A new cistron in the murine hepatitis virus replicase gene

Abstract

We report an RNA-negative, temperature-sensitive (ts) mutant of Murine hepatitis virus, Bristol ts31 (MHV-Brts31), that defines a new complementation group within the MHV replicase gene locus. MHV-Brts31 has near-normal levels of RNA synthesis at the permissive temperature of 33 degrees C but is unable to synthesize viral RNA when the infection is initiated and maintained at the nonpermissive temperature of 39.5 degrees C. Sequence analysis of MHV-Brts31 RNA indicated that a single G-to-A transition at codon 1307 in open reading frame 1a, which results in a replacement of methionine-475 with isoleucine in nonstructural protein 3 (nsp3), was responsible for the ts phenotype. This conclusion was confirmed using a vaccinia virus-based reverse genetics system to produce a recombinant virus, Bristol tsc31 (MHV-Brtsc31), which has the same RNA-negative ts phenotype and complementation profile as those of MHV-Brts31. The analysis of protein synthesis in virus-infected cells showed that, at the nonpermissive temperature, MHV-Brtsc31 was not able to proteolytically process either p150, the precursor polypeptide of the replicase nonstructural proteins nsp4 to nsp10, or the replicase polyprotein pp1ab to produce nsp12. The processing of replicase polyprotein pp1a in the region of nsp1 to nsp3 was not affected. Transmission electron microscopy showed that, compared to revertant virus, the number of double-membrane vesicles in MHV-Brts31-infected cells is reduced at the nonpermissive temperature. These results identify a new cistron in the MHV replicase gene locus and show that nsp3 has an essential role in the assembly of a functional MHV replication-transcription complex.

FIG. 1.

(A) Plaque morphology of MHV-A59, MHV-Brts31, MHV-Br31R1, MHV-inf-1, MHV-Brtsc31, and MHV-Br31cR1. 17Cl-1 cells were infected with 10 to 20 PFU of virus, overlaid with solid medium, and maintained at 33°C for 3 days or 37°C and 39.5°C for 2 days. The overlay was removed, and the cells were fixed and stained as described in Materials and Methods. (B) Virus RNA accumulation in MHV-Brts31 and MHV-Br31R1 virus-infected cells at permissive and nonpermissive temperatures. RNA was extracted from cells that had been infected with MHV-A59, MHV-Brts31, or MHV-Br31R1 and maintained at either 33°C or 39.5°C. After electrophoresis and transfer to membrane, the virus RNA was detected with an α-³²P-random-prime-labeled PCR probe derived from the MHV-A59 nucleocapsid protein gene. (C) Virus RNA accumulation in MHV-inf-1 and MHV-Brtsc31 virus-infected cells at permissive and nonpermissive temperatures. RNA was extracted from cells that had been infected with MHV-inf-1 or MHV-Brtsc31 and maintained at either 33°C or 39.5°C. After electrophoresis and transfer to membrane, the virus RNA was detected with an α-³²P-random-prime-labeled PCR probe derived from the MHV-A59 nucleocapsid protein gene.

FIG. 2.

(A) RNA synthesis phenotype of the MHV-Brts31 mutant. RNA synthesis was determined using 1-h pulse-labels with [³H]uridine in the presence of actinomycin D, given to mock (▪)-, MHV-A59 (▴)-, and MHV-Brts31 (○)-infected cells 4 to 10 h postinfection at 33°C (left panel) or mock (▪)-, MHV-A59 (▴)-, and MHV-Br31R1 (⋄)-infected cells 1 to 7 h postinfection at 39.5°C (right panel). Duplicate samples of 5 × 10⁴ cells were precipitated with trichloroacetic acid, and the incorporated radioactivity was determined. The duplicates were reproducible to within 2% of each other. (B) Temperature sensitivity of RNA synthesis in MHV-Brts31 and MHV-Br31R1 virus-infected cells. RNA synthesis was determined using 30-min pulse-labels with [³H]uridine in the presence of actinomycin D, with or without the addition of cycloheximide (CH), after shifting the incubation temperature of MHV-Brts31- or MHV-Br31R1-infected cells from 33°C to 39.5°C at 4.75 h postinfection (p.i.). Dark gray bars, 0- to 30-min pulse; light gray bars, 30- to 60-min pulse. Triplicate samples of 5 × 10⁴ cells were precipitated with trichloroacetic acid, and the incorporated radioactivity was determined. The triplicates were reproducible to within 2% of each other.

FIG. 3.

Regions recognized by MHV nsp-specific antisera and location of the MHV-Brts31 M475I mutation. The MHV replicase gene comprises two ORFs, ORF1a and ORF1b, connected by a (−1) ribosomal frameshift, which encode the viral replicase polyproteins. The replicase polyproteins are processed by papain-like cysteine protease activities 1 and 2 (PLP1 and PLP2, respectively, in nsp3) and the 3C-like cysteine protease activity (3CLpro in nsp5) to generate intermediates and mature nonstructural proteins (nsp's), which assemble to generate functional replication-transcription complexes. The site of the ts lesion in the MHV-Brts31 nsp3 open reading frame is indicated by the star. Domains recognized by the polyclonal antibodies used in the immunoprecipitation assays are indicated by black bars.

FIG. 4.

Analysis of proteolytic products generated in MHV-Brtsc31 and MHV-Br31cR1 virus-infected cells at permissive and nonpermissive temperatures. Newly synthesized proteins were radiolabeled with ³⁵S-trans label in MHV-Brtsc31- (A) or MHV-Br31cR1-infected cells (B) at either the permissive (33°C) or nonpermissive (39.5°C) temperature and subjected to immunoprecipitation with polyclonal antisera specific to nsp1 (lanes 1 and 6), nsp2 and nsp3 (lanes 2 and 7), nsp4 (lanes 3 and 8), nsp5 (lanes 4 and 9), and nsp8 (lanes 5 and 10). The products were analyzed by 5 to 12.5% SDS-PAGE and visualized by autoradiography. ¹⁴C molecular mass markers (GE Healthcare) are shown in lanes M.

FIG. 5.

Analysis of proteolytic products generated in MHV-Brtsc31 and MHV-Br31cR1 virus-infected cells at permissive and nonpermissive temperatures. Newly synthesized proteins were radiolabeled with ³⁵S-trans label in MHV-Brtsc31- (A) or MHV-Br31cR1-infected cells (B) at either the permissive (33°C) or nonpermissive (39.5°C) temperature and subjected to immunoprecipitation with polyclonal antisera specific to nsp5 (odd-numbered lanes) and nsp12 (even-numbered lanes). The products were analyzed by 5 to 12.5% SDS-PAGE and visualized by autoradiography.

FIG. 6.

Analysis of double-membrane vesicles in MHV-Brts31 and MHV-Br31R1 virus-infected cells at permissive and nonpermissive temperatures. 17Cl-1 cells were infected with either MHV-Brts31 or MHV-Br31R1 at an MOI of 20.0 and incubated at 33°C for 5.5 h (A, C, and E) or initially incubated at 33°C and then shifted to 39.5°C at 3.5 to 5.5 h postinfection (B, D, and F). At 5.5 h postinfection, cells were harvested and processed for TEM analysis. DMVs can be visualized by TEM as dark-ringed vesicles in the cytoplasm of MHV-infected cells (arrows). Asterisks denote rare aberrant vesicles detected at the nonpermissive temperature in MHV-Brts31-infected cells. Bars, 1 μm (A to D) or 0.1 μm (E and F).