Mutant Fusion Proteins with Enhanced Fusion Activity Promote Measles Virus Spread in Human Neuronal Cells and Brains of Suckling Hamsters

Vero cells expressing human SLAM (Vero/hSLAM cells) (14), Vero cells, and IMR-32 cells were maintained in Dulbecco's minimum essential medium (DMEM) (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS). SYM-1 cells were maintained in 1:1 DMEM/RPMI medium supplemented with 10% FBS. Vero/hSLAM cells constitutively expressing the T7 RNA polymerase (Vero/hSLAM-T7 cells) were established using a retrovirus vector as follows. To generate the retrovirus expressing the T7 RNA polymerase, the cDNA encoding the T7 RNA polymerase was cloned into pMX-IRES-Puro (a gift from M. Shimojima and T. Kitamura) (40–42), producing pMX-T7pol-IRES-Puro. PLAT-gp cells (Invivogen, San Diego, CA) cultured in 6-well cluster plates were transfected with 7.5 μg of pMX-T7pol-IRES-Puro and 0.5 μg of pCVSV-G using Lipofectamine 2000 (Invitrogen, Carlsbad, CA), and the retrovirus was recovered. Vero/hSLAM cells in 24-well plates were infected with this retrovirus, and at 72 h after transduction, the culture medium was replaced with complete DMEM containing 2 μg/ml puromycin (Invivogen). A single clone was isolated after several passages with puromycin and used for experiments. Vero/hSLAM-T7 cells were maintained in DMEM supplemented with 10% fetal bovine serum and 2 μg/ml puromycin.

The F genes of mutant viruses were amplified by PCR using the primer pair 5′-TTGAATTCGCCACCATGGGTCTCAAGGTGAACGT-3′ and 5′-TTGCGGCCGCTCAGAGCGACCTTACATAGG-3′. After digestion with EcoRI and NotI, the PCR products were cloned into pCA7, the eukaryotic expression vector (43), producing pCA7-ICF(I87T), pCA7-ICF(M94V), pCA7-ICF(S262R), pCA7-ICF(L354M), and pCA7-ICF(N462K). pCA7-ICH and pCA7-ICF, which encode the H and F proteins of the IC-B strain of MV, respectively, have been described previously (19). Six different sets of mutations were introduced independently into pCA7-ICF by site-directed mutagenesis using complementary primer pairs, producing pCA7-ICF(T461I), pCA7-ICF(S103I), pCA7-ICF(N462S), pCA7-ICF(N465S), pCA7-ICF(N462S/N465S), and pCA7-ICF(S103I/N462S/N465S). All full-length-genome plasmids were derived from pHHRz(+)MV323-EGFP (44, 45), which carries the antigenomic full-length cDNA of the wild-type IC-B strain together with enhanced green fluorescent protein (EGFP). The F gene of pHHRz(+)MV323-EGFP was replaced with those of pCA7-ICF-derived plasmids with different mutations, producing pHHRz(+)MV323-F(S262R)-EGFP, pHHRz(+)MV323-F(L354M)-EGFP, pHHRz(+)MV323-F(N462K)-EGFP, pHHRz(+)MV323-F(T461I)-EGFP, and pHHRz(+)MV323-F(S103I/N462S/N465S)-EGFP. To construct the plasmid pHHRz(+)MV323-ΔM-EGFP, the gene encoding the 5′ and 3′ untranslated regions (UTR) of the M protein but lacking the coding sequence of the M protein was amplified by the overlapping-PCR method with the primer set 5′-ATCCGCGGTCAGGGATCTGG-3′, 5′-CTGGGCACTGCGGTTGTGGAACTTAGGAGGCAATC-3′, 5′-CTCCTAAGTTCCACAACCGCAGTGCCCAGCAATACC-3′, and 5′-TGCCGCGGGCCGGGGTCTG-3′ and ligated to the plasmid pHHRz(+)MV323-EGFP after digestion with SacII. The plasmids pCITE-RL and pTKminusT7-FL used in the quantitative fusion assay were constructed as follows. To construct the plasmid pCITE-RL, the gene encoding Renilla luciferase was amplified by PCR from p18MGKLuc01 (46) and ligated to the plasmid pCITE-2a(+) (Novagen, Madison, WI). To construct the plasmid pTKminusT7-FL, the gene encoding firefly luciferase was amplified by PCR from p18MGFLuc01 (46) and ligated to the plasmid pTK-RL (Promega, Madison, WI), from which the T7 promoter and Renilla luciferase sequence had been removed.

Recombinant MVs were generated as reported previously (45). Briefly, BHK/T7-9 cells, which constitutively express T7 RNA polymerase, were transfected with full-length-genome plasmids carrying the antigenomes of MV and three support plasmids, pCITE-IC-N, pCITE-IC-PΔC, and pCITEko-9301B-L. At 2 days after transfection, the cells were cocultured with Vero/hSLAM cells. The recombinant viruses IC323-F(S262R)-EGFP, IC323-F(L354M)-EGFP, IC323-F(N462K)-EGFP, IC323-F(T461I)-EGFP, IC323-F(S103I/N462S/N465S)-EGFP, and IC323-ΔM-EGFP were generated from pHHRz(+)MV323-F(S262R)-EGFP, pHHRz(+)MV323-F(L354M)-EGFP, pHHRz(+)MV323-F(N462K)-EGFP, pHHRz(+)MV323-F(T461I)-EGFP, pHHRz(+)MV323-F(S103I/N462S/N465S)-EGFP, and pHHRz(+)MV323-ΔM-EGFP, respectively. The generated MVs were propagated in Vero/hSLAM cells. The number of PFU of each recombinant virus was determined in Vero/hSLAM cells.

293T cells cultured in a 12-well plate were transfected with pCA7-ICH (0.4 μg), an F-protein-encoding plasmid (pCA7-ICF or pCA7 encoding each mutant F protein; 0.1 μg) and pCITE-RL encoding Renilla luciferase driven by T7 polymerase (1.2 μg), using Polyethylenimine (PEI) “Max” high-potency linear PEI (Polysciences, Warrington, PA). These transfectants were used as effector cells. As an internal control, pTKminusT7-FL encoding firefly luciferase driven by the herpes simplex virus (HSV) thymidine kinase (TK) promoter was cotransfected into the effector cells. Vero/hSLAM-T7 cells were cultured in a 12-well plate and used as target cells. At 6 h posttransfection, effector cells were washed with PBS 3 times, detached with 0.05% EDTA in PBS, and centrifuged (400 × g; 5 min; 4°C). The cell pellet was resuspended in 500 μl of growth medium, and 100 μl of cell suspension was overlaid onto the target cells. After 18 h of incubation, cell-cell fusion was quantified by measuring the luciferase activity. Firefly and Renilla luciferase activities were independently assayed by using the dual-luciferase reporter assay system (Promega) with a Mithras LB940 plate reader (Berthold Technologies, Pforzheim, Germany). The relative fusion activity (Renilla luciferase activity divided by firefly luciferase activity) was calculated. The value obtained with pCA7-ICH and pCA7-ICF was set to 100%.

Vero/hSLAM cells cultured in a 12-well cluster plate were transfected with pCA7-ICH (0.2 μg) plus pCA7 encoding the wild-type or each mutant F protein (1.5 μg), using Lipofectamine 2000 (Invitrogen). One day after transfection, the cells were observed under a light microscope after Giemsa staining.

Vero cells cultured in a 24-well cluster plate were transfected with pCA7-ICH (0.75 μg) plus pCA7 encoding each mutant F protein (0.75 μg). Plasmid DNAs were incubated with 1.5 μl of Lipofectamine LTX Plus reagent (Invitrogen) for 5 min at room temperature and incubated with 4.5 μl of Polyethylenimine “Max” high-potency linear PEI for 30 min at room temperature, followed by dropping the mixture onto Vero cells. At 48 h posttransfection, the cells were observed under a light microscope after Giemsa staining. Then, the number of nuclei in syncytia per visual field was determined with a 10× objective lens.

Recombinant viruses were rescued in BHK-T7/9 cells as described above. The transfected BHK-T7/9 cells were maintained in growth medium containing fusion block peptide (Z-D-Phe-Phe-Gly; Peptide Institute Inc., Osaka, Japan) to prevent cell-to-cell fusion. At 24 h posttransfection, the transfected BHK-T7/9 cells were washed 3 times with PBS and detached by trypsin digestion. The preparation, containing 200 EGFP-positive cells, was overlaid onto a confluent monolayer of Vero or IMR-32 cells cultured in a 10-cm dish. At 1 or 3 days after overlay, EGFP fluorescence was observed under a fluorescence microscope.

Vero/hSLAM cells were transfected with 4 μg of plasmid DNA encoding MV F variants as indicated. After washing with PBS three times, cells were incubated in PBS with 0.5 mg of NHS-SS-Biotin (Thermo Scientific, Rockford, IL)/ml for 20 min at 4°C, followed by washing and quenching for 5 min at 4°C in DMEM. The cells were scraped in 1 ml immunoprecipitation buffer (10 mM HEPES [pH 7.4], 50 mM sodium pyrophosphate, 50 mM sodium fluoride, 50 mM sodium chloride, 5 mM EDTA, 5 mM EGTA, 1% Triton X-100) containing protease inhibitor cocktail (Nacalai Tesque, Inc., Kyoto, Japan), and the lysates were cleared by centrifugation for 20 min at 20,000 × g and 4°C. The biotinylated proteins were adsorbed to Sepharose-coupled streptavidin (GE Healthcare, Waukesha, WI) for 90 min at 4°C, washed once in immunoprecipitation buffer, buffer 1 (100 mM Tris [pH 7.6], 500 mM lithium chloride, 0.1% Triton X-100) and buffer 2 (20 mM HEPES [pH 7.2], 2 mM EGTA, 10 mM magnesium chloride, 0.1% Triton X-100). Finally the proteins were incubated in urea buffer for 25 min at 50°C and subjected to Western blot analysis using antibodies specific for the MV-F tail. As a loading control, 10 μl of cell lysates was mixed with urea buffer and subjected to Western blot analysis using antibodies specific for beta-actin (Santa Cruz Biotechnology, Santa Cruz, CA). For densitometric quantification of F proteins, blots were analyzed using a VersaDoc digital imaging system (Bio-Rad, Richmond, CA), and the signals were quantified with QuantityOne software (Bio-Rad).

Ten-day-old Syrian golden hamsters (SLC-Japan, Shizuoka, Japan) were anesthetized with sevoflurane. Then, 25 μl of diluted viruses was inoculated into the right or left hemisphere of the brains of hamsters. After the inoculation, clinical symptoms were observed every day, and moribund hamsters were euthanized. The brains were collected from dead and moribund hamsters. Three hamsters inoculated with wild-type MV or IC323-F(L354M)-EGFP were euthanized 6 days postinoculation, and the brains were also collected. EGFP autofluorescence of the MV-infected cells in each brain was observed under a fluorescence stereomicroscope. All animal experiments were reviewed by the Institutional Committee of Ethics on Animal Experiments and carried out according to the Guidelines for Animal Experiments of the Faculty of Medicine, Kyushu University, Fukuoka, Japan. For histopathological analysis, the collected brain was fixed in 10% buffered formalin and processed into paraffin sections. Sections were stained with hematoxylin and eosin (HE). Immunohistochemical analysis was performed, using the following antibodies: mouse anti-NeuN (1:100; Chemicon, Temecula, CA), mouse anti-glial fibrillary acidic protein (GFAP) (1:1,000; Dako A/S, Glostrup, Denmark), rabbit anti-green fluorescent protein (GFP) (1:500; Invitrogen), and anti-MV-N (1:100; Novus Biological, Littleton, CO). Fluorescence images were captured with a confocal microscope (A1 Confocal Laser Microscope; Nikon Corporation, Tokyo, Japan).

In two different experiments, we identified substitutions in the F protein of IC323-EGFP (the EGFP-expressing recombinant MV based on the wild-type IC-B strain) (44) that enhanced its fusion activity. First, in our attempt to modify IC323-EGFP so that it possessed the H protein with an epitope tag at its C terminus, we found that the virus did not induce syncytia in Vero/hSLAM cells (14). This presumably occurred because the added tag adversely affected the interaction of the H protein with the receptor SLAM and/or the F protein. However, after a few passages in Vero/hSLAM cells, several mutant viruses emerged, which regained the ability to induce syncytia. These mutant viruses were plaque purified, and their H, F, and M genes, encoding the proteins involved in membrane fusion, were subjected to sequencing analysis. Three mutant viruses had single-amino-acid substitutions in the F protein but no substitutions in the H and M proteins (Table 1). Second, Vero cells (SLAM and nectin 4 negative) were infected with IC323-EGFP. As expected, no syncytia were detected at the beginning, but after a few passages, we found two syncytium-producing viruses that had single-amino-acid substitutions in the F protein but none in the H and M proteins (Table 1).

To determine whether these single amino acid substitutions indeed endow the F protein with enhanced fusion activity, Vero/hSLAM cells were cotransfected with the expression plasmid encoding the F protein with each substitution and the plasmid encoding the H protein (without the epitope tag). Compared with the wild-type F protein, all mutant F proteins produced larger syncytia in Vero/hSLAM cells (Fig. 1A). To further evaluate the fusion activities of these F-protein mutants, a quantitative plasmid-mediated fusion assay was established. The S262R, L354M, and N462K substitutions allowed the F protein to exhibit more than 2-times-higher fusion activity than the wild-type F protein, while the M94V substitution slightly enhanced its fusion activity (Fig. 1B).

The fusion activities of these F-protein mutants were also examined in Vero cells by expressing them together with the wild-type H protein. While the wild-type F protein did not induce syncytium formation in Vero cells, all mutant proteins did so (Fig. 1C). Since the sizes of syncytia observed in Vero cells were much smaller than those in Vero/hSLAM cells, the quantitative fusion assay did not give large values so as to reliably evaluate fusion activity. Thus, fusion activity in Vero cells was evaluated by counting the nuclei in syncytia (Fig. 1D). The S262R and N462K substitutions conferred on the F protein more enhanced fusion activity than the other substitutions. When these mutant F proteins were expressed alone (without the H protein), they did not induce syncytia in Vero or Vero/hSLAM cells (data not shown).

Figure 2 shows the locations of these 5 substitutions within the full-length F-protein structure. The substitutions I87T, M94V, and S262R are located in the “microdomain,” the region near the HR-A domain that is involved in the initiation of fusion (47–49). The L354M substitution is located in the cysteine-rich region, which is thought to interact with the H protein (50). Mutant MVs resistant to a fusion-inhibitory peptide contain amino acid substitutions in this region (51). The N462K substitution is located in the HR-B domain. Mutant viruses resistant to fusion-inhibitory compounds contain substitutions at position 462 of the F protein, including the N462K substitution (47, 52). These three regions (the microdomain, cysteine-rich region, and HR-B domain) in the F protein have been implicated in controlling its fusion activity.

Although the amino acid sequences of the F protein are highly conserved among clinical isolates and vaccine strains (53), SSPE strains have many substitutions throughout the F protein. There are no apparently characteristic substitutions, but it is notable that most SSPE strains have substitutions in the microdomain, as well as the N-terminal region of the HR-B domain. We noted that the T461I and S103I/N462S/N465S substitutions are present in multiple SSPE strains and located in the fusion-controlling regions described above (Fig. 2). Using virus-like particles, Ayata et al. have reported that the neurovirulence of one SSPE strain (Osaka-2) is related to the ability of its F protein to induce syncytia in Vero cells, which is attributed to a single substitution (T461I) (54).

We examined whether the T461I and S103I/N462S/N465S substitutions confer enhanced fusion activity on the wild-type F protein. When expressed together with the wild-type H protein, the F protein containing the T461I or S103I/N462S/N465S substitutions induced larger syncytia in Vero/hSLAM cells than the wild-type F protein (Fig. 1A). The quantitative plasmid-mediated fusion assay also showed that these two mutant F proteins have 2.5- to 5-times-higher fusion activity in Vero/hSLAM cells than the wild-type F protein (Fig. 1B). The single substitution N462S or N465S slightly increased the fusion activity of the F protein, but the combined substitutions S103I/N462S/N465S additively enhanced fusion activity. The F protein containing the T461I or S103I/N462S/N465S substitutions also induced syncytia in receptor-negative (SLAM- and nectin 4-negative) Vero cells (Fig. 1C and D).

To determine why these mutant F proteins exhibit enhanced fusion activity, their cell surface expression levels were examined (Fig. 3). All mutant F proteins had surface expression levels comparable to or slightly lower than that of the wild-type F protein, unlike the F protein with C68S/C195S substitutions, which is known to exhibit markedly decreased surface expression (48). The results indicate that the enhanced fusion activity of the mutant F proteins is due to their intrinsic fusogenicity, not to an increase in their surface expression levels.

To examine the effect of enhanced fusion activity of the F protein on MV pathogenicity, we selected five different F-protein substitutions (S262R, L354M, N462K, T461I, and S103I/N462S/N465S) that can induce high levels of fusion in Vero cells (Fig. 1D). These substitutions were incorporated into IC323-EGFP by using reverse genetics, producing five recombinant viruses expressing the wild-type H protein (without the epitope tag) and the respective mutant F proteins. The parental IC323-EGFP induced syncytia in nectin 4-positive cells, as well as in SLAM-positive cells. However, IC323-EGFP never produced syncytia in Vero cells lacking effective receptors (SLAM and nectin 4) for wild-type MV, although it occasionally infected single cells (Fig. 4A). In contrast, all five recombinant viruses with the mutant F proteins induced syncytia even in Vero cells.

These recombinant viruses were also examined for the ability to infect the human neuroblastoma cell lines IMR-32 and SYM-1, which do not express SLAM and nectin 4. The absence of nectin 4 in these neuroblastoma cell lines was confirmed by reverse transcription (RT)-PCR to detect its transcripts (data not shown). Infection of IMR-32 and SYM-1 cells with IC323-EGFP resulted in some infected cells, but syncytia were never produced (Fig. 4B and C). Anti-human nectin 4 monoclonal antibody, which completely blocked infection of H358 cells with IC323-EGFP, did not affect that of IMR-32 and SYM-1 cells (data not shown). The five viruses with the mutant F proteins again induced syncytia in IMR-32 and SYM-1 cells, although the virus with the L354M substitution [IC-F(L354M)-EGFP] did not spread as efficiently as the other mutant viruses (Fig. 4B and C). Moreover, these five viruses induced larger syncytia in H358 cells (nectin 4-positive cells) than IC323-EGFP (data not shown).

To evaluate the neurovirulence of recombinant MVs with the mutant F proteins, we used a hamster model for MV infection with minor modifications (54). Six 10-day-old suckling hamsters were inoculated with IC323-EGFP or each mutant virus intracerebrally (10,000 PFU/brain). No hamsters inoculated with IC323-EGFP showed any symptoms for 4 weeks postinoculation (Fig. 5). In contrast, most of the hamsters inoculated with the recombinant viruses with the mutant F proteins, except IC-F(L354M)-EGFP, died or became moribund approximately 1 week postinoculation. Neurological signs (abnormal gait, convulsions, and continuous tremors) were observed 1 day before the hamsters became moribund. The L354M substitution has the weakest enhancing effect on the fusion activity of the F protein among the five substitutions (Fig. 1B and D and 4B and C). IC-F(L354M)-EGFP exhibited no neurovirulence in hamsters, while the other 4 mutant viruses showed high lethality (66 to 100%) (Fig. 5). Fusion activity in SLAM- and nectin 4-negative cells appeared to correlate well with the survival rate after virus challenge.

The brains were collected from moribund and dead animals, and the spread of EGFP-expressing recombinant viruses was examined under a fluorescence stereomicroscope (Fig. 6). Three hamsters were additionally inoculated with IC323-EGFP or IC-F(L354M)-EGFP and euthanized 6 days postinoculation for microscopic observation. While EGFP was not detected in the brains of hamsters inoculated with IC323-EGFP, IC-F(L354M)-EGFP was found to spread locally in the cerebrum. In sharp contrast, hamsters inoculated with the other 4 mutant viruses showed strong EGFP expression widely in their brains. After the observation, the brains were cocultured with Vero/hSLAM cells to recover viruses from hamsters inoculated with the mutant viruses. No amino acid substitution was found in the H, F, and M proteins of any recovered mutant virus compared with those of the virus used for challenge (data not shown), excluding the possibility that some mutations newly acquired in vivo account for the observed phenotype.

After observation with a fluorescence stereomicroscope, some of the brains were also subjected to histopathological examination (Fig. 7). Inflammatory cell infiltration was observed widely in the cerebra of hamsters inoculated with recombinant viruses possessing the mutant F proteins, except IC-F(L354M)-EGFP. In some lesions, monocytic or histiocytic cells predominantly invaded the inflamed sites, whereas the infiltration of lymphoid cells was observed in others. Severe lesions were found, especially in the cerebral cortex (Fig. 7A, B, and E) and the pyramidal layer and dentate gyrus of the hippocampus (Fig. 7C, D, and F). In these lesions, nuclear fragmentation, indicative of apoptotic cell death, was also observed. Syncytial giant cells were not observed in any brain samples. In contrast, few changes were found in the brains of hamsters inoculated with IC323-EGFP or IC-F(L354M)-EGFP. Immunohistochemical analysis (data not shown) confirmed that the region where EGFP was detected also expressed MV proteins. Moreover, EGFP colocalized with an astrocyte marker, GFAP, or with a neuronal marker, NeuN, showing that the mutant viruses infected astrocytes, as well as neurons, although the number of EGFP-positive astrocytes was smaller than that of EGFP-positive neurons.

Taken together, the results indicate that neurovirulence is correlated with the severity of lesions induced by viruses [IC323-EGFP and IC-F(L354M)-EGFP versus recombinant viruses with the other mutant F proteins].

To compare the contributions of fusion-enhancing mutations in the extracellular domain of the F protein to SLAM- and nectin 4-independent MV infection with that of defects of the M protein, Vero and IMR-32 cells were infected with the recombinant virus lacking the M protein or the cytoplasmic domain of the F protein. First, the recombinant MV lacking the M protein (M-less MV) was generated by using reverse genetics, and the absence of the M protein was confirmed in virus-infected cells. Consistent with a previous report (37), M-less MV induced larger syncytia in Vero/hSLAM cells than wild-type MV (data not shown). As M-less MV could reach only low titers, it was not possible to perform infection of SLAM- and nectin 4-negative cells at sufficiently high multiplicities of infection (MOIs). Instead, BHK-T7/9 cells used for rescue of the recombinant M-less MV were detached by trypsin digestion and overlaid onto Vero or IMR-32 cells. Unexpectedly, apparent syncytium formation was not observed in either cell line (Fig. 8). Although small syncytium-like cells were occasionally observed in the cells cocultured with the rescued M-less MV, they were much smaller than syncytia formed by recombinant MVs possessing the mutant F proteins that were similarly rescued and processed (Fig. 8) (syncytia were observed 3 days, but not 1 day, after overlay). Moreover, expansion of these small syncytium-like cells was not found, even when the cells were observed 7 days after overlay.

Second, we examined the recombinant MV carrying the F protein lacking the cytoplasmic tail (IC323-EGFP-FΔ30) (39). Although we found that IC323-EGFP-FΔ30 induced larger syncytia in SLAM-positive cells than wild-type MV (39), the virus did not induce syncytia in Vero and IMR-32 cells (Fig. 9A and B). In addition, 8 hamsters were inoculated with IC323-EGFP-FΔ30, and 3 of them were euthanized 6 days postinoculation for microscopic observation (Fig. 9C). EGFP was detected in the brains of hamsters inoculated with IC323-EGFP-FΔ30 or IC323-EGFP only when observed in a high-sensitivity mode (with a long exposure time). The spread of IC323-EGFP-FΔ30 was locally restricted in the cerebrum, although it was more widespread than that of IC323-EGFP. The remaining 5 hamsters were examined daily for clinical symptoms of infection for 4 weeks postinoculation. No hamsters showed any symptoms, and EGFP was not detected in the brains of the hamsters at 4 weeks postinoculation (data not shown). The recombinant M-less MV could not be tested for in vivo infection experiments because of its low titers.