Role of matrix and fusion proteins in budding of Sendai virus

Abstract

Paramyxoviruses are assembled at the surface of infected cells, where virions are formed by the process of budding. We investigated the roles of three Sendai virus (SV) membrane proteins in the production of virus-like particles. Expression of matrix (M) proteins from cDNA induced the budding and release of virus-like particles that contained M, as was previously observed with human parainfluenza virus type 1 (hPIV1). Expression of SV fusion (F) glycoprotein from cDNA caused the release of virus-like particles bearing surface F, although their release was less efficient than that of particles bearing M protein. Cells that expressed only hemagglutinin-neuraminidase (HN) released no HN-containing vesicles. Coexpression of M and F proteins enhanced the release of F protein by a factor greater than 4. The virus-like particles containing F and M were found in different density gradient fractions of the media of cells that coexpressed M and F, a finding that suggests that the two proteins formed separate vesicles and did not interact directly. Vesicles released by M or F proteins also contained cellular actin; therefore, actin may be involved in the budding process induced by viral M or F proteins. Deletion of C-terminal residues of M protein, which has a sequence similar to that of an actin-binding domain, significantly reduced release of the particles into medium. Site-directed mutagenesis of the cytoplasmic tail of F revealed two regions that affect the efficiency of budding: one domain comprising five consecutive amino acids conserved in SV and hPIV1 and one domain that is similar to the actin-binding domain required for budding induced by M protein. Our results indicate that both M and F proteins are able to drive the budding of SV and propose the possible role of actin in the budding process.

FIG. 1

Release of viral proteins into culture medium. Cells were transfected with cDNAs encoding SV M, F, or HN and were labeled with [³⁵S]Met and [³⁵S]Cys for 16 h. (A) Protein expression in transfected cells. Cells were lysed in 1 ml of TNE buffer, and 100 μl of the clarified lysate was used for immunoprecipitation with specific MAbs. (B) Proteins released into culture medium were collected, purified through 30% glycerol in PBS, and analyzed by SDS-PAGE. The upper bands observed in the material of the M vesicles are charge-induced M aggregates (28). The arrows indicate cellular actin.

FIG. 2

Electron micrographs of virus-like particles released into medium. Supernatants from cultures of cells transfected with SV M (A) or SV F (B and C) cDNAs were concentrated by membrane filtration. The samples were adsorbed to carbon-coated grids, negatively stained, and examined by electron microscopy. Bars, 86 nm.

FIG. 3

Density gradient analysis of the vesicles released from cells expressing viral proteins. Cells infected with SV or transfected with cDNAs encoding M, F, or HN alone or in combination were labeled with [³⁵S]Met and [³⁵S]Cys. The clarified cell culture medium was centrifuged through a 5 to 40% sucrose gradient. Eight fractions were collected, and the proteins in each fraction were analyzed by SDS-PAGE.

FIG. 4

Efficiency of protein release from transfected cells. Cells transfected with expression vectors containing M, F, or HN cDNAs alone or in combination were labeled with [³⁵S]Met and [³⁵S]Cys. (A) Vesicles released into the medium were purified and analyzed by SDS-PAGE. (B) Protein expression in cells was analyzed by immunoprecipitation and SDS-PAGE. (C) Proteins in cell lysates and in culture medium were quantified from SDS-PAGE gels (A and B) by using a STORM 860 imaging system, and the efficiency of protein release was calculated. Each value represents the mean of three independent experiments.

FIG. 5

Cellular actin was present in vesicles that contained M or F. Cells expressing SV M or F protein were labeled with [³⁵S]Met and [³⁵S]Cys, and the clarified cell culture supernatants were centrifuged on 5 to 40% sucrose gradients. Eight density fractions were collected, and Western blots of proteins in each fraction were analyzed with an anti-actin MAb. After chemiluminescent signals dissipated, the membrane was exposed to X-ray film overnight to detect ³⁵S-labeled proteins.

FIG. 6

C-terminal regions of SV and hPIV1 M proteins are required for the release of M-containing vesicles. (A) Sequences of the C-terminal region of SV and hPIV1 M proteins. (B and C) Release of the deletion mutant of SV M (B) or of a series of deletion mutants of hPIV1 M (C) into the culture medium was determined as described in the legend to Fig. 1. Expression of M proteins in the transfected cells was quantified by immunoprecipitation with an anti-M MAb.

FIG. 7

Identification of SV F residues that affect budding of F-containing vesicles. (A) Sequence of the F protein cytoplasmic domain. SV and hPIV1 F share five consecutive amino acids (TYTLE). Sequences of the F mutants used are also shown. (B) Release of the SV F mutants into the medium was assessed as described in the legend to Fig. 1. (C) Surface expression of the mutant F proteins by transfected cells was quantified by cell-surface enzyme-linked immunosorbent assay with a cocktail of anti-F MAbs.