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. 2003 Apr;77(8):4597-608.
doi: 10.1128/jvi.77.8.4597-4608.2003.

The small envelope protein E is not essential for murine coronavirus replication

Lili Kuo  1 , Paul S Masters
Affiliations

The small envelope protein E is not essential for murine coronavirus replication

Lili Kuo et al. J Virol. 2003 Apr.

Abstract

The importance of the small envelope (E) protein in the assembly of coronaviruses has been demonstrated in several studies. While its precise function is not clearly defined, E is a pivotal player in the morphogenesis of the virion envelope. Expression of the E protein alone results in its incorporation into vesicles that are released from cells, and the coexpression of the E protein with the membrane protein M leads to the assembly of coronavirus-like particles. We have previously generated E gene mutants of mouse hepatitis virus (MHV) that had marked defects in viral growth and produced virions that were aberrantly assembled in comparison to wild-type virions. We have now been able to obtain a viable MHV mutant in which the entire E gene, as well as the nonessential upstream genes 4 and 5a, has been deleted. This mutant (Delta E) was obtained by a targeted RNA recombination method that makes use of a powerful host range-based selection system. The Delta E mutant produces tiny plaques with an unusual morphology compared to plaques formed by wild-type MHV. Despite its low growth rate and low infectious titer, the Delta E mutant is genetically stable, showing no detectable phenotypic changes after several passages. The properties of this mutant provide further support for the importance of E protein in MHV replication, but surprisingly, they also show that E protein is not essential.

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Figures

FIG.1.
FIG.1.
Selection of the ΔE mutant. (A) Construction of a transcription vector for synthesis of donor RNA. Plasmid pLK70 was derived from pMH54 (17), as detailed in Materials and Methods. In each plasmid schematic, the arrow indicates the T7 promoter, and the solid circle represents the linker between cDNA segments corresponding to the 5′ and 3′ ends of the MHV genome. The restriction sites shown are those relevant to plasmid construction (Sse8387I, BssHII, and NheI), in vitro transcription (PacI), or mutant analysis (SmlI). The DNA sequences shown are as follows: 1 and 2, the boundaries of the region that was deleted from the wild type (wt); and 3, the newly created junction in the deletion mutant. The underlined nucleotides are the three base changes made in pMH54 that generated an Sse8387I site (17). Also indicated are the TRSs governing synthesis of sgRNA4 and the M mRNA. Shown beneath the DNA sequences are translations of the S, M, and E ORFs (asterisks indicate stop codons). (B) Scheme for generation of the ΔE mutant by targeted RNA recombination between the interspecies chimera fMHV (17) and donor RNA transcribed from plasmid pLK70. fMHV contains the ectodomain-encoding region of the FIPV S gene (hatched rectangle) and is able to grow in feline cells but not in murine cells. A single crossover (solid line), within the HE gene, should generate a recombinant that has simultaneously reacquired the MHV S ectodomain and the ability to grow in murine cells and has also incorporated the deletion of the E gene. A potential second crossover (broken line), in the distal portion of the S gene, would produce a recombinant retaining the E gene. At the bottom are shown the mixed progeny of two independent targeted recombination experiments, forming tiny and large plaques on mouse L2 cells. The monolayers were stained with neutral red 75 h postinfection and were photographed 19 h later.
FIG. 2.
FIG. 2.
Titration of passage 2 stocks obtained from purified tiny plaques from independent infection-transfection experiments. Two dilutions each of the ΔE mutants Alb289 and Alb291 are shown (top, 10−2; bottom, 10−3) to emphasize the atypical morphology of the tiny plaques. Also shown, for comparison, are plaques of Alb240, a wild-type (wt) recombinant derived from pMH54 and fMHV (17), and mock-infected cells (mock). The titers of the viruses were determined on mouse L2 cell monolayers, which were stained with neutral red 75 h postinfection and photographed 19 h later.
FIG. 3.
FIG. 3.
Analysis of large-plaque and small-plaque progeny from targeted recombination. (A) RNA was isolated from cells infected with plaque-purified recombinants, reverse transcribed with random primer p(N)6, and amplified with primers CK4 and PM147. The PCR products were analyzed by agarose gel electrophoresis. Large plaques (#1 to 4) were purified from independent infection-transfection experiments. The small-plaque recombinants Alb289 through Alb296 represent four independent sets of mutants in which consecutively numbered odd-even pairs are siblings. Lanes M, DNA fragment size markers. wt, wild type; mock, mock-infected cells. (B) Sequence of the S gene-M gene junction in the ΔE mutant Alb291. The junction sequences of Alb289, Alb290, and Alb292 to Alb296 were identical.
FIG. 4.
FIG. 4.
RT-PCR analysis to rule out the presence of fMHV in purified ΔE recombinants. The random-primed RT product obtained with RNA isolated from infected cells was amplified with primer pairs either unique to fMHV or common to fMHV, wild-type (wt) MHV, and the ΔE mutant. In the genomic schematics at the top, the hatched region indicates the ectodomain-encoding portion of the FIPV S gene; primer positions are not drawn strictly to scale. The PCR products were analyzed by agarose gel electrophoresis. Lanes M, DNA fragment size markers; mock, mock-infected cells.
FIG. 5.
FIG. 5.
RT-PCR analysis to rule out the presence of the E gene elsewhere in the genome. The random-primed RT product obtained with RNA isolated from infected cells was amplified with primer pairs internal to the wild-type (wt) E ORF or including the E ORF and the upstream gene 5a. The PCR products were analyzed by agarose gel electrophoresis. Lanes M, DNA fragment size markers; mock, mock-infected cells.
FIG. 6.
FIG. 6.
Analysis of RNA species synthesized by the ΔE mutant. (A) Virus-specific RNA was labeled with [33P]orthophosphate in the presence of actinomycin D in cells infected with wild-type (wt) recombinant Alb240 or ΔE mutant Alb291 or in mock-infected (mock) control cells. Purified RNA was analyzed in a formaldehyde-agarose gel, as detailed in Materials and Methods. (B) Northern blot analysis of unlabeled RNA isolated from cells infected with wild-type recombinant Alb240 or ΔE mutants Alb291 and Alb293 or from mock-infected control cells. Purified RNA was separated in a formaldehyde-agarose gel and transferred to a nylon filter. Alternatively, RNA was directly dot blotted onto nylon filters; each set of dots corresponds to serial twofold dilutions, starting with 5 μg of total cellular RNA. The RNA was hybridized with a 32P-labeled DNA probe specific either for the E gene or for the 3′ UTR of the MHV genome; bound probe was visualized by fluorography. The Alb240 lane marked with an asterisk is a short exposure of the adjacent, overexposed lane.
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