Mutations in the endodomain of Sindbis virus glycoprotein E2 define sequences critical for virus assembly

Abstract

Envelopment of Sindbis virus at the plasma membrane is a multistep process in which an initial step is the association of the E2 protein via a cytoplasmic endodomain with the preassembled nucleocapsid. Sindbis virus is vectored in nature by blood-sucking insects and grows efficiently in a number of avian and mammalian vertebrate hosts. The assembly of Sindbis virus, therefore, must occur in two very different host cell environments. Mammalian cells contain cholesterol which insect membranes lack. This difference in membrane composition may be critical in determining what requirements are placed on the E2 tail for virus assembly. To examine the interaction between the E2 tail and the nucleocapsid in Sindbis virus, we have produced substitutions and deletions in a region of the E2 tail (E2 amino acids 408 to 415) that is initially integrated into the endoplasmic reticulum. This sequence was identified as being critical for nucleocapsid binding in an in vitro peptide protection assay. The effects of these mutations on virus assembly and function were determined in both vertebrate and invertebrate cells. Amino acid substitutions (at positions E2: 408, 410, 411, and 413) reduced infectious virus production in a position-dependent fashion but were not efficient in disrupting assembly in mammalian cells. Deletions in the E2 endodomain (delta406-407, delta409-411, and delta414-417) resulted in the failure to assemble virions in mammalian cells. Electron microscopy of BHK cells transfected with these mutants revealed assembly of nucleocapsids that failed to attach to membranes. However, introduction of these deletion mutants into insect cells resulted in the assembly of virus-like particles but no assayable infectivity. These data help define protein interactions critical for virus assembly and suggest a fundamental difference between Sindbis virus assembly in mammalian and insect cells.

FIG. 1.

Sequence of the SV E2 endodomain and related alphaviruses. Residues in boldface were deleted, and underlined residues were substituted as described in Materials and Methods. Dots indicate conservation of the sequence of the E2 tail between these alphaviruses.

FIG. 2.

Iodination of capsid residue Y180 as an indicator of E2 protein binding. Capsid protein was iodinated in the presence (A) or absence (B) of a peptide representing the 33-aa E2 endodomain. The reaction was quenched and then treated (+) or not treated (−) with V8 protease to release a peptide containing capsid Y180.

FIG. 3.

Production of infectious virus after transfection of BHK cells with E2 endodomain mutants. (A) Production of infectious virus by E2 endodomain substitution mutants. Virus was harvested 20 to 24 h posttransfection, and titers were determined by plaque assay on BHK cells as described in Materials and Methods. (B) Production of infectious virus by E2 endodomain deletion mutants. Virus was harvested 20 to 24 h posttransfection, and titers were determined by plaque assay on BHK cells as described in Materials and Methods.

FIG. 4.

Protein synthesis in deletion mutant-transfected cells. (A) Proteins released into the media of BHK cells transfected with E2 endodomain deletion mutants. Equal volumes of media surrounding equal numbers of transfected cells was applied directly to PAGE. Arrows indicate the positions of the virus structural proteins E1, E2, capsid, and E3. (B) Proteins immune precipitated from cells infected with deletion mutant Δ414-417. Lanes: 1, wild-type RNA; 2, mock transfected; 3, transfected with RNA from deletion mutant Δ414-417. PE2, E1, E2, and C are SV structural proteins. Arrows A and B indicate aberrant proteins produced in deletion mutant Δ414-417.

FIG. 5.

Electron micrographs of BHK cells transfected with E2 endodomain deletion mutants. (A) Wild type; (B) Δ406-407 mutant; (C) Δ409-411 mutant; (D) Δ414-417 mutant. (E) Enlargement of an area in panel D. Arrows indicate nucleocapsids. Bars, 200 nm.

FIG. 6.

Growth of E2 endodomain deletion mutants in insect cells. Aedes albopictus (clone U4.4) cells were transfected with wild type or mutant strains containing virus RNA as described in Materials and Methods.

FIG. 7.

Electron micrographs of Aedes albopictus cells transfected with E2 endodomain deletion mutants. (A) Wild type (arrows indicate virions); (B and C) mutant Δ409-411. Bars, 100 nm.