Analysis of constructed E gene mutants of mouse hepatitis virus confirms a pivotal role for E protein in coronavirus assembly

Abstract

Expression studies have shown that the coronavirus small envelope protein E and the much more abundant membrane glycoprotein M are both necessary and sufficient for the assembly of virus-like particles in cells. As a step toward understanding the function of the mouse hepatitis virus (MHV) E protein, we carried out clustered charged-to-alanine mutagenesis on the E gene and incorporated the resulting mutations into the MHV genome by targeted recombination. Of the four possible clustered charged-to-alanine E gene mutants, one was apparently lethal and one had a wild-type phenotype. The two other mutants were partially temperature sensitive, forming small plaques at the nonpermissive temperature. Revertant analyses of these two mutants demonstrated that the created mutations were responsible for the temperature-sensitive phenotype of each and provided support for possible interactions among E protein monomers. Both temperature-sensitive mutants were also found to be markedly thermolabile when grown at the permissive temperature, suggesting that there was a flaw in their assembly. Most significantly, when virions of one of the mutants were examined by electron microscopy, they were found to have strikingly aberrant morphology in comparison to the wild type: most mutant virions had pinched and elongated shapes that were rarely seen among wild-type virions. These results demonstrate an important, probably essential, role for the E protein in coronavirus morphogenesis.

FIG. 1

Amino acid sequence comparison of the E proteins of seven MHV strains. The deduced MHV-1, MHV-2, MHV-3, and MHV-DVIM E sequences were determined in the present work. The E sequences of MHV-A59 (8), MHV-JHM (40), and MHV-S (31, 46) have been reported previously (GenBank accession numbers M16602, X04997, and M64835, respectively). Spaces in the alignment indicate positions at which the amino acid is identical to that of MHV-A59; dots denote every 10th residue.

FIG. 2

Strategy for mutagenesis of the MHV-A59 E gene. The putative membrane anchor, charged amino acid residues, and four possible clustered charged-to-alanine mutants are indicated above the E protein sequence. Beneath are shown the base changes made in transcription vectors to change charged residues to alanines. The EcoRV site near the end of the E coding sequence and the FspI site created by the mutations in mutant 3 and mutant 4 are underlined.

FIG. 3

Incorporation of clustered charged-to-alanine mutations into the MHV genome by targeted recombination. Sets of mutations engineered into subclones pCFS9, pCFS10, pCFS11, and pCFS15 were transferred to pCFS8 (14) to produce transcription vectors pFF45, pFF46, pFF47, and pFF48, respectively, which encode pseudo-DI RNAs comprising a 5′ segment of the MHV genome fused, at the start of the S gene, to the entire 3′ end of the genome. E gene mutations are represented by a star; the solid rectangle indicates the 19-nt tag at the start of gene 4. Restriction sites shown are those relevant to plasmid construction, as detailed in Materials and Methods. Brackets designate gene fragments rather than entire genes.

FIG. 4

Portions of genomic RNA sequence within the E genes of clustered charged-to-alanine mutant 2 (Alb153), mutant 3 (Alb154), and mutant 4 (Alb183). The lane markers indicate the terminating dideoxynucleoside triphosphate for each reaction. The segments of sequence show the derived positive-sense RNA sequence with its corresponding translation adjacent. Stars denote mutated nucleotides.

FIG. 5

Revertant analysis of mutant 3 (Alb154: K63A, K67A) and mutant 4 (Alb183: D60A, R61A, K63A). For each mutant or revertant, only amino acid residues differing from the wild-type MHV-A59 residue (top line) are shown; second-site mutations in the revertants are in boldface type.

FIG. 6

Thermal inactivation of mutant 3 (Alb154: K63A, K67A), mutant 4 (Alb183: D60A, R61A, K63A), and the isogenic wild-type strain (Alb129). (A) Passage 3 stocks of each virus were grown at 33°C and thermally inactivated at 40°C (pH 6.5) for the indicated times (22). Titers of surviving viruses were determined at 37°C. (B) Same as panel A but with virus stocks grown at 39°C. The titers of virus stocks prior to heat treatment are indicated at the bottom.

FIG. 7

Electron microscopy of wild-type (Alb129) virions released from infected mouse 17Cl1 cells at 33°C. Freshly passaged virus was viewed by electron microscopy following negative staining with sodium phosphotungstate.

FIG. 8

Electron microscopy of virions of E protein mutant 4 (Alb183: D60A, R61A, K63A) released from infected mouse 17Cl1 cells at 33°C. Freshly passaged virus was viewed by electron microscopy following negative staining with sodium phosphotungstate.