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. 1998 Aug;72(8):6689-98.
doi: 10.1128/JVI.72.8.6689-6698.1998.

ORF1a-encoded replicase subunits are involved in the membrane association of the arterivirus replication complex

Y van der Meer  1 , H van Tol, J K Locker, E J Snijder
Affiliations

ORF1a-encoded replicase subunits are involved in the membrane association of the arterivirus replication complex

Y van der Meer et al. J Virol. 1998 Aug.

Abstract

Among the functions of the replicase of equine arteritis virus (EAV; family Arteriviridae, order Nidovirales) are important viral enzyme activities such as proteases and the putative RNA polymerase and RNA helicase functions. The replicase is expressed in the form of two polyproteins (open reading frame 1a [ORF1a] and ORF1ab), which are processed into 12 nonstructural proteins by three viral proteases. In immunofluorescence assays, the majority of these cleavage products localized to the perinuclear region of the cell. A dense granular and vesicular staining was observed, which strongly suggested membrane association. By using confocal microscopy and double-label immunofluorescence, the distribution of the EAV replicase was shown to overlap with that of PDI, a resident protein of the endoplasmic reticulum and intermediate compartment. An in situ labeling of nascent viral RNA with bromo-UTP demonstrated that the membrane-bound complex in which the replicase subunits accumulate is indeed the site of viral RNA synthesis. A number of ORF1a-encoded hydrophobic domains were postulated to be involved in the membrane association of the arterivirus replication complex. By using various biochemical methods (Triton X-114 extraction, membrane purification, and sodium carbonate treatment), replicase subunits containing these domains were shown to behave as integral membrane proteins and to be membrane associated in infected cells. Thus, contribution to the formation of a membrane-bound scaffold for the viral replication-transcription complex appears to be an important novel function for the arterivirus ORF1a replicase polyprotein.

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Figures

FIG. 1
FIG. 1
Proteolytic processing scheme, hydrophobicity plot, and subunit nomenclature of the EAV ORF1a and ORF1ab replicase polyproteins (39, 49, 52). The three EAV protease domains (papainlike cysteine protease [PCP], cysteine protease [CP], and serine protease [SP]) and their cleavage sites (arrows and arrowheads) are shown. In the ORF1b-encoded polypeptide, the four major domains conserved in nidoviruses are depicted: POL, putative RNA-dependent RNA polymerase; M, putative metal-binding domain; HEL, putative RNA helicase; C, conserved C-terminal domain specific for nidoviruses. The hydrophobicity plot was generated by the method of Kyte and Doolittle (24). Above the axis is hydrophobic. aa, amino acid.
FIG. 2
FIG. 2
Immunofluorescence analysis showing the time course of the intracellular distribution of the EAV replicase in Vero cells. Cells were mock infected (A) or EAV infected and fixed at 4 h (B), 6 h (C), 8 h (D), 10 h (E), and 12 h (F) p.i. Subsequently, the cells were processed for indirect immunofluorescence analysis with an anti-nsp2 antiserum (39). Photographs were generated with the same exposure times for recording and printing. Essentially identical results were obtained with antisera recognizing nsp4, nsp7–8, nsp8, nsp9, nsp10, and nsp12 (reference and data not shown).
FIG. 3
FIG. 3
Colocalization of the EAV replicase and the cellular protein PDI, a marker for ER and IC. Three different cell lines were used: BHK-21 (A to C), RK-13 (D to F), and Vero (G to I). These were EAV infected, fixed at 8 h p.i., and processed for double-label immunofluorescence analysis with a mouse MAb directed against PDI (50), an anti-nsp2 rabbit antiserum (39), and appropriate secondary antibodies conjugated to fluorescent tags. PDI is shown in red (A, D, and G), and EAV nsp2 is shown in green (B, E, and H). For each sample, the differentially fluorescing images were recorded from the same optical section by using a confocal microscope. A computer-generated overlay of the PDI and EAV nsp2 images is shown (C, F, and I).
FIG. 4
FIG. 4
Intracellular distribution of the EAV replicase (nsp2) and the major EAV glycoprotein GL, which can, at the early stages of infection, be used as a marker for the Golgi complex (see the text). Vero cells were EAV infected, fixed at 6 h (A to C) and 10 h (D to F) p.i., and processed for double-label immunofluorescence analysis with a mouse MAb directed against GL (18), the anti-nsp2 rabbit antiserum (39), and appropriate fluorescent conjugates. GL is shown in red (A and D), and EAV nsp2 is shown in green (B and E). For each sample, the differentially fluorescing images were recorded from the same optical section by using a confocal microscope. Computer-generated overlays of the GL and nsp2 images are also shown (C and F).
FIG. 5
FIG. 5
Colocalization of the EAV replicase (nsp2) and viral RNA synthesis. BHK-21 (A and B) and RK-13 (C and D) cells were EAV infected, and de novo-synthesized viral RNA was labeled in situ by using BrUTP. Dactinomycin was used to shut off host cell RNA synthesis, and BrUTP was subsequently introduced into the cells by using liposomes. The cells were fixed at 7.5 h p.i. and processed for double-label immunofluorescence analysis with the anti-nsp2 rabbit antiserum (39), a rat MAb recognizing BrUTP-labeled RNA, and appropriate fluorescent conjugates. The labeling of the same cells for nsp2 (A and C) and BrUTP-labeled RNA (B and D) is shown. The presence of nsp2-positive but BrUTP-negative cells is explained by the fact that BrUTP could be introduced into only 20 to 40% of the cells (see the text).
FIG. 6
FIG. 6
TX-114 extraction analysis of ORF1a- and ORF1b-encoded EAV replicase subunits. The replicase processing scheme and nsp nomenclature are shown at the top of the figure, with solid boxes representing hydrophobic domains (Fig. 1). RK-13 cells were EAV infected, and viral proteins were 35S labeled from 5 to 8 h p.i. Cells were lysed with 1% TX-114, and the lysate was subjected to three rounds of TX-114 extraction (see Materials and Methods) (4). Detergent pellets (p) and supernatants (s) were pooled and diluted in immunoprecipitation buffer. EAV replicase subunits were immunoprecipitated with a set of previously described rabbit antisera (39, 49) recognizing nsp1 (α1), nsp2 (α2), nsp4 (α4), nsp7–8 (α78), nsp9 (α9), and nsp10 (α10). The samples were analyzed by SDS-PAGE and autoradiography.
FIG. 7
FIG. 7
Membrane association of ORF1a- and ORF1b-encoded EAV replicase subunits. The replicase-processing scheme and nsp nomenclature are shown at the top of the figure, with solid boxes representing hydrophobic domains (Fig. 1). RK-13 cells were EAV infected, and viral proteins were 35S labeled from 5 to 8 h p.i. Cells were broken with a Dounce cell homogenizer, and a PNS was prepared. Half of the PNS was kept at pH 7, whereas the other half was treated with 100 mM sodium carbonate at pH 11 to remove integral and peripheral proteins from the membranes (16). Subsequently, membrane (m) and cytoplasmic (c) fractions were prepared by ultracentrifugation, diluted in immunoprecipitation buffer, and used for an immunoprecipitation analysis with the same set of antisera described in the legend to Fig. 6. The immunoprecipitation samples were analyzed by SDS-PAGE and autoradiography.
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