institute Inc., Osaka, Japan) (34) per ml was added to each well to inhibit the second round of infection by progeny virions. At 36 h p.i., the number of EGFP-expressing cells was counted under a fluorescence microscope. The number was expressed as cell infectious units (CIU). CIU were comparable to PFU.
Replication kinetics. Vero/hSLAM and B95a cells on six-well cluster plates were infected with recombinant MVs at a multiplicity of infection (MOI) of 0.01 per cell. Vero cells on six-well cluster plates were infected with MVs at an MOI of 0.08 per cell. At various time intervals, cells were harvested with culture medium, and PFU were determined on Vero/hSLAM cells.
Immunoprecipitation. Monolayers of Vero/hSLAM cells on six-well cluster plates were infected with recombinant MVs. At 20 h p.i., the cells were cultured in methionine- and cysteine-deficient medium for 1 h. The cells were pulse-labeled with [35S]methionine-cysteine by using Promix 35S in vitro cell labeling mix (Amersham Biosciences, Piscataway, NJ) and then lysed in radioimmunoprecipitation assay buffer (0.15 mM NaCl, 0.05 mM Tris-HCl [pH 7.2], 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS]). Polypeptides in the cell lysate were immunoprecipitated with a rabbit polyclonal antibody raised against the MV Toyoshima stain (a gift from T. Kohama), separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and then detected with a Fuji BioImager 1000 (Fuji, Tokyo, Japan).
RESULTS
Construction and recovery of recombinant chimeric MVs. We previously generated an EGFP-expressing recombinant MV (IC323-EGFP) based on the IC-B strain isolated using B95a cells (12, 42). The recombinant Edmonston tag strain is derived largely from the Edmonston B vaccine strain, with some modifications (32). Since these strains belong to distinct genotypes, A and D3 (41) (the IC-B strain is referred to as 84-I in the paper), and have undergone many passages in laboratories, there exist 413 nucleotide (98 amino acid) differences in their genomes. In order to determine which genetic differences are responsible for the characteristic phenotype of the Edmonston strain, we generated recombinant chimeric MVs in which part of the IC323-EGFP genome was replaced with the corresponding sequences from the Edmonston tag strain (m1 to m9 in Fig. 1A). We exchanged individual genes (m7, m8, and m9) or sequences larger (m1, m4, m5, and m6) or smaller (m2 and m3) than single genes, depending on the availability of suitable restriction enzymes. When combined, the replacements covered the whole MV genome except for the 127 nt of the 3' genome terminus, in which three nucleotide differences between the two strains exist at nt 26 (T to A), 42 (C to A), and 50 (G to A). All chimeric MVs were successfully recovered and were propagated in B95a cells for further analysis. Since the H and M proteins had different molecular weights in the IC-B and Edmonston tag strains (44), their identities could be readily confirmed by SDS-PAGE. All chimeric MVs recovered had H and M proteins of the expected sizes (Fig. 1B and data not shown). All replacements of genome segments were further confirmed by using appropriate restriction enzymes or sequencing.
Contributions of individual genes of the Edmonston strain to efficient viral growth in cultured cells. We first examined cell entry efficiencies of chimeric viruses on SLAM-negative versus SLAM-positive cells (Fig. 2A). Vero and Vero/hSLAM cells were infected with serially diluted virus stocks. In order to prevent the second round of infection, a fusion block peptide was added to the medium following infection. CIU were determined at 36 h after infection by counting the number of EGFP-positive cells, and CIU on Vero cells were compared with those on Vero/hSLAM cells. The chimeric virus m8 entered Vero and Vero/hSLAM cells at almost the same efficiency, as it possessed the H protein of the Edmonston strain, which can use both SLAM and CD46 as receptors. As expected, the other chimeric viruses (m1 to m7 and m9) entered Vero cells much less efficiently, like IC323-EGFP, as reported previously (12).
We then studied growth kinetics of the chimeric viruses in Vero and Vero/hSLAM cells (Fig. 2B). In Vero/hSLAM cells, most chimeric viruses were comparable in replication to IC323-EGFP, while m4 and m6 reached very high titers (more than 107 PFU/ml at 3 days p.i.). In Vero cells, IC323-EGFP hardly replicated, but m8 grew as efficiently as in Vero/hSLAM cells, as reported previously (12, 44). m1, m2, m3, and m7 also replicated poorly, like IC323-EGFP. Surprisingly, m4, m5, and m6 replicated in Vero cells almost as efficiently as m8, despite the fact that they could not enter Vero cells by using CD46, unlike m8 (Fig. 2A). m9 also replicated in Vero cells better than IC323-EGFP but not as well as m4, m5, m6, and m8. Notably, m4, m5, and m6 possess in common the M gene from the Edmonston strain.
Various cell lines were infected with these recombinant viruses and examined for virus spread at 4 days p.i. (Fig. 2C) (data not shown for m2 and m3, as they showed almost the same phenotypes in all cell lines examined as m1; Vero/hSLAM was observed at 1 day p.i.). As expected, all viruses caused syncytia in SLAM-positive cells such as Vero/hSLAM and B95a cells. While m4 induced smaller and irregular syncytia in these cells, m5, m6, and m7 induced slightly larger syncytia than IC323-EGFP. In Vero cells, infection with IC323-EGFP, m1, m2, m3, or m7 led to some individual cells exhibiting green autofluorescence, although no syncytium formation was observed. A similar finding has been reported previously for IC323 and IC323-EGFP (12, 21, 44). m8 induced large syncytia, expanding to almost the entire monolayer of Vero cells. Unexpectedly, m5 and m6 also induced syncytia (m6 induced larger syncytia than m5), albeit to much lesser extent than m8. By contrast, m4 did not cause syncytia in Vero cells although it efficiently spread in the culture, and almost all cells were infected by 6 days p.i. It was notable that m9 also spread slowly in the Vero cell culture. In 293T cells (SLAM negative and CD46 positive), m5, m6, and m8 produced syncytia, although m5-induced syncytia were very small. Surprisingly, in RK13 cells (a rabbit kidney cell line), m6 and m7 induced syncytia, and m5 produced syncytia comprised of several cells, which readily detached from the plate. Although GFP-positive cells were not numerous initially because of these viruses' low entry efficiencies in RK13 cells, most of initially infected cells developed into syncytia after 4 days of incubation. Since m8 did not induce syncytia at all, CD46 is probably not involved in this cell-cell fusion observed in RK13 cells. In MDCK (dog kidney), chicken embryo fibroblast, CHO, and BHK (hamster kidney) cells, few productive infections were observed with all recombinant MVs (data not shown).
These data indicate that the M and L genes or trailer region of the Edmonston strain may allow recombinant viruses to grow efficiently in Vero and 293T cells and that its F gene may confer on recombinant viruses the ability to induce syncytia in RK13 cells.
Identification of amino acid residues in the M protein that are important for efficient viral growth in Vero cells. The recombinant viruses that contained the Edmonston M gene (m4, m5, and m6) replicated efficiently in Vero cells, even though they could not enter the cells by using an efficient cellular receptor such as SLAM or CD46 (Fig. 2). Although the involvement of the P and/or F gene in this finding cannot be completely ruled out, the M gene of the Edmonston strain appears to allow the recombinant viruses to replicate efficiently in Vero cells at a postentry step(s).
The IC-B and Edmonston tag M proteins differ at four amino acid positions (positions 64, 89, 175, and 209) (Fig. 3A). (We reexamined the Edmonston tag M gene sequence. There were two nucleotide differences from the GenBank sequence under accession number Z66517, one of which caused the amino acid substitution at amino acid position 64. The other was a synonymous substitution.) In order to determine whether any of these differences in the M protein are responsible for efficient replication of recombinant viruses in Vero cells, substitutions were introduced either singly or in combination into the IC323-EGFP genome (Fig. 3A). Since the glycine at position 175 (175G) present in the Edmonston tag stain is not found in other Edmonston lineage vaccine strains (28), we examined the residues at only three positions other than 175G. We designate these M protein mutants according to their mutated amino acid residues and positions. We studied the growth kinetics of these recombinant MVs in Vero cells (Fig. 3B). P64S apparently replicated better than IC323-EGFP, whereas E89K grew even more efficiently. A209T replicated very poorly, like IC323-EGFP. The recombinant virus having both P64S and E89K substitutions replicated as efficiently as m4. The addition of the A209T substitution did not affect growth kinetics of recombinant viruses (P64S/A209T, E89K/A209T, and P64S/E89K/A209T). These results indicate that the Edmonston M protein is indeed (at least in part) responsible for the ability of m4, m5, and m6 to replicate efficiently in Vero cells. The E89K substitution in the M protein facilitates viral replication in Vero cells without much affecting entry efficiency, and the P64S substitution also does so to a lesser degree.
Vero and B95a cells were infected with the recombinant viruses having mutated M proteins and examined for virus spread at 4 days p.i. (Fig. 3C). In Vero cells, which these recombinant viruses could not enter using SLAM or CD46 as a receptor, P64S and E89K spread in the culture without producing syncytia. The virus having both substitutions (P64S/E89K) spread more extensively in Vero cells, while A209T behaved like IC323-EGFP. These results were consistent with the data in Fig. 3B. In B95a cells, P64S induced slightly smaller syncytia than IC323-EGFP. E89K produced only very small syncytia, and P64S/E89K did not induce apparent syncytia. Nevertheless, E89K and P64S/E89K disseminated very efficiently in the culture. Thus, under conditions in which recombinant viruses could enter cells efficiently using SLAM, those possessing the Edmonston M and F proteins (m5 and m6), but not those possessing the Edmonston-type M protein and the IC-B F protein (m4, E89K, and P64S/E89K), induced large syncytia (Fig. 2C and Fig. 3C). Nevertheless, it seems that the latter still spread efficiently without much producing cell-cell fusion.
Region of the L gene responsible for efficient growth in Vero cells. To determine which region(s) of the Edmonston L gene or 5' trailer sequence allows better replication in Vero cells, we generated recombinant viruses having the chimeric L gene or trailer sequence by using conveniently located restriction enzyme sites (m10 to m12 in Fig. 4A). These recombinant viruses were examined for their growth in Vero cells. The recombinant m10 and m11 viruses replicated as efficiently as m9 having the Edmonston L gene, while m12 grew poorly like IC323-EGFP (Fig. 4B). These data indicate that the first half (defined by the SpeI-NheI fragment) of the Edmonston L gene, but not the trailer sequence or adjacent untranslated region of the L gene, is responsible for better replication in Vero cells. Sixteen amino acid differences were found in the first half of the L protein between the IC-B and Edmonston tag strains (Table 1).
Growth of recombinant viruses in B95a cells. The above data show that the Edmonston M and L genes confer on viruses the ability to replicate efficiently in Vero cells. To learn how those viruses which have acquired a growth advantage in Vero cells would replicate in lymphoid cells, we studied growth kinetics of IC323-EGFP, m9, and P64S/E89K in B95a cells (Fig. 5). Both m9 and P64S/E89K replicated more slowly than IC323-EGFP, indicating that better replication in Vero cells due to substitutions in the M and L genes results in less efficient growth in B95a cells.
DISCUSSION
The Edmonston strain, the prototype MV, is known to grow well in most primate cell lines. We here studied contributions of individual genes of the Edmonston strain to its growth in cultured cells by generating and characterizing recombinant chimeric viruses based on the wild-type IC-B strain, which has more restricted tropism (16, 42). The recombinant virus with the Edmonston H gene encoding the receptor-binding protein grew efficiently in SLAM-negative primate cell lines, as it could utilize ubiquitously expressed CD46 as a receptor. Unexpectedly, the recombinant viruses bearing the Edmonston M or L gene also replicated well in Vero cells, although they could not enter the cells efficiently like the parental IC-B strain. Thus, our results clearly indicate that not only the H gene but also the M and L genes contribute to efficient replication of the Edmonston strain in cultured cells.
There were four amino acid differences between the M proteins of the IC-B and Edmonston tag strains. We demonstrated that the P64S and E89K substitutions were critical for the ability of the Edmonston M protein to allow virus to grow efficiently in Vero cells. The nucleotide sequences of protein-coding regions from an early-passage laboratory isolate of the original Edmonston virus (the Edmonston "wild type") and five vaccine strains have been determined (28). Ten coding changes have been identified in the M genes of the vaccine strains compared with that of the Edmonston "wild-type" strain. One of the two changes common to all vaccine strains was the E89K substitution, thus reinforcing the validity of our finding. At position 64 in the M protein, the Edmonston "wild type" and two vaccine strains have proline like the IC-B strain, whereas three other vaccine strains have serine at that position (28). Furthermore, Takeuchi et al. reported that there were two nucleotide differences in the P/C/V and M genes between B95a cell- and Vero cell-isolated MVs from the same patient (43). They further showed that the P64H substitution in the M protein was important for efficient virus growth in Vero cells (21). These results are consistent with the present observation that the residue at position 64 is important for efficient growth in Vero cells. Previous studies have suggested that the M protein plays an important role in MV assembly (4, 36), regulation of membrane fusion (4), and transcription (38). In order to determine how the P64S and E89K substitutions confer on virus the growth advantage in cultured cells, we are currently examining the replication efficiency of the recombinant virus having those substitutions in the M protein at different steps of virus life cycle.
While all recombinant viruses possessing the Edmonston M gene (m4 to m6) replicated efficiently in Vero cells, only m6, and not m4 or m5, grew well in 293T cells (Fig. 2C). Unlike 293T cells, Vero cells cannot produce interferons upon virus infection because of a genetic defect (8). m4 and m5 possessed the P gene, in addition to the M gene, from the Edmonston tag strain (the only difference between m5 and m6 is the presence of the Edmonston P gene sequence in the former). We have previously shown that the V protein (encoded within the P gene) of the Edmonston tag strain does not exhibit interferon antagonist activity because of inadvertent mutations (25). Thus, it seemed that m4 and m5 failed to grow in 293 T cells because of their inability to counteract interferon action.
We demonstrated that the substitutions in the first half of the L gene found in the Edmonston strain compared with the IC-B strain were also advantageous for growth in Vero cells. This is consistent with the previous finding that the L proteins of MV vaccine strains exhibit higher transcription and replication activity than those of wild-type MVs in monkey kidney-derived CV-1 cells, as revealed by a minigenome reporter assay (2). Homologous domains exist among various RNA-dependent RNA polymerases, including the paramyxovirus L proteins (31). It has been proposed that these regions (RNA-dependent RNA polymerase domains I to VI) represent functional domains (31). The first half of the MV L protein contains domain I to III, which are responsible for P protein binding, RNA binding, and catalytic activity (6, 13, 28, 31), respectively. Only one amino acid coding change has been found in the first half of the L protein from the Edmonston lineage vaccine strains compared with that of the Edmonston "wild-type" strain (28), which is not the same as any of the differences between the IC-B and Edmonston tag strains. It is possible that the substitutions in the first half of the L protein found in the Edmonston strain confers on virus better RNA polymerase activity in cultured cells, thus allowing virus to replicate more efficiently.
Notably, among the recombinant viruses possessing the Edmonston M gene, m5 and m6 (both having the Edmonston F gene) replicated more efficiently in Vero cells than m4 (having the IC-B F gene) (Fig. 2B). Furthermore, m5 and m6 produced syncytia in Vero cells, while m4 did not (Fig. 2C). In SLAM-positive cells (Vero/hSLAM and B95a), recombinant viruses having the Edmonston F gene (m5 to m7) tended to produce larger syncytia (Fig. 2C). Since the reported sequence data for the infectious cDNA clone of the Edmonston B strain (Edmonston tag strain) (32) used in this study (GenBank accession number Z66517) show no amino acid difference in the F protein from that of the IC-B strain (43) (accession number NC_001498), we reexamined the Edmonston tag F gene sequence and found the M94V substitution in its F protein sequence. These results therefore suggest that when expressed in association with the IC-B H protein, the Edmonston tag F protein (94V) is more efficient in inducing cell-cell fusion than the IC-B F protein (94M), consistent with previous studies (3, 5, 15, 29, 30, 33).
In this study, we did not assess the effect of the three nucleotide differences found in the 3' leader sequence on growth in cultured cells. Bankamp et al. suggested that the two nucleotide substitutions at positions 26 and 42 in the Edmonston vaccine strains induced better transcription and genome replication in a minigenome reporter assay (2). Detailed analysis of the function of the leader sequence is now in progress.
The vaccine strains derived from the original Edmonston virus (which is no longer available) must have undergone many mutations during passages in a variety of human, chicken, and other cells, having adapted to efficient growth in them (27, 28). Since these cells did not express the authentic MV receptor SLAM, viruses may have come to use alternate receptors such as CD46 by introducing changes in the H gene. In fact, all vaccine strains whose genomic sequences have been determined, as well as the Edmonston "wild-type" strain, have the changes (481Y and/or 546G) in the H protein that allow virus to bind CD46 (19, 20, 28, 47). However, as our present study shows, even without those mutations that enable virus to enter cells efficiently, virus may be able to grow well in SLAM-negative cells by introducing changes in genes other than the H gene. Thus, when viruses were isolated in SLAM-negative cells (21, 43) or when viruses first isolated in SLAM-positive B95a cells were subsequently adapted to growth in SLAM-negative cells (18, 24, 37, 39), they may not have had any mutation in the H gene but instead possessed mutations in other genes, including the M and L genes.
ACKNOWLEDGMENTS
We thank M. A. Billeter, B. Moss, and T. Kohama for providing reagents. We are grateful to K. Takeuchi for his contribution to the establishment of the reverse genetics of the IC-B strain.
This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology and the Ministry of Health, Labor and Welfare of Japan; from the Japan Society for the Promotion of Science; and from the Takeda Science Foundation.
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ABSTRACT
The Edmonston strain of measles virus (MV) was obtained by sequential passages of the original isolate in various cultured cells. Although attenuated in vivo, it grows efficiently in most primate cell lines. Previous studies have revealed that MV tropism cannot be solely explained by the use of CD150 and/or CD46 as a cellular receptor. In order to evaluate the contributions of individual genes of the Edmonston strain to growth in cultured cells, we generated a series of recombinant viruses in which part of the genome of the clinical isolate IC-B (which uses CD150 as a receptor) was replaced with the corresponding sequences of the Edmonston strain. The recombinant virus possessing the Edmonston hemagglutinin (H) gene (encoding the receptor-binding protein) grew as efficiently in Vero cells as the Edmonston strain. Those viruses having either the matrix (M) or large (L) protein gene from the Edmonston strain could also replicate well in Vero cells, although they entered them at low efficiencies. P64S and E89K substitutions were responsible for the ability of the M protein to make virus grow efficiently in Vero cells, while the first half of the Edmonston L gene was important for better replication. Despite efficient growth in Vero cells, the recombinant viruses with these mutations had growth disadvantage in CD150-positive lymphoid B95a cells. Thus, not only the H gene but also the M and L genes contribute to efficient replication of the Edmonston strain in some cultured cells.
INTRODUCTION
Measles virus (MV), a member of the genus Morbillivirus of the family Paramyxoviridae, is an enveloped virus with a nonsegmented negative-strand RNA genome. The MV genome has six genes that encode the nucleocapsid (N), phospho- (P), matrix (M), fusion (F), hemagglutinin (H), and large (L) proteins. The P gene encodes additional accessory proteins, the V and C proteins, by a process of RNA editing and by an alternative translational initiation in a different reading frame, respectively (11).
MV was first isolated in a primary culture of human kidney cells inoculated with a sample from a child with measles (9). This isolate, the original Edmonston strain, was subsequently adapted to a variety of cultured cells, giving rise to currently used live vaccines (35). The Edmonston strain grows well in continuous cell lines, including Vero cells derived from the African green monkey kidney, and has become the most extensively studied MV strain in laboratories. In 1990, Kobune etal. reported that the marmoset B-lymphoid cell line B95a is highly sensitive to MV, allowing efficient MV isolation from clinical specimens (16). Whereas the Edmonston strain replicates efficiently in almost any primate cell line, B95a cell-isolated strains were found to grow in a restricted number of cell lines (16, 45). This riddle was resolved when it was demonstrated that the signaling lymphocyte activation molecule (SLAM) (also called CD150) acts as a cellular receptor for MV (10, 14, 46, 48). SLAM is expressed on certain types of cells of the immune system (23). On the other hand, besides SLAM, the Edmonston strain can use as a receptor CD46, which is expressed on all nucleated cells (7, 22, 46). Thus, the ubiquitous expression of CD46 appears to explain the ability of the Edmonston strain to replicate in most primate cell lines.
However, there have been reports that the receptor usage cannot readily explain MV cell tropism. The recombinant Edmonston MV expressing the H protein of the B cell-isolated WTF strain spread in Vero cells, although the parental WTF strain did not (15). Since the H protein is responsible for the receptor binding, this finding suggests that cell tropism of therecombinant virus may be determined at a level other than the H protein-receptor interaction. Takeuchi et al. reported a similar finding (44). Furthermore, an MV strain, isolated in peripheral blood mononuclear cells, which uses SLAM but not CD46 as a receptor was successfully adapted to Vero cells without the acquisition of the ability to interact with CD46 (18). Takeuchi et al. also reported that there was no sequence difference in the H gene between MV strains isolated in B95a and in Vero cells from the same measles patient, although they exhibited distinct tropisms (43). We and others have previously demonstrated the presence of SLAM- and CD46-independent cell entry of MV (1, 12). However, as its efficiency is 2 to 3 log units lower than that of SLAM- or CD46-dependent entry, this inefficient entry can only partly account for the above observations. Thus, there must be some mechanism(s) which allows virus to replicate efficiently at a postentry step(s) even when it enters cells inefficiently.
In an attempt to determine the roles of individual genes of the Edmonston strain in growth in cultured cells, we here generated a series of recombinant MVs in which part of the genome of the B95a cell-isolated IC-B strain (16, 17, 42) was replaced with the corresponding sequences of the Edmonston strain (32). Our results showed that the M and L genes, in addition to the H gene, are important for efficient replication of the Edmonston strain in Vero cells. We have also determined the amino acid residues of the Edmonston M protein and the region of the L gene that are responsible for efficient growth in Vero cells.
MATERIALS AND METHODS
Cells and viruses. Vero, 293T (a human embryonic fibroblast cell line), and RK13 (a rabbit kidney cell line) cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (ICN Biomedicals, Aurora, Ohio) supplemented with 7.5% fetal bovine serum (FBS). B95a cells (a marmoset B-cell line transformed with Epstein-Barr virus) (16) were maintained in RPMI 1640 medium (ICN Biomedicals) supplemented with 10% FBS. Vero cells constitutively expressing human SLAM (Vero/hSLAM) (26) were maintained in DMEM supplemented with 7.5% FBS and 500 μg of Geneticin (G418) (Nacalai Tesque, Tokyo, Japan) per ml. Chinese hamster ovary (CHO) cells constitutively expressing human SLAM (CHO/hSLAM) (46) were maintained in RPMI 1640 medium supplemented with 7.5% FBS and 500 μg of G418 per ml. Recombinant MVs were generated from cDNAs by using CHO/hSLAM cells and the vaccinia virus encoding T7 RNA polymerase, vTF7-3 (a gift from B. Moss), as reported previously (40). Generated MVs were propagated in B95a cells, and virus stocks at two or three passages in B95a cells were used for experiments.
Construction of full-length cDNAs of recombinant virus genomes. The plasmid p(+)MV323-EGFP, which encodes the full-length antigenomic cDNA of the B95a-isolated IC-B strain (16, 17, 42) with an additional transcriptional unit of the enhanced green fluorescent protein (EGFP), was described previously (12). The plasmid p(+)MV2A (a gift from M. A. Billeter) encodes the full-length antigenomic cDNA of the Edmonston B vaccine strain with some modifications (GenBank accession number Z66517) (32). The rescued virus from p(+)MV2A is referred to as the Edmonston tag strain. By using appropriate restriction enzymes, a series of the genomic regions of p(+)MV323-EGFP were replaced with the corresponding regions of p(+)MV2A, generating 12 plasmids carrying the full-length genomes of recombinant chimeric viruses. The chimeric construct p(+)MV-EGFP-m1 was generated by replacing the BlpI-BlpI fragment (nucleotides [nt] 127 to 3146) containing most of the N and P genes of p(+)MV323-EGFP with the corresponding fragment of p(+)MV2A (Fig. 1). In this paper, nucleotide position numbers are shown in accordance with the sequence of the parental IC-B strain genome (42, 43) (GenBank accession number NC_001498). The SapI-SapI fragment [the first SapI site exists just upstream of the T7 promoter sequence of the pBluescriptII-KS(+) vector (42) and the second at nt 1813 in the IC-B sequence] was exchanged between p(+)MV323-EGFP and p(+)MV-EGFP-m1, generating two chimeric constructs, p(+)MV-EGFP-m2 and p(+)MV-EGFP-m3. Thus, p(+)MV-EGFP-m2 and p(+)MV-EGFP-m3 contain the BlpI-SapI fragment (nt 127 to 1813) and SapI-BlpI fragment (nt 1813 to 3146) of p(+)MV2A, respectively. Chimeric constructs p(+)MV-EGFP-m4, p(+)MV-EGFP-m5, p(+)MV-EGFP-m6, and p(+)MV-EGFP-m7 were generated by replacing SacII-BsmBI (nt 2040 to 5475), SacII-PacI (nt 2040 to 7238), BlpI-PacI (nt 3146 to 7238), and BstEII-PacI (nt 4807 to 7238) fragments of p(+)MV323-EGFP with the corresponding fragments of p(+)MV2A, respectively. The plasmid p(+)MV323/EdH-EGFP was described previously (12) and contains a PacI-SpeI fragment (nt 7238 to 9175) which covers the entire open reading frame (ORF) of the H protein of p(+)MV2A. p(+)MV323/EdH-EGFP is referred to as p(+)MV-EGFP-m8 in this paper. Chimeric constructs p(+)MV-EGFP-m9 and -m12 were generated by replacing SpeI-NotI and PmlI-NotI fragments, respectively, of the p(+)MV323-EGFP plasmid (the SpeI and PmlI sites exist in the IC-B sequence at nt 9175 and 14795, while the NotI site is just downstream of the ribozyme sequence by which the IC-B genome sequence is flanked [42]). p(+)MV-EGFP-m10 and p(+)MV-EGFP-m11 were generated by replacing SpeI-NheI (nt 9175 to 12216) and SpeI-PmlI (nt 9175 to 14795) fragments of p(+)MV323-EGFP with the corresponding fragments of p(+)MV2A, respectively.
Three amino acid substitutions (P64S, E89K, and A209T) found in the M protein of the Edmonston tag strain were introduced either independently or in combination into p(+)MV323-EGFP. To produce the P64S and E89K substitutions, nucleotide substitutions C to T at nt 3627 and G to A at nt 3702 were introduced into the p(+)MV323-EGFP plasmid by site-directed mutagenesis using the complementary primer pairs M-P64Sf (5'-TTGAGGACAGCGATTCCCTAGGGCCTCC-3') plus M-P64Sr (5'-GGAGGCCCTAGGGAATCGCTGTCCTCAA-3') and M-E89Kf (5'-AGCAAAACCCGAGAAACTCCTCAAAGAGG-3') plus M-E89Kr (5'-CCTCTTTGAGGAGTTTCTCGGGTTTTGCT-3'), respectively (substituted nucleotides are underlined), and KOD Plus DNA polymerase (Toyobo, Osaka, Japan). To introduce the A209T substitution, an EcoRI-EcoRI fragment (nt 3972 to 4383) of the p(+)MV323-EGFP plasmid was replaced with the corresponding fragment of p(+)MV2A. This fragment contains four synonymous substitutions in addition to the A209T substitution. The resulting plasmids having either the P64S, E89K, or A209T substitution were named p(+)MV-IC-EGFP-M/P64S, -M/E89K, and -M/A209T, respectively, and those having two of the three substitutions (P64S and E89K, P64S and A209T, and E89K and A209T) were named p(+)MV-IC-EGFP-M/P64S/E89K, -M/P64S/A209T, and -M/E89K/A209T, respectively. The plasmid having all three substitutions was named p(+)MV-IC-EGFP-M/P64S/E89K/A209T.
Virus titration. Monolayers of Vero/hSLAM cells on six-well cluster plates were infected with serially diluted virus samples and incubated for 1 h at 37°C. After the inoculum was removed, cells were washed with phosphate-buffered saline and overlaid with DMEM containing 5% FBS and 1% agarose. At 3 days postinfection (p.i.), PFU was determined by counting the number of plaques under a fluorescence microscope. Alternatively, monolayers of Vero/hSLAM cells on 24-well cluster plates were infected with 50 μl of serially diluted virus samples and incubated for 1 h at 37°C. After 1 h of incubation, 150 μl of DMEM supplemented with 7.5% FBS and 100 μg of the fusion block peptide Z-D-Phe-Phe-Gly (Peptide Institute Inc., Osaka, Japan) (34) per ml was added to each well to inhibit the second round of infection by progeny virions. At 36 h p.i., the number of EGFP-expressing cells was counted under a fluorescence microscope. The number was expressed as cell infectious units (CIU). CIU were comparable to PFU.
Replication kinetics. Vero/hSLAM and B95a cells on six-well cluster plates were infected with recombinant MVs at a multiplicity of infection (MOI) of 0.01 per cell. Vero cells on six-well cluster plates were infected with MVs at an MOI of 0.08 per cell. At various time intervals, cells were harvested with culture medium, and PFU were determined on Vero/hSLAM cells.
Immunoprecipitation. Monolayers of Vero/hSLAM cells on six-well cluster plates were infected with recombinant MVs. At 20 h p.i., the cells were cultured in methionine- and cysteine-deficient medium for 1 h. The cells were pulse-labeled with [35S]methionine-cysteine by using Promix 35S in vitro cell labeling mix (Amersham Biosciences, Piscataway, NJ) and then lysed in radioimmunoprecipitation assay buffer (0.15 mM NaCl, 0.05 mM Tris-HCl [pH 7.2], 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS]). Polypeptides in the cell lysate were immunoprecipitated with a rabbit polyclonal antibody raised against the MV Toyoshima stain (a gift from T. Kohama), separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and then detected with a Fuji BioImager 1000 (Fuji, Tokyo, Japan).
RESULTS
Construction and recovery of recombinant chimeric MVs. We previously generated an EGFP-expressing recombinant MV (IC323-EGFP) based on the IC-B strain isolated using B95a cells (12, 42). The recombinant Edmonston tag strain is derived largely from the Edmonston B vaccine strain, with some modifications (32). Since these strains belong to distinct genotypes, A and D3 (41) (the IC-B strain is referred to as 84-I in the paper), and have undergone many passages in laboratories, there exist 413 nucleotide (98 amino acid) differences in their genomes. In order to determine which genetic differences are responsible for the characteristic phenotype of the Edmonston strain, we generated recombinant chimeric MVs in which part of the IC323-EGFP genome was replaced with the corresponding sequences from the Edmonston tag strain (m1 to m9 in Fig. 1A). We exchanged individual genes (m7, m8, and m9) or sequences larger (m1, m4, m5, and m6) or smaller (m2 and m3) than single genes, depending on the availability of suitable restriction enzymes. When combined, the replacements covered the whole MV genome except for the 127 nt of the 3' genome terminus, in which three nucleotide differences between the two strains exist at nt 26 (T to A), 42 (C to A), and 50 (G to A). All chimeric MVs were successfully recovered and were propagated in B95a cells for further analysis. Since the H and M proteins had different molecular weights in the IC-B and Edmonston tag strains (44), their identities could be readily confirmed by SDS-PAGE. All chimeric MVs recovered had H and M proteins of the expected sizes (Fig. 1B and data not shown). All replacements of genome segments were further confirmed by using appropriate restriction enzymes or sequencing.
Contributions of individual genes of the Edmonston strain to efficient viral growth in cultured cells. We first examined cell entry efficiencies of chimeric viruses on SLAM-negative versus SLAM-positive cells (Fig. 2A). Vero and Vero/hSLAM cells were infected with serially diluted virus stocks. In order to prevent the second round of infection, a fusion block peptide was added to the medium following infection. CIU were determined at 36 h after infection by counting the number of EGFP-positive cells, and CIU on Vero cells were compared with those on Vero/hSLAM cells. The chimeric virus m8 entered Vero and Vero/hSLAM cells at almost the same efficiency, as it possessed the H protein of the Edmonston strain, which can use both SLAM and CD46 as receptors. As expected, the other chimeric viruses (m1 to m7 and m9) entered Vero cells much less efficiently, like IC323-EGFP, as reported previously (12).
We then studied growth kinetics of the chimeric viruses in Vero and Vero/hSLAM cells (Fig. 2B). In Vero/hSLAM cells, most chimeric viruses were comparable in replication to IC323-EGFP, while m4 and m6 reached very high titers (more than 107 PFU/ml at 3 days p.i.). In Vero cells, IC323-EGFP hardly replicated, but m8 grew as efficiently as in Vero/hSLAM cells, as reported previously (12, 44). m1, m2, m3, and m7 also replicated poorly, like IC323-EGFP. Surprisingly, m4, m5, and m6 replicated in Vero cells almost as efficiently as m8, despite the fact that they could not enter Vero cells by using CD46, unlike m8 (Fig. 2A). m9 also replicated in Vero cells better than IC323-EGFP but not as well as m4, m5, m6, and m8. Notably, m4, m5, and m6 possess in common the M gene from the Edmonston strain.
Various cell lines were infected with these recombinant viruses and examined for virus spread at 4 days p.i. (Fig. 2C) (data not shown for m2 and m3, as they showed almost the same phenotypes in all cell lines examined as m1; Vero/hSLAM was observed at 1 day p.i.). As expected, all viruses caused syncytia in SLAM-positive cells such as Vero/hSLAM and B95a cells. While m4 induced smaller and irregular syncytia in these cells, m5, m6, and m7 induced slightly larger syncytia than IC323-EGFP. In Vero cells, infection with IC323-EGFP, m1, m2, m3, or m7 led to some individual cells exhibiting green autofluorescence, although no syncytium formation was observed. A similar finding has been reported previously for IC323 and IC323-EGFP (12, 21, 44). m8 induced large syncytia, expanding to almost the entire monolayer of Vero cells. Unexpectedly, m5 and m6 also induced syncytia (m6 induced larger syncytia than m5), albeit to much lesser extent than m8. By contrast, m4 did not cause syncytia in Vero cells although it efficiently spread in the culture, and almost all cells were infected by 6 days p.i. It was notable that m9 also spread slowly in the Vero cell culture. In 293T cells (SLAM negative and CD46 positive), m5, m6, and m8 produced syncytia, although m5-induced syncytia were very small. Surprisingly, in RK13 cells (a rabbit kidney cell line), m6 and m7 induced syncytia, and m5 produced syncytia comprised of several cells, which readily detached from the plate. Although GFP-positive cells were not numerous initially because of these viruses' low entry efficiencies in RK13 cells, most of initially infected cells developed into syncytia after 4 days of incubation. Since m8 did not induce syncytia at all, CD46 is probably not involved in this cell-cell fusion observed in RK13 cells. In MDCK (dog kidney), chicken embryo fibroblast, CHO, and BHK (hamster kidney) cells, few productive infections were observed with all recombinant MVs (data not shown).
These data indicate that the M and L genes or trailer region of the Edmonston strain may allow recombinant viruses to grow efficiently in Vero and 293T cells and that its F gene may confer on recombinant viruses the ability to induce syncytia in RK13 cells.
Identification of amino acid residues in the M protein that are important for efficient viral growth in Vero cells. The recombinant viruses that contained the Edmonston M gene (m4, m5, and m6) replicated efficiently in Vero cells, even though they could not enter the cells by using an efficient cellular receptor such as SLAM or CD46 (Fig. 2). Although the involvement of the P and/or F gene in this finding cannot be completely ruled out, the M gene of the Edmonston strain appears to allow the recombinant viruses to replicate efficiently in Vero cells at a postentry step(s).
The IC-B and Edmonston tag M proteins differ at four amino acid positions (positions 64, 89, 175, and 209) (Fig. 3A). (We reexamined the Edmonston tag M gene sequence. There were two nucleotide differences from the GenBank sequence under accession number Z66517, one of which caused the amino acid substitution at amino acid position 64. The other was a synonymous substitution.) In order to determine whether any of these differences in the M protein are responsible for efficient replication of recombinant viruses in Vero cells, substitutions were introduced either singly or in combination into the IC323-EGFP genome (Fig. 3A). Since the glycine at position 175 (175G) present in the Edmonston tag stain is not found in other Edmonston lineage vaccine strains (28), we examined the residues at only three positions other than 175G. We designate these M protein mutants according to their mutated amino acid residues and positions. We studied the growth kinetics of these recombinant MVs in Vero cells (Fig. 3B). P64S apparently replicated better than IC323-EGFP, whereas E89K grew even more efficiently. A209T replicated very poorly, like IC323-EGFP. The recombinant virus having both P64S and E89K substitutions replicated as efficiently as m4. The addition of the A209T substitution did not affect growth kinetics of recombinant viruses (P64S/A209T, E89K/A209T, and P64S/E89K/A209T). These results indicate that the Edmonston M protein is indeed (at least in part) responsible for the ability of m4, m5, and m6 to replicate efficiently in Vero cells. The E89K substitution in the M protein facilitates viral replication in Vero cells without much affecting entry efficiency, and the P64S substitution also does so to a lesser degree.
Vero and B95a cells were infected with the recombinant viruses having mutated M proteins and examined for virus spread at 4 days p.i. (Fig. 3C). In Vero cells, which these recombinant viruses could not enter using SLAM or CD46 as a receptor, P64S and E89K spread in the culture without producing syncytia. The virus having both substitutions (P64S/E89K) spread more extensively in Vero cells, while A209T behaved like IC323-EGFP. These results were consistent with the data in Fig. 3B. In B95a cells, P64S induced slightly smaller syncytia than IC323-EGFP. E89K produced only very small syncytia, and P64S/E89K did not induce apparent syncytia. Nevertheless, E89K and P64S/E89K disseminated very efficiently in the culture. Thus, under conditions in which recombinant viruses could enter cells efficiently using SLAM, those possessing the Edmonston M and F proteins (m5 and m6), but not those possessing the Edmonston-type M protein and the IC-B F protein (m4, E89K, and P64S/E89K), induced large syncytia (Fig. 2C and Fig. 3C). Nevertheless, it seems that the latter still spread efficiently without much producing cell-cell fusion.
Region of the L gene responsible for efficient growth in Vero cells. To determine which region(s) of the Edmonston L gene or 5' trailer sequence allows better replication in Vero cells, we generated recombinant viruses having the chimeric L gene or trailer sequence by using conveniently located restriction enzyme sites (m10 to m12 in Fig. 4A). These recombinant viruses were examined for their growth in Vero cells. The recombinant m10 and m11 viruses replicated as efficiently as m9 having the Edmonston L gene, while m12 grew poorly like IC323-EGFP (Fig. 4B). These data indicate that the first half (defined by the SpeI-NheI fragment) of the Edmonston L gene, but not the trailer sequence or adjacent untranslated region of the L gene, is responsible for better replication in Vero cells. Sixteen amino acid differences were found in the first half of the L protein between the IC-B and Edmonston tag strains (Table 1).
Growth of recombinant viruses in B95a cells. The above data show that the Edmonston M and L genes confer on viruses the ability to replicate efficiently in Vero cells. To learn how those viruses which have acquired a growth advantage in Vero cells would replicate in lymphoid cells, we studied growth kinetics of IC323-EGFP, m9, and P64S/E89K in B95a cells (Fig. 5). Both m9 and P64S/E89K replicated more slowly than IC323-EGFP, indicating that better replication in Vero cells due to substitutions in the M and L genes results in less efficient growth in B95a cells.
DISCUSSION
The Edmonston strain, the prototype MV, is known to grow well in most primate cell lines. We here studied contributions of individual genes of the Edmonston strain to its growth in cultured cells by generating and characterizing recombinant chimeric viruses based on the wild-type IC-B strain, which has more restricted tropism (16, 42). The recombinant virus with the Edmonston H gene encoding the receptor-binding protein grew efficiently in SLAM-negative primate cell lines, as it could utilize ubiquitously expressed CD46 as a receptor. Unexpectedly, the recombinant viruses bearing the Edmonston M or L gene also replicated well in Vero cells, although they could not enter the cells efficiently like the parental IC-B strain. Thus, our results clearly indicate that not only the H gene but also the M and L genes contribute to efficient replication of the Edmonston strain in cultured cells.
There were four amino acid differences between the M proteins of the IC-B and Edmonston tag strains. We demonstrated that the P64S and E89K substitutions were critical for the ability of the Edmonston M protein to allow virus to grow efficiently in Vero cells. The nucleotide sequences of protein-coding regions from an early-passage laboratory isolate of the original Edmonston virus (the Edmonston "wild type") and five vaccine strains have been determined (28). Ten coding changes have been identified in the M genes of the vaccine strains compared with that of the Edmonston "wild-type" strain. One of the two changes common to all vaccine strains was the E89K substitution, thus reinforcing the validity of our finding. At position 64 in the M protein, the Edmonston "wild type" and two vaccine strains have proline like the IC-B strain, whereas three other vaccine strains have serine at that position (28). Furthermore, Takeuchi et al. reported that there were two nucleotide differences in the P/C/V and M genes between B95a cell- and Vero cell-isolated MVs from the same patient (43). They further showed that the P64H substitution in the M protein was important for efficient virus growth in Vero cells (21). These results are consistent with the present observation that the residue at position 64 is important for efficient growth in Vero cells. Previous studies have suggested that the M protein plays an important role in MV assembly (4, 36), regulation of membrane fusion (4), and transcription (38). In order to determine how the P64S and E89K substitutions confer on virus the growth advantage in cultured cells, we are currently examining the replication efficiency of the recombinant virus having those substitutions in the M protein at different steps of virus life cycle.
While all recombinant viruses possessing the Edmonston M gene (m4 to m6) replicated efficiently in Vero cells, only m6, and not m4 or m5, grew well in 293T cells (Fig. 2C). Unlike 293T cells, Vero cells cannot produce interferons upon virus infection because of a genetic defect (8). m4 and m5 possessed the P gene, in addition to the M gene, from the Edmonston tag strain (the only difference between m5 and m6 is the presence of the Edmonston P gene sequence in the former). We have previously shown that the V protein (encoded within the P gene) of the Edmonston tag strain does not exhibit interferon antagonist activity because of inadvertent mutations (25). Thus, it seemed that m4 and m5 failed to grow in 293 T cells because of their inability to counteract interferon action.
We demonstrated that the substitutions in the first half of the L gene found in the Edmonston strain compared with the IC-B strain were also advantageous for growth in Vero cells. This is consistent with the previous finding that the L proteins of MV vaccine strains exhibit higher transcription and replication activity than those of wild-type MVs in monkey kidney-derived CV-1 cells, as revealed by a minigenome reporter assay (2). Homologous domains exist among various RNA-dependent RNA polymerases, including the paramyxovirus L proteins (31). It has been proposed that these regions (RNA-dependent RNA polymerase domains I to VI) represent functional domains (31). The first half of the MV L protein contains domain I to III, which are responsible for P protein binding, RNA binding, and catalytic activity (6, 13, 28, 31), respectively. Only one amino acid coding change has been found in the first half of the L protein from the Edmonston lineage vaccine strains compared with that of the Edmonston "wild-type" strain (28), which is not the same as any of the differences between the IC-B and Edmonston tag strains. It is possible that the substitutions in the first half of the L protein found in the Edmonston strain confers on virus better RNA polymerase activity in cultured cells, thus allowing virus to replicate more efficiently.
Notably, among the recombinant viruses possessing the Edmonston M gene, m5 and m6 (both having the Edmonston F gene) replicated more efficiently in Vero cells than m4 (having the IC-B F gene) (Fig. 2B). Furthermore, m5 and m6 produced syncytia in Vero cells, while m4 did not (Fig. 2C). In SLAM-positive cells (Vero/hSLAM and B95a), recombinant viruses having the Edmonston F gene (m5 to m7) tended to produce larger syncytia (Fig. 2C). Since the reported sequence data for the infectious cDNA clone of the Edmonston B strain (Edmonston tag strain) (32) used in this study (GenBank accession number Z66517) show no amino acid difference in the F protein from that of the IC-B strain (43) (accession number NC_001498), we reexamined the Edmonston tag F gene sequence and found the M94V substitution in its F protein sequence. These results therefore suggest that when expressed in association with the IC-B H protein, the Edmonston tag F protein (94V) is more efficient in inducing cell-cell fusion than the IC-B F protein (94M), consistent with previous studies (3, 5, 15, 29, 30, 33).
In this study, we did not assess the effect of the three nucleotide differences found in the 3' leader sequence on growth in cultured cells. Bankamp et al. suggested that the two nucleotide substitutions at positions 26 and 42 in the Edmonston vaccine strains induced better transcription and genome replication in a minigenome reporter assay (2). Detailed analysis of the function of the leader sequence is now in progress.
The vaccine strains derived from the original Edmonston virus (which is no longer available) must have undergone many mutations during passages in a variety of human, chicken, and other cells, having adapted to efficient growth in them (27, 28). Since these cells did not express the authentic MV receptor SLAM, viruses may have come to use alternate receptors such as CD46 by introducing changes in the H gene. In fact, all vaccine strains whose genomic sequences have been determined, as well as the Edmonston "wild-type" strain, have the changes (481Y and/or 546G) in the H protein that allow virus to bind CD46 (19, 20, 28, 47). However, as our present study shows, even without those mutations that enable virus to enter cells efficiently, virus may be able to grow well in SLAM-negative cells by introducing changes in genes other than the H gene. Thus, when viruses were isolated in SLAM-negative cells (21, 43) or when viruses first isolated in SLAM-positive B95a cells were subsequently adapted to growth in SLAM-negative cells (18, 24, 37, 39), they may not have had any mutation in the H gene but instead possessed mutations in other genes, including the M and L genes.
ACKNOWLEDGMENTS
We thank M. A. Billeter, B. Moss, and T. Kohama for providing reagents. We are grateful to K. Takeuchi for his contribution to the establishment of the reverse genetics of the IC-B strain.
This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology and the Ministry of Health, Labor and Welfare of Japan; from the Japan Society for the Promotion of Science; and from the Takeda Science Foundation.
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