Characterization of an equine arteritis virus replicase mutant defective in subgenomic mRNA synthesis

Abstract

Equine arteritis virus (EAV) is a positive-stranded RNA virus that synthesizes a 5'- and 3'-coterminal nested set of six subgenomic mRNAs. These mRNAs all contain a common leader sequence which is derived from the 5' end of the genome. Subgenomic mRNA transcription and genome replication are directed by the viral replicase, which is expressed in the form of two polyproteins and subsequently processed into smaller nonstructural proteins (nsps). During the recent construction of an EAV infectious cDNA clone (pEAV030 [L. C. van Dinten, J. A. den Boon, A. L. M. Wassenaar, W. J. M. Spaan, and E. J. Snijder, Proc. Natl. Acad. Sci. USA 94:991-996, 1997]), a mutant cDNA clone (pEAV030F) which carries a single replicase point mutation was obtained. This substitution (Ser-2429-->Pro) is located in the nsp10 subunit and renders the EAV030F virus deficient in subgenomic mRNA synthesis. To obtain more insight into the role of nsp10 in transcription and the nature of the transcriptional defect, we have now analyzed the EAV030F mutant in considerable detail. The Ser-2429-->Pro mutation does not affect the proteolytic processing of the replicase but apparently affects the function of nsp10 in transcription. Furthermore, our study showed that EAV030F still produces subgenomic positive and negative strands, albeit at a very low level. Both subgenomic positive-strand synthesis and negative-strand synthesis are equally affected by the Ser-2429-->Pro mutation, suggesting that nsp10 plays an important role in an early step of EAV mRNA transcription.

FIG. 1

EAV genome organization and replication strategy. The EAV genome with its genes and the replicase ORF1a and ORF1ab translation products are outlined at the top. The nested set of sg mRNAs is depicted below the genome. The black box represents the common 5′ leader sequence. The ORFs that are translated from each of the sg mRNAs are indicated in grey.

FIG. 2

(A) Processing scheme of the EAV replicase ORF1ab protein. The grey, white, and black triangles depict the cleavage sites of the ORF1a-encoded papainlike nsp1 cysteine protease (P), the nsp2 cysteine protease (CP), and the nsp4 serine protease (SP), respectively. The other boxes represent the polymerase domain (POL), the helicase domain (HEL), a putative metal-binding domain (M), and a C-terminal domain conserved in all nidoviruses (C). Also indicated are the nomenclature for the nsps, the location of Ser-2429 in nsp10, and the approximate positions of the epitopes for the anti-nsp7-8 and anti-nsp10 antisera. (B) Immunoprecipitation analysis of cells infected with EAV (lanes I), cells transfected with EAV030F (lanes F) or EAV030H (lanes H), or mock-transfected cells (lanes M). Cells were labeled from 6 to 8, 8 to 10, or 10 to 12 h. The results of the immunoprecipitation analysis using antisera directed against nsp10 (anti-nsp10) and nsp7-8 (anti-nsp7-8; see panel A) are shown.

FIG. 2

(A) Processing scheme of the EAV replicase ORF1ab protein. The grey, white, and black triangles depict the cleavage sites of the ORF1a-encoded papainlike nsp1 cysteine protease (P), the nsp2 cysteine protease (CP), and the nsp4 serine protease (SP), respectively. The other boxes represent the polymerase domain (POL), the helicase domain (HEL), a putative metal-binding domain (M), and a C-terminal domain conserved in all nidoviruses (C). Also indicated are the nomenclature for the nsps, the location of Ser-2429 in nsp10, and the approximate positions of the epitopes for the anti-nsp7-8 and anti-nsp10 antisera. (B) Immunoprecipitation analysis of cells infected with EAV (lanes I), cells transfected with EAV030F (lanes F) or EAV030H (lanes H), or mock-transfected cells (lanes M). Cells were labeled from 6 to 8, 8 to 10, or 10 to 12 h. The results of the immunoprecipitation analysis using antisera directed against nsp10 (anti-nsp10) and nsp7-8 (anti-nsp7-8; see panel A) are shown.

FIG. 3

(A) Analysis of [³H]uridine-labeled intracellular RNA isolated 12 h after infection or transfection from mock-transfected cells (lane M), cells infected with EAV (lane I), or cells transfected with EAV030F RNA (lane F) or EAV030H RNA (lane H). The RNAs were separated in a denaturing 1% agarose gel. (B) Schematic representation of the conversion of RIs into RFs by RNase treatment. (C) Analysis of [3H]uridine-labeled RF RNAs obtained 12 h postinfection or transfection from cells infected with EAV (lane I) and cells transfected with EAV030F RNA (lane F) or EAV030H RNA (lane H). The RFs corresponding to EAV RNA1 to RNA7 are indicated with I to VII, respectively. The RF RNAs were separated in a nondenaturing 1% agarose gel.

FIG. 4

(A) RT-PCR strategy for the detection of positive-stranded (I) and negative-stranded (II) genomic RNA and sg RNA7. Leader and antileader sequence are indicated with white and black boxes, respectively. Oligonucleotides E125 and E160 are directed against the body sequences of RNA1 and sg RNA7, respectively, and are used for priming cDNA synthesis (dashed line) on the positive strands of RNA1 and RNA7. Oligonucleotide E157 is directed against the antileader sequence (black box) and is used for cDNA synthesis on the negative strand. For the PCR, oligonucleotides E157 and E125 are used to amplify regions specific for positive- and negative-stranded RNA1. Oligonucleotides E157 and E160 are used for the PCR specific for positive- and negative-stranded sg RNA7. (B) RT-PCR analysis for the positive and negative strands of RNA1 (top panel) and positive and negative strands of sg RNA7 (bottom panel). The RNA was isolated from cells transfected with EAV030H (lanes H), EAV030F (lanes F), or EAV030SGA (lanes SGA) or from mock-transfected cells. The plus and minus signs indicate specificity of the RT-PCR for positive and negative strands, respectively.

FIG. 5

(A) Schematic representation of the EAV030H and EAV030F clones containing the CAT gene inserted upstream of ORF7 (EAV030HCAT7 and EAV030FCAT7) and the clones containing the CAT gene at the same position in the reverse orientation (EAV030HTAC7 and EAV030FTAC7). (B) CAT assays of lysates obtained from mock-transfected cells and cells transfected with EAV030HCAT7, EAV030FCAT7, EAV030HTAC7, EAV030FTAC7, EAV030H, or EAV030F. (C) Amount of acetylated chloramphenicol (in counts per minute as determined by liquid scintillation counting) for CAT assays using 100- and 1,000-fold dilutions of lysates of EAV030HCAT7-transfected cells or undiluted lysates obtained from cells transfected with EAV030FCAT7 and mock-transfected cells.

FIG. 6

Semiquantitative RT-PCR analysis to estimate the amounts of positive (+) and negative (−) strands for RNA1 (A) and sg RNA7 (B). In the RT reaction, serial 10-fold dilutions of RNA isolated from cells transfected with EAV030H (rows labeled H) or EAV030F (rows labeled F) were used. The subsequent PCRs (Fig. 4A) were identical for all samples.