Measles viruses with altered envelope protein cytoplasmic tails gain cell fusion competence

Abstract

The cytoplasmic tail of the measles virus (MV) fusion (F) protein is often altered in viruses which spread through the brain of patients suffering from subacute sclerosing panencephalitis (SSPE). We transferred the coding regions of F tails from SSPE viruses in an MV genomic cDNA. Similarly, we constructed and transferred mutated tail-encoding regions of the other viral glycoprotein hemagglutinin (H) gene. From the mutated genomic cDNAs, we achieved rescue of viruses that harbor different alterations of the F tail, deletions in the membrane-distal half of the H tail, and combinations of these mutations. Viruses with alterations in any of the tails spread rapidly through the monolayer via enhanced cell-cell fusion. Double-tail mutants had even higher fusion competence but slightly decreased infectivity. Analysis of the protein composition of released mutant viral particles indicated that the tails are necessary for accurate virus envelope assembly and suggested a direct F tail-matrix (M) protein interaction. Since even tail-altered glycoproteins colocalized with M protein in intracellular patches, additional interactions may exist. We conclude that in MV infections, including SSPE, the glycoprotein tails are involved not only in virus envelope assembly but also in the control of virus-induced cell fusion.

FIG. 1

Structure of the MV genome and of the plasmids used for virus rescue, and amino acid sequence of the cytoplasmic tails of the MV glycoproteins. (A) Structure of p(+)MV and of the plasmid intermediates used in the construction of mutant viruses. Plasmid p(+)MV encoding the MV Edmonston B strain antisense genome is shown at the top (32). Solid boxes represent the reading frames of the six MV cistrons. The T7 promoter and relevant restriction sites are indicated. The structures of the plasmids used for subcloning are shown below. Shaded boxes represent gene segments coding for the altered F and H tails. The nucleotide numbering is that used for EMBL accession no. Z66517. (B) Predicted amino acid sequence of the standard (std) and altered MV glycoprotein tails. Boldface letters represent residues conserved within five members of the morbillivirus genus. Residues in the SSPE-derived F protein tails differing from that of the Edmonston strain are indicated by lowercase letters. TM, transmembrane; aa, amino acids.

FIG. 2

Intracellular transport and fusogenic activity of glycoproteins with altered tails. (A) F proteins with altered tails. HeLa T4 cells were infected with a recombinant vaccinia virus expressing the T7 polymerase and subsequently lipofected with the plasmids as indicated above each lane. At 20 h posttransfection, the cells were metabolically labelled for 1 h, chased for 3 h, and lysed, and clarified supernatants were used for immunoprecipitation with an MV-specific antiserum. Precipitated proteins were digested with (+) or without (−) endo H and then separated by reducing SDS-PAGE (8% polyacrylamide). The positions of F₀ and F₁ are indicated on the right. Below the gel, fusion activities are indicated as a percentage of the nuclei found in syncytia. The extent of fusion was determined after cotransfection of the indicated plasmid with a plasmid coding for standard H protein by counting the nuclei found in syncytia and in nonfused cells. (B) H proteins with altered tails. The methods are the same as for panel A. R, endo H-resistant and partially resistant forms; S, endo H-sensitive forms. The exact nature of the minor faster-migrating band in the HcΔ24 lanes is not known. At the bottom the percentage of nuclei found in syncytia, the ratio of endo H-resistant to -sensitive forms (R/S) as determined by densitometric analyses of autoradiographs, and the surface expression level of the H proteins as determined by FACScan analysis are indicated. Surface expression was normalized to the standard H protein expression.

FIG. 3

Protein produced by nine rescued mutant viruses in Vero cells, immunoprecipitated by an MV-specific antiserum. Vero cells were infected with the cytoplasmic tail mutants as indicated above the lanes. At 16 h postinfection, the cells were pulsed for 1 h in the presence of Tran³⁵S-label and chased for 4 h. Immunoprecipitated proteins were subjected to SDS-PAGE (8% polyacrylamide). The positions of H, N, F₁, and M proteins are indicated on the left.

FIG. 4

Time course of cell-associated and cell-free virus production in Vero cells infected with standard and mutant viruses. Vero cells were infected at an MOI of 3, and virus titers were determined by 50% end-point dilution at the indicated time points postinfection (p.i.). The titer of cell-associated infectivity is indicated by solid triangles, and that of released virus is indicated by open triangles. The continuous lines join the average points of two representative experiments. The CPE corresponds to the extent of fusion. +, 5 to 25% of nuclei were found in syncytia; ++, 26 to 50% of nuclei were found in syncytia; +++, 51 to 75% of nuclei were found in syncytia; ++++, ≥76% of nuclei were found in syncytia.

FIG. 5

CPEs in Vero cells infected with standard virus or with one of three mutant viruses. Vero cells were infected (MOI, 3) with standard MV (B, G, and L), MV-Fc+28 (C, H, and M), MV-HcΔ14 (D, I, and N), or MV-HcΔ14/Fc+28 (E, J, and O) or left uninfected (A, F, and K). CPEs were monitored by phase-contrast microscopy 12 h (A to E), 36 h (F to J), and 60 h (K to O) after infection.

FIG. 6

Localization of F, H, and M proteins in Vero cells infected with standard virus or with one of three mutant viruses. Infected Vero cells were permeabilized 50 h after infection and reacted with F, H, or M protein-specific antibodies. Immunoreactivity was monitored by confocal microscopy. Micrographs represent a 0.5-μm optical section of a syncytium induced either by infection with standard MV (A to C and G to I), MV-HcΔ14 (D and J), MV-FcSeV (E and K), or MV-HcΔ14/FcSeV (F and L). Image stacks were obtained in double excitation mode and shown for standard MV infection: F (A) or H protein (G) in green and M protein (B and H) in red. Recombining the two single images revealed colocalization of either F and M protein (C to F) or H and M protein (I to L) in yellow. For the mutant viruses, only the recombined single images are shown. The arrow indicates colocalization at the cell surface, whereas the arrowhead indicates intracellular colocalization of the F and M proteins. Bar, 20 μm.

FIG. 7

Protein composition of standard and mutant virus particles. Standard MV (A), MV-HcΔ14 (B), MV-FcSeV (C), and MV-HcΔ14/FcSeV (D) were grown in Vero cells in the presence of Tran³⁵S-label. Supernatant virus was first pelleted by sedimentation onto a 60% sucrose cushion and then purified by equilibrium centrifugation in a 20 to 60% sucrose step gradient. Virus particles from 10 gradient fractions were pelleted, disrupted in lysis buffer, and directly analyzed by SDS-PAGE (8% polyacrylamide). Marker proteins in lanes (a) of panels A to C were immunoprecipitated proteins from a standard MV infection, whereas immunoprecipitated proteins from MV-HcΔ14/FcSeV infected cells were applied in panel D. Lanes (b) show proteins immunoprecipitated from mock-infected cells. The individual fractions are marked at the top (1 to 10 from top to bottom). The positions of the H (or HcΔ14), P, N, X, F₁ (or FcSeV₁), and M proteins are indicated. X may be cellular actin (47).