Enhancement of murine coronavirus replication by severe acute respiratory syndrome coronavirus protein 6 requires the N-terminal hydrophobic region but not C-terminal sorting motifs

Abstract

Severe acute respiratory syndrome coronavirus encodes several accessory proteins of unknown function. We previously showed that one such protein, encoded by ORF6, enhanced the growth of mouse hepatitis virus in tissue culture cells and in mice. Protein 6 consists of an N-terminal hydrophobic peptide and a C-terminal region containing intracellular protein sorting motifs. Herein, we show that mutation of the hydrophobic region but not the sorting motifs affected the ability of protein 6 to enhance virus growth. Collectively, these results support the notion that the 6 protein interacts with membrane-bound viral replication or assembly machinery to directly enhance virus replication and virulence in animals.

FIG. 1.

Generation of rJ2.2 expressing SARS-CoV ORF6 variants. (A) Amino acid sequence of protein 6 showing the N-terminal amphipathic portion with interspersed charged residues (boldface) and the C terminus containing a tyrosine sorting motif (italics) and four diacidic motifs (underlining). (B) Helical wheel projection of the predicted N-terminal amphipathic helix (residues 1 to 38). Shaded and open circles indicate hydrophobic and hydrophilic residues, respectively. Red and blue letters indicates acidic and basic residues, respectively. (C) Protein 6 variants containing alanine substitutions to abrogate the function of the sorting motifs or deletions within the hydrophobic portion were generated and introduced into ORF4 of rJ2.2 by targeted recombination as previously described (14). HE, hemagglutinin-esterase; S, spike; E, envelope; M, transmembrane; N, nucleocapsid. (D) Virally encoded protein 6 variants were detected by Western blot assay using anti-HA MAb HA.11 (Covance, Berkeley, CA). (E) Confocal microscopy of 17Cl-1 cells infected with J2.2.6 and all variant viruses. Cells were infected at a multiplicity of infection of 0.1, fixed at 10 to 11 h postinfection, and stained with mouse anti-MHV M MAb (MAb 5B11.5; provided by M. Buchmeier, The Scripps Research Institute, La Jolla, CA) and fluorescein isothiocyanate-conjugated rat anti-HA (MAb 3F10; Roche Molecular Biochemicals, Mannheim, Germany) to detect protein 6. Nuclei were labeled with TO-PRO3 (Invitrogen, Carlsbad, CA).

FIG. 2.

Virus titers in cells infected with rJ2.2 expressing mutated SARS-CoV protein 6. (A to C) 17Cl-1 cells were infected (multiplicity of infection, 1.0) in triplicate with (A) rJ2.2, J2.2.6 or J2.2.6^KO, or (B) J2.2, rJ2.2.6^Δ3-10, rJ2.2.6^Δ11-18, or rJ2.2.6^Δ3-18 or (C) J2.2, rJ2.2.6^0(DxE), or rJ2.2.6^1(DxE). (D and E) L929 cells were infected with (D) rJ2.2.6, rJ2.2.6^KO, rJ2.2.6^Δ3-10, rJ2.2.6^Δ11-18 or rJ2.2.6^Δ3-18 or (E) rJ2.2.6, rJ2.2.6^KO, rJ2.2.6^0(DxE), and rJ2.2.6^1(DxE). Cells were harvested at the indicated times, and viral titers were determined by plaque assay on HeLa-MHVR cells. Error bars are included but are too small to be visible.

FIG. 3.

Morbidity, mortality, and viral titers in infected mice. B6 mice were infected intranasally with 1,000 PFU of protein 6 variant viruses or controls and monitored daily for mortality (A and B) and weight loss (C and D). (A and C) Mortality (A) and weight loss (C) after infection with rJ2.2.6 (n = 35), rJ2.2.6^KO (n = 31), or viruses expressing protein 6 with mutated sorting motifs [rJ2.2.6^0(DxE) (n = 19) or rJ2.2.6^1(DxE)] (n = 26). (B and D) Mortality (B) and weight loss (D) after infection with rJ2.2.6, rJ2.2.6^KO, or viruses expressing 6 protein with hydrophobic domain deletions (rJ2.2.6^Δ3-10 [n = 10], rJ2.2.6^Δ11-18 [n = 10], or rJ2.2.6^Δ3-18 [n = 18]). (E and F) Viral titers in brain homogenates from infected mice were determined by plaque assay on HeLa-MHVR cells. (E) Virus titers in mice infected with rJ2.2.6 (n = 5), rJ2.2.6^1(DxE) (n = 5), or rJ2.2.6^0(DxE) (n = 5) were greater than those infected with rJ2.2.6^KO (n = 5) (P < 0.01) at day 3 p.i. Titers for rJ2.2.6^1(DxE)-infected mice (n = 7) were greater than those infected with rJ2.2.6 (n = 5), rJ2.2.6^KO (n = 5), or rJ2.2.6^0(DxE) (n = 10) at day 5 p.i. (P < 0.05). (F) Titers in the brains of mice infected with rJ2.2.6^Δ3-18 (n = 4) were significantly lower than those found for mice infected with rJ2.2.6 (n = 5), rJ2.2.6^Δ3-10 (n = 6), or rJ2.2.6^Δ11-18 (n = 4) at day 7 p.i. (P < 0.01).

FIG. 4.

Subcellular positions of GFP appends on protein 6-GFP and GFP-protein 6 chimeras. HeLa cells were transfected with plasmids encoding p6-GFP (A) or GFP-p6 (B) and photographed 24 h later by confocal microscopy. Panels depict GFP fluorescence (left), positions of anti-GFP (α-GFP) antibodies after digitonin permeabilizations (middle), and a merging of the two images (right). Images in part C depict 293 cells transfected 24 h earlier with plasmids encoding MHV strain A59 M proteins. Cells were permeabilized with methanol (top panels) or digitonin (bottom panels) and incubated with anti-M (α-M) antibody J.1.3 (provided by J. Fleming, University of Wisconsin, Madison, WI), which binds an epitope in Golgi lumen (2, 3). Photographs reveal immunofluorescent (left), differential interference contrast (DIC; middle), and merged images (right). These experiments were conducted three times with identical results. Part D schematizes two putative configurations of protein 6 relative to intracellular membranes. endo, cytoplasmic; ecto, luminal.