L* protein of Theiler's murine encephalomyelitis virus is required for virus growth in a murine macrophage-like cell line

Abstract

We sought to confirm the importance of L* protein for growth of Theiler's murine encephalomyelitis virus (TMEV) in a macrophage-like cell line, J774-1. The protein is out of frame with the polyprotein and synthesized in DA but not GDVII subgroup strains of TMEV. A recombinant virus, DANCL*/GD, which substitutes the DA 5' noncoding and L* coding regions for the corresponding regions of GDVII and synthesizes L* protein, grew with little restriction in J774-1 cells. In contrast, another recombinant virus, DANCL*-1/GD, which has an ACG rather than an AUG as the starting codon of L* protein at nucleotide 1079, resulting in no synthesis of L* protein, did not grow well. No significant difference between the rates of adsorption to J774-1 cells of these viruses was observed. RNase protection assay demonstrated that DANCL*/GD viral RNA significantly increased, whereas only a minimal increase was observed for DANCL*-1/GD. The present study suggests that L* protein is required for virus growth in macrophages.

FIG. 1

Parental and recombinant cDNAs generated to delineate the role of L* protein for virus growth in J774-1 cells. The positions of the TMEV noncoding and coding areas are shown at the top. The DA genome and segments from it are shown as open bars, and the GDVII genome is shown as closed bars. The stars indicate point mutations of U to C in the L* starting codon at nt 1080. NheI indicates an NheI site introduced at the nt 1547 L* stop codon.

FIG. 2

L* protein synthesis of the parental and the recombinant viruses. (A) BHK-21 cells infected with virus at an MOI of 10 PFU per cell were radiolabeled with l-[³⁵S]methionine as described in the text. Synthesized proteins were analyzed by SDS–15% PAGE. (B) Electrophoresed proteins were analyzed by Western blotting with a rabbit polyclonal antibody to a synthetic peptide and horseradish peroxidase-linked anti-rabbit immunoglobulin G as the first and the second antibodies, respectively. In both SDS-PAGE and Western blotting analyses, a distinct 17-kDa band was clearly demonstrated in DA- and DANCL*/GD-infected cells, but not in GDVII-, DANCL*-1/GD-, and mock-infected cells. Lanes: 1, DA-infected cells; 2, GDVII-infected cells; 3, DANCL*/GD-infected cells; 4, DANCL*-1/GD-infected cells; 5, mock-infected cells. The arrow in panel A indicates a 17-kDa protein.

FIG. 3

Kinetics of recombinant DANCL*/GD and DANCL*-1/GD viruses in BHK-21 (A) and J774-1 (B) cells. The culture supernatants and cell lysates of infected cells were harvested at the indicated times and subjected to titer determination by a standard plaque assay on BHK-21 cells. Data are the means of three independent experiments. Details of the construction are provided in the text and shown in Fig. 1. □, culture supernatant of DANCL*/GD-infected cells; ■, cell lysate of DANCL*/GD-infected cells; ○, culture supernatant of DANCL*-1/GD-infected cells; ●, cell lysate of DANCL*-1/GD-infected cells.

FIG. 4

Genomic RNA synthesis in the recombinant virus-infected J774-1 cells. Total RNA was extracted from the cells of DANCL*/GD or DANCL*-1/GD virus at 3 and 9 h p.i. A 2-μg quantity of RNA was hybridized with [α-³²P]UTP-labeled riboprobe and treated with RNase solution as described in the text. The RNase-resistant fragments were denatured and electrophoresed on a 5% polyacrylamide–8 M urea gel. Lanes P and 1, [α-³²P]UTP-labeled probe and mock-infected J774-1 cells, respectively; lanes 2 and 3, J774-1 cells infected with DANCL*/GD at 3 and 9 h p.i., respectively; lanes 4 and 5, J774-1 cells infected with DANCL*-1/GD at 3 and 9 h p.i., respectively.