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. 2002 Apr;76(8):3697-708.
doi: 10.1128/jvi.76.8.3697-3708.2002.

RNA replication of mouse hepatitis virus takes place at double-membrane vesicles

Rainer Gosert  1 , Amornrat Kanjanahaluethai, Denise Egger, Kurt Bienz, Susan C Baker
Affiliations

RNA replication of mouse hepatitis virus takes place at double-membrane vesicles

Rainer Gosert et al. J Virol. 2002 Apr.

Abstract

The replication complexes (RCs) of positive-stranded RNA viruses are intimately associated with cellular membranes. To investigate membrane alterations and to characterize the RC of mouse hepatitis virus (MHV), we performed biochemical and ultrastructural studies using MHV-infected cells. Biochemical fractionation showed that all 10 of the MHV gene 1 polyprotein products examined pelleted with the membrane fraction, consistent with membrane association of the RC. Furthermore, MHV gene 1 products p290, p210, and p150 and the p150 cleavage product membrane protein 1 (MP1, also called p44) were resistant to extraction with Triton X-114, indicating that they are integral membrane proteins. The ultrastructural analysis revealed double-membrane vesicles (DMVs) in the cytoplasm of MHV-infected cells. The DMVs were found either as separate entities or as small clusters of vesicles. To determine whether MHV proteins and viral RNA were associated with the DMVs, we performed immunocytochemistry electron microscopy (IEM). We found that the DMVs were labeled using an antiserum directed against proteins derived from open reading frame 1a of MHV. By electron microscopy in situ hybridization (ISH) using MHV-specific RNA probes, DMVs were highly labeled for both gene 1 and gene 7 sequences. By combined ISH and IEM, positive-stranded RNA and viral proteins localized to the same DMVs. Finally, viral RNA synthesis was detected by labeling with 5-bromouridine 5'-triphosphate. Newly synthesized viral RNA was found to be associated with the DMVs. We conclude from these data that the DMVs carry the MHV RNA replication complex and are the site of MHV RNA synthesis.

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Figures

FIG. 1.
FIG. 1.
Identification of MHV ORF1a products recognized by anti-D14 serum. (A) Detection of p150 and p22 ORF1a proteolytic products. HeLa-MHVR cells were infected with MHV A59, and newly synthesized proteins were labeled with Trans35S-label from 3.5 to 5.5 hpi. Total lysates of uninfected (U) or infected (I) cells were subjected to immunoprecipitation with preimmune (Pre) or anti-D14 serum. Products were analyzed by electrophoresis on an SDS-5.0 to 12.5% polyacrylamide gel and subjected to autoradiography. (B) Pulse-chase analysis of MHV JHM-x-infected cells. Proteins were pulse-labeled with Trans35S-label from 4.5 to 5.0 hpi. Labeling medium was then replaced with complete medium containing 10× methionine and cysteine. Cells were lysed at the chase times indicated, and total lysates were subjected to immunoprecipitation with anti-D14 serum. Products were analyzed on an SDS-7.5 to 12.5% polyacrylamide gel and subjected to autoradiography. Sizes are shown on the left in kilodaltons.
FIG. 2.
FIG. 2.
Identification of polyprotein ORF1ab precursors and products of MHV A59-infected HeLa-MHVR cells labeled in the absence (−) or presence (+) of protease inhibitor E64d. (A) Infected cells were either untreated or treated with 400 μg of protease inhibitor E64d per ml at 3 hpi for 30 min. Then, proteins were labeled with Trans35S-label in the presence or absence of E64d for 2 h. Total lysates were prepared and subjected to immunoprecipitation with the indicated antisera. Products were analyzed by electrophoresis as in Fig. 1. Lanes 5 and 6 provide a longer exposure of the results depicted in lanes 3 and 4. Sizes are shown on the left in kilodaltons. (B) Schematic diagram depicting the MHV A59 genome and our current understanding of the proteolytic processing cascade of the RNA-dependent RNA polymerase (ORF1ab) polyprotein. The locations of antiserum determinants and riboprobes is indicated above the MHV genome. aa's, amino acids.
FIG. 3.
FIG. 3.
MHV ORF1ab products detected from the postnuclear supernatant subjected to carbonate buffer at pH 7.0 (A) and pH 11.0 (B) and fractionated by centrifugation. HeLa-MHVR cells were infected with MHV A59, and proteins were metabolically labeled with Trans35S-label from 3.0 to 5.0 hpi. Cells were harvested and broken open with a Dounce cell homogenizer, and a postnuclear supernatant was prepared as described in Materials and Methods. One half of the sample was kept at pH 7.0, while the other half was treated with 100 mM sodium carbonate (pH 11.0) to remove peripheral proteins from the membranes (19). Samples were then subjected to ultracentrifugation to distinguish the soluble (S) and membranous pellet (P)-associated proteins. Soluble and pellet fractions were subjected to immunoprecipitation with anti-ORF1a and -ORF1b sera. Coprecipitating products are indicated by open arrowheads. Products were analyzed as described for Fig. 1. Sizes are shown on the left in kilodaltons.
FIG. 4.
FIG. 4.
Identification of MHV A59 ORF1a integral membrane proteins from infected-cell lysates treated with Triton X-114. HeLa-MHVR cells were infected with MHV A59, and proteins were metabolically labeled with Trans35S-label from 3.5 to 5.5 hpi. Cells were lysed in PBS containing 1% Triton X-114, and nuclei were removed by low-speed centrifugation. The cytosolic fraction was separated by centrifugation through a 6% (wt/vol) sucrose cushion as described in Materials and Methods. The detergent pellet fraction (P) and the soluble fraction (S) were subjected to immunoprecipitation with the indicated antisera. Products were analyzed as described for Fig. 1. Coprecipitating products are indicated by open arrowheads. Sizes are shown on the left in kilodaltons.
FIG. 5.
FIG. 5.
EM analysis of membrane alterations in MHV A59-infected cells. (A) Electron micrograph showing multiple DMVs in the cytoplasm of MHV-infected HeLa-MHVR cells at 5 hpi. Bar, 1 μm. (B) DMVs seen in MHV-infected 17Cl-1 cells at 7 hpi. The double membrane is fused into a trilayer. Inset: For comparison, Golgi apparatus composed of a bilayer membrane. Arrowheads indicate the thickness of the membranes. Bar, 100 nm.
FIG. 5.
FIG. 5.
EM analysis of membrane alterations in MHV A59-infected cells. (A) Electron micrograph showing multiple DMVs in the cytoplasm of MHV-infected HeLa-MHVR cells at 5 hpi. Bar, 1 μm. (B) DMVs seen in MHV-infected 17Cl-1 cells at 7 hpi. The double membrane is fused into a trilayer. Inset: For comparison, Golgi apparatus composed of a bilayer membrane. Arrowheads indicate the thickness of the membranes. Bar, 100 nm.
FIG. 6.
FIG. 6.
IEM and ISH analysis of MHV proteins and RNA in infected HeLa-MHVR cells at 5 hpi. (A) MHV ORF1a proteins are associated with DMVs. MHV ORF1a proteins were detected using IEM with rabbit anti-D3 serum and anti-rabbit Ig-15-nm gold conjugate. The majority of the protein detected is associated with DMVs, with some label associated with electron-dense regions. (B) MHV RNA is associated with DMVs. RNA was detected using a DIG-labeled riboprobe to MHV gene 7 and 10-nm-gold-tagged anti-DIG antibody. The majority of the RNA is associated with the DMVs, with some RNA labeled in a chain-like pattern. The riboprobe entirely hybridized to the target RNA covers a distance of approximately 160 nm. Bars, 200 nm.
FIG. 7.
FIG. 7.
Combined IEM-ISH and double ISH performed on MHV-infected HeLa-MHVR cells at 5 hpi. (A) MHV ORF1a proteins detected by anti-D3 serum and secondary antibody coupled to 15-nm gold particles, and viral RNA detected by DIG-labeled riboprobe complementary to MHV gene 7 and anti-DIG antibody coupled to 10-nm gold particles localize to the same DMVs. (B) Subgenomic and genomic RNAs exist on the same DMVs. Probe 1, complementary to MHV gene 7 sequences, was detected using 10-nm-gold-tagged sheep anti-DIG antibody. Probe 2, complementary to a region of gene 1 sequences, was detected using 6-nm-gold-tagged antibiotin antibody. Some of the DMVs carrying both labels are indicated by arrowheads. Bars, 200 nm.
FIG. 8.
FIG. 8.
Detection of newly synthesized viral RNA in MHV-infected HeLa-MHVR cells at 5 hpi. MHV-infected cells were incubated with BrUTP in the presence of actinomycin D for 1 h to allow incorporation of label into newly synthesized MHV RNA. BrU-RNA was detected using an anti-BrdU antibody and goat anti-mouse Ig antibody coupled to 10-nm gold particles. The BrU-RNA was detected in association with the DMVs. Bar, 200 nm.
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