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Rubella virus (RV), a small enveloped positive-strand RNA virus, is the sole member of the genus Rubivirus in the family Togaviridae (3). The RV virion contains a nucleocapsid consisting of a 40S genomic RNA molecule and a single species of capsid protein (33 kDa) (17). The nucleocapsid is enveloped within a host-derived lipid bilayer containing two viral glycoproteins, E1 (58 kDa) and E2 (42 to 46 kDa) (16). The structural protein genes are expressed from a 24S subgenomic RNA and are translated in the order NH₂-C-E2-E1-COOH (18). RV matures by budding at the plasma membrane of infected cells (1) and enters uninfected cells by a membrane fusion process in the endosome (6). Both processes are directed by E2-E1 heterodimers (4, 27). Formation of an E2-E1 heterodimer is required for transport of E1 out of the endoplasmic reticulum lumen to the Golgi and plasma membrane (4).

Semliki Forest virus (SFV), a well-characterized alphavirus, uses an acid-triggered membrane fusion to infect host cells. Its fusion with cellular membranes is mediated by the viral spike proteins, heterotrimers of two transmembrane subunits, E2 and E1, and a peripheral protein, E3 (8, 25). Mutagenesis of an SFV spike protein cDNA indicates that the internal hydrophobic domain of E1 is closely involved in membrane fusion (12). Like SFV, RV infects cells via a low-pH-dependent pathway (6, 9, 23). Although little is known about the fusion process of RV, available evidence suggests that RV E1 plays a dominant role in membrane fusion (6, 27).

In previous studies, we constructed and characterized mutations within the putative E1 fusion domain, 29 amino acid residues, extending from amino acids 81 to 109 (Fig. 1). Mutants generated in this domain were based on the rationale that substitution of Cys82 by serine (C82S) would affect E2-E1 interaction, substitution of a charged aspartic acid at Gly93 (G93D) would inhibit the ability of E1 to induce membrane fusion, and substitution of glycine for Pro104 (P104G) would disrupt the amphipathic and helix of fusion peptide. Using a cDNA clone coding for E2 and E1 structural proteins, we demonstrated that C82S mutation or deletion of this hydrophobic domain (dt) of E1 (Fig. 1) resulted in disruption of E1 conformation that ultimately impaired E1-E2 heterodimer formation and cell surface expression of both E1 and E2 (27). However, both G93D and P104G mutations were found to block neither E1-E2 heterodimer formation nor the transport of E1 and E2 to the cell surface. No syncytium formation was detected in cells expressing C82S, dt, and G93D mutations, whereas the wild-type (wt) virus and P104G mutant exhibited fusogenic activity, with 60% and 20 to 40%, respectively, of cells fused at pH 4.8 (27). These studies were performed on RV E2 or E1 structural protein cloned into a plasmid, and the effects of these mutations on virus assembly and infectivity could not be determined.

View larger version (13K): [in this window] [in a new window]   FIG. 1.   Schematic diagram of RV cDNA constructs and the mutations introduced in the hydrophobic region of RV E1. E1, E2, and C genes are indicated at the top. Signal peptides are indicated by open boxes, and the transmembrane regions are shown by solid boxes. The E1 hydrophobic domain is shown. Beneath the arrows are the single amino acid changes and the deletion mutation. 24S, the cDNA containing all three structural protein genes of RV inserted in pNUT vector (19) under the control of an inducible mouse metallothionein promoter (mMT-1); E2E1, the cDNA containing the E1 and E2 genes in pNUT vector. Restriction endonuclease sites: E, EcoRI; H, HindIII; B, BamHI; N, NcoI; S, SphI; and Bs, BstEII. nt, nucleotides.

In the present study, we incorporated G93D and P104G mutations into an infectious RV cDNA clone and examined their effects on virus infectivity. The effects of mutation on virus assembly were studied using a system in which RV spike proteins can be assembled into RV-like particles in the absence of virus replication (5, 20). We found that virus assembly was not affected by G93D or P104G mutation, but the mutants were 10-fold (P104G virus) and over 1,000-fold (G93D virus) less infective than the wt virus. Passage experiments were carried out to examine the reversion of mutant viruses harvested from BHK cells transfected with RNA transcripts from mutants. In revertants from G93D mutant virus, the substituted aspartic acid residue was found to have changed to the original glycine residue. No revertant was observed with P104G mutant virus.

Effects of mutations on virus assembly. In SFV, mutations in the putative fusion peptide of E1 glycoprotein confer a strong and heat-reversible budding effect (2). The assembly of mutant spike proteins into mature virions is severely impaired, and a cleaved soluble fragment of E1 is released into the medium (2). In our previous studies, we showed that in the fusion defective G93D mutant, the E2-E1 heterodimers at the cell surface are unstable, resulting in the release of cleaved E2 and E1 into the medium (27). It is of interest to examine whether the assembly of virus particles is also affected by the G93D mutation. Since G93D and P104G mutations reside within the putative fusion peptide, we chose to study virus assembly in the absence of virus replication, using the capacity of RV structural proteins to form virus-like particles (VLPs) (20). In this system, coordinated expression of capsid, E2, and E1 proteins in stable transformed BHK cells (BHK-24S) results in their assembly into VLPs in the absence of genomic RNA.

View larger version (78K): [in this window] [in a new window]   FIG. 2.   Assembly of RV structural proteins into VLPs in BHK cells. Transformed BHK cells were induced with ZnSO4 (40 µM) for 4 h prior to labeling. Induced BHK cells were labeled with [35S]methionine and chased with unlabeled methionine for 2, 4, and 8 h. Labeled RV proteins from cellular lysates (A) and culture media (B) were immunoprecipitated with human anti-RV serum, followed by SDS-PAGE. Lanes: B, BHK cells; Wt, BHK-24S (wt); G93D, BHK-24S (G93D); P104G, BHK-24S (P104G). Positions of E1, E2, C, and apparent molecular weight markers (in kilodaltons) are shown.

Effect of mutations on virus infectivity. In our previous studies (27), substitution of Gly93 residue by an aspartic acid in the E1 hydrophobic region blocked syncytium formation without affecting the transport of spike proteins to the cell surface. We speculate that this mutation will be lethal in a virion, resulting in the assembly of a nonfusogenic and noninfectious virion. However, it has been shown in Moloney murine leukemia virus that fusion accompanying viral entry and fusion responsible for syncytium formation are independent processes in NIH3T3/DTras cells (26). Therefore, the failure to induce syncytium formation does not necessarily correspond to the complete lack of fusion activity in G93D mutant. To examine whether the G93D mutant could function in the viral envelope to bring about cell infection, we introduced the G93D and P104G mutations separately into an RV infectious full-length cDNA clone, pBR/M33 (28). Initially the plasmid, pBR/M33 (XbaI/HindIII) containing part of the RV nonstructural protein genes and the entire structural protein genes (nucleotides 4952 to 9762) was digested with restriction enzymes SphI and BamHI, and the SphI/BamHI fragment containing G93D or P104G mutation (Fig. 1) was recloned into plasmid pBR/M33 (XbaI/HindIII) (minus the original SphI/BamHI fragment). The resulting recombinant plasmids containing G93D and P104G mutations were digested with enzymes XbaI and HindIII, and the XbaI/HindIII fragments containing G93D and P104G mutations were isolated and inserted into pBR/M33 that had its original XbaI/HindIII fragment deleted. DNA sequencing and restriction analysis were performed to confirm the introduced mutations in the recombinant plasmids (pBR/M33/G93D and pBR/M33/P104G).

View larger version (75K): [in this window] [in a new window]   FIG. 3.   Synthesis of RV structural proteins in BHK cells transfected with full-length RNA transcripts containing G93D and P104G mutations by electroporation. Monolayers of BHK cells (35 mm) were transfected with 2 µg of RNA transcript by electroporation. Culture medium was harvested daily and replaced with fresh medium. Transfected BHK cells were labeled with [35S]methionine for 3 h and chased with unlabeled methionine for 18 h. Labeled RV proteins from cellular lysates (A) and media (B) were immunoprecipitated with human anti-RV serum. wt, wild-type M33 full-length infectious clone; P104G, full-length infectious clone containing P104G mutation; G93D, full-length infectious clone containing P104G mutation. Positions of E1, E2, C, and apparent molecular weight markers (in kilodaltons) are shown.

Analysis of G93D revertants. We found that P104G virus formed small plaques (0.5 mm in diameter) on Vero cells, whereas the plaques of G93D virus were similar to those of wt (3 mm). It is likely that G93D virus formed tiny plaques (due to defects in infectivity), and the observed large-plaque morphology in plaque assay was due to the occurrence of revertants after 6 days of incubation. RV replicates slowly and forms microfocal plaques which are visible after 6 to 8 days of incubation.

View larger version (56K): [in this window] [in a new window]   FIG. 4.   Synthesis of RV structural proteins in Vero cells infected with wt, P104G, and G93D viruses. Monolayers of Vero cells (35 mm) were infected with wt, P104G, or G93D virus harvested from BHK cells at day 3 posttransfection at a multiplicity of 0.1 PFU/cell. Infected cells were labeled with [35S]methionine for 3 h and chased with unlabeled methionine for 18 h. Labeled RV proteins from cellular lysates and media were immunoprecipitated with human anti-RV serum. (A) Lysates from infected Vero cells at day 1, 2, and 3 postinfection. (B) Labeled RV structural proteins from lysates and media at day 5 postinfection. wt, wild-type M33 virus; P104G, P104G virus; G93D, G93D virus. Positions of E1, E2, C, and apparent molecular weight markers (in kilodaltons) are shown.

Fusion activities of the mutant viruses. Since a low level of infectious G93D virus was produced in BHK cells transfected with full-length RNA transcript carrying the G93D mutation, it is possible that the inability of G93D/E2E1 mutant protein to induce syncytium formation (27) does not reflect a complete lack of fusion activity. Therefore, we examined the pH dependence of virus-induced polykaryon formation using mutant viruses harvested from BHK cells transfected with infectious RNA transcripts. Vero cells were infected with wt, G93D, or P104G virus, harvested at day 3 posttransfection at 1 PFU/cell, and incubated for 40 or 64 h at 37°C. The infected cells were treated with fusion medium (pH 4.8) for 20 min at 37°C, washed with growth medium (pH 7.0), and incubated with the growth medium at 37°C for an additional 4 h. The polykaryons formed were viewed under a phase-contrast microscope. At 40 h postinfection, cultures of cells expressing the wt and P104G mutant had 60 and 10%, respectively, of fused cells, but no polykaryons were observed with the G93D mutant (Fig. 5A to C). At 64 h postinfection, cell lysis occurred in wt-infected cells, and about 15 and 10% of fused cells were observed in the cell cultures of P104G and G93D mutants, respectively (Fig. 5D). The syncytium formation observed in G93D virus-infected cells could be attributed to the presence of G93D revertants in virus stock. To investigate this possibility, we analyzed the pH-dependent syncytium formation of BHK cells expressing VLPs carrying the G93D mutation (BHK-24S/G93D). Transformed BHK cells were incubated with growth medium containing 40 µM zinc sulfate for 16 h to induce expression of RV structural proteins (20). Induced BHK cells were treated with fusion medium and incubated with growth medium as described above. The percentages of fused cells observed were 50 to 60, 20, and 5% for BHK-24S (wt), BHK-24S (P104G), and BHK-24S (G93D), respectively (data not shown). It is interesting that a low level of polykaryons was detected in BHK-24S (G93D) cells. The likely explanation for not detecting polykaryon formation in E2E1 (G93D) mutant in the previous studies (27) could be the instability of E2-E1 heterodimers in the absence of capsid protein, as soluble E2 and E1 proteins were observed in the medium after a longer time of chase (27). These results indicate that G93D virus possesses a very limited fusogenic activity that correlates well with its low virus infectivity.

View larger version (216K): [in this window] [in a new window]   FIG. 5.   Syncytium formation in Vero cells infected with wt, P104G, and G93D viruses. Infected cells were exposed to fusion medium at pH 4.8 for 20 min, incubated in regular growth medium for 4 h, fixed, and photographed. Vero cells were infected with wt virus (A), P104G virus (B), or G93D virus (C) at 40 h postinfection or with G93D virus at 64 h postinfection (D).