Characterization of a murine coronavirus defective interfering RNA internal cis-acting replication signal.

The mouse hepatitis virus (MHV) sequences required for replication of the JHM strain of MHV defective interfering (DI) RNA consist of three discontinuous genomic regions: about 0.47 kb from both terminal sequences and a 0.13-kb internal region present at about 0.9 kb from the 5' end of the DI genome. In this study, we investigated the role of the internal 0.13-kb region in MHV RNA replication. Overall sequences of the 0.13-kb regions from various MHV strains were similar to each other, with nucleotide substitutions in some strains; MHV-A59 was exceptional, with three nucleotide deletions. Computer-based secondary-structure analysis of the 0.13-kb region in the positive strand revealed that most of the MHV strains formed the same or a similar main stem-loop structure, whereas only MHV-A59 formed a smaller main stem-loop structure. The RNA secondary structures in the negative strands were much less uniform among the MHV strains. A series of DI RNAs that contained MHV-JHM-derived 5'- and 3'-terminal sequences plus internal 0.13-kb regions derived from various MHV strains were constructed. Most of these DI RNAs replicated in MHV-infected cells, except that MRP-A59, with a 0.13-kb region derived from MHV-A59, failed to replicate. Interestingly, replication of MRP-A59 was temperature dependent; it occurred at 39.5 degrees C but not at 37 or 35 degrees C, whereas a DI RNA with an MHV-JHM-derived 0.13-kb region replicated at all three temperatures. At 37 degrees C, synthesis of MRP-A59 negative-strand RNA was detected in MHV-infected and MRP-A59 RNA-transfected cells. Another DI RNA with the internal 0.13-kb region deleted also synthesized negative-strand RNA in MHV-infected cells. MRP-A59-transfected cells were shifted from 39.5 to 37 degrees C at 5.5 h postinfection, a time when most MHV negative-strand RNAs have already accumulated; after the shift, MRP-A59 positive-strand RNA synthesis ceased. The minimum sequence required for maintenance of the positive-strand major stem-loop structure and biological function of the MHV-JHM 0.13-kb region was about 57 nucleotides. Function was lost in the 50-nucleotide sequence that formed a positive-strand stem-loop structure identical to that of MHV-A59. These studies suggested that the RNA structure made by the internal sequence was important for positive-strand MHV RNA synthesis.     

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

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.     

The Putative Helicase of the Coronavirus Mouse Hepatitis Virus Is Processed from the Replicase Gene Polyprotein and Localizes in Complexes That Are Active in Viral RNA Synthesis

The coronavirus mouse hepatitis virus (MHV) translates its replicase gene (gene 1) into two co-amino-terminal polyproteins, polyprotein 1a and polyprotein 1ab. The gene 1 polyproteins are processed by viral proteinases to yield at least 15 mature products, including a putative RNA helicase from polyprotein 1ab that is presumed to be involved in viral RNA synthesis. Antibodies directed against polypeptides encoded by open reading frame 1b were used to characterize the expression and processing of the MHV helicase and to define the relationship of helicase to the viral nucleocapsid protein (N) and to sites of viral RNA synthesis in MHV-infected cells. The antihelicase antibodies detected a 67-kDa protein in MHV-infected cells that was translated and processed throughout the virus life cycle. Processing of the 67-kDa helicase from polyprotein 1ab was abolished by E64d, a known inhibitor of the MHV 3C-like proteinase. When infected cells were probed for helicase by immunofluorescence laser confocal microscopy, the protein was detected in patterns that varied from punctate perinuclear complexes to large structures that occupied much of the cell cytoplasm. Dual-labeling studies of infected cells for helicase and bromo-UTP-labeled RNA demonstrated that the vast majority of helicase-containing complexes were active in viral RNA synthesis. Dual-labeling studies for helicase and the MHV N protein showed that the two proteins almost completely colocalized, indicating that N was associated with the helicase-containing complexes. This study demonstrates that the putative RNA helicase is closely associated with MHV RNA synthesis and suggests that complexes containing helicase, N, and new viral RNA are the viral replication complexes.     

Specific binding of host cellular proteins to multiple sites within the 3' end of mouse hepatitis virus genomic RNA.

The initial step in mouse hepatitis virus (MHV) RNA replication is the synthesis of negative-strand RNA from a positive-strand genomic RNA template. Our approach to begin studying MHV RNA replication is to identify the cis-acting signals for RNA synthesis and the proteins which recognize these signals at the 3' end of genomic RNA of MHV. To determine whether host cellular and/or viral proteins interact with the 3' end of the coronavirus genome, an RNase T1 protection/gel mobility shift electrophoresis assay was used to examine cytoplasmic extracts from mock- and MHV-JHM-infected 17Cl-1 murine cells for the ability to form complexes with defined regions of the genomic RNA. We demonstrated the specific binding of host cell proteins to multiple sites within the 3' end of MHV-JHM genomic RNA. By using a set of RNA probes with deletions at either the 5' or 3' end or both ends, two distinct binding sites were located. The first protein-binding element was mapped in the 3'-most 42 nucleotides of the genomic RNA [3' (+42) RNA], and the second element was mapped within an 86-nucleotide sequence encompassing nucleotides 171 to 85 from the 3' end of the genome (171-85 RNA). A single potential stem-loop structure is predicted for the 3' (+)42 RNA, and two stem-loop structures are predicted for the 171-85 RNA. Proteins interacting with these two elements were identified by UV-induced covalent cross-linking to labeled RNAs followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The RNA-protein complex formed with the 3'-most 42 nucleotides contains approximately five host polypeptides, a highly labeled protein of 120 kDa and four minor species with sizes of 103, 81, 70, and 55 kDa. The second protein-binding element, contained within a probe representing nucleotides 487 to 85 from the 3' end of the genome, also appears to bind five host polypeptides, 142, 120, 100, 55, and 33 kDa in size, with the 120-kDa protein being the most abundant. The RNA-protein complexes observed with MHV-infected cells in both RNase protection/gel mobility shift and UV cross-linking assays were identical to those observed with uninfected cells. The possible involvement of the interaction of host proteins with the viral genome during MHV replication is discussed.     

Methylation of newly synthesized ribonucleic acid by isolated rat liver nuclei. Characterization of the ribonucleic acid synthesized by nuclei from starved animals

1. Nuclei from rat liver incubated with S-adenosyl[methyl-(14)C]methionine incorporated radioactivity into RNA and into lipid and protein. 2. All of the labelled RNA was extracted from the nuclei with trichloroacetic acid at 90 degrees C. 3. The [(14)C]methyl-group incorporation into the hot-trichloroacetic acid extract was 30% inhibited by the addition of actinomycin D (100mug/mg of DNA) or by the omission of CTP, GTP and UTP. 4. Assuming that the main substrate for this triphosphate-dependent methylation was newly synthesized precursor rRNA containing one methyl group/30 uridylate residues, it was calculated that approx. 60% of the [(14)C]UMP incorporated under similar conditions represented precursor rRNA synthesis. 5. In agreement with this, low concentrations of actinomycin D (approx. 1mug/mg of DNA) sufficient to abolish the triphosphate-dependent incorporation of [(14)C]methyl group inhibited 68% of the [(14)C]UMP incorporation. 6. The incorporation of [(14)C]UMP by nuclei from starved animals decreased progressively with increasing periods of starvation, whereas the triphosphate-dependent [(14)C]methyl-group incorporation was not further decreased after 1 day of starvation. 7. This suggests that precursor rRNA synthesis decreased within 1 day whereas other species of RNA were affected only after longer periods of starvation.     

Isolation and characterization of nonhistone chromosomal protein C-14 which stimulates RNA synthesis.

The nonhistone chromatin protein, C-14, was extracted from chromatin of Novikoff hepatoma ascites cells and isolated in high purity as shown by its migration as a single dense spot on two-dimensional polyacrylamide gels. Its mobility on sodium dodecyl sulfate gels is consistent with a molecular weight of approximately 70 000. The amino acid composition shows that protein C-14 has an acidic:basic amino acid ratio of 1.8. Its amino terminal amino acid is lysine. Protein C-14 stimulated the incorporation of [3H]UMP into RNA by approximately 30% when added to naked DNA and homologous RNA polymerase I. A 30% stimulation of [3H]UMP incorporation into RNA was also found when protein C-14 was added to an E. coli RNA polymerase system containing either E. coli or Novikoff hepatoma DNA.     

Translation and processing of mouse hepatitis virus virion RNA in a cell-free system.

The first event after infection with mouse hepatitis virus strain A59 (MHV-A59) is presumed to be the synthesis of an RNA-dependent RNA polymerase from the input genomic RNA. The synthesis and processing of this putative polymerase protein was studied in a cell-free translation system utilizing 60S RNA from MHV-A59 virions. The polypeptide products of this reaction included two major species of 220 and 28 kilodaltons. Kinetics experiments indicated that both p220 and p28 appeared after 60 min of incubation and that protein p28 was synthesized initially as the N-terminal portion of a larger precursor protein. When the cell-free translation products were labeled with N-formyl[35S]methionyl-tRNAi, p28 was the predominant radioactive product, confirming its N-terminal location within a precursor protein. Translation in the presence of the protease inhibitors leupeptin and ZnCl2 resulted in the disappearance of p28 and p220 and the appearance of a new protein, p250. This product, which approached the maximal size predicted for a protein synthesized from genomic RNA, was not routinely detected in the absence of inhibitors even under conditions which optimized the translation reaction for elongation of proteins. Subsequent chelation of ZnCl2 resulted in the partial cleavage of the precursor protein and the reappearance of p28. One-dimensional peptide mapping with Staphylococcus aureus V-8 protease confirmed the precursor-product relationship of p250 and p28. The results show that MHV virion RNA, like many other viral RNAs, is translated into a large polyprotein, which is cleaved soon after synthesis into smaller, presumably functional proteins. This is in marked contrast to the synthesis of other MHV proteins, in which minimal proteolytic processing occurs.     

Coronavirus minus-strand RNA synthesis and effect of cycloheximide on coronavirus RNA synthesis.

The temporal sequence of coronavirus plus-strand and minus-strand RNA synthesis was determined in 17CL1 cells infected with the A59 strain of mouse hepatitis virus (MHV). MHV-induced fusion was prevented by keeping the pH of the medium below pH 6.8. This had no effect on the MHV replication cycle, but gave 5- to 10-fold-greater titers of infectious virus and delayed the detachment of cells from the monolayer which permitted viral RNA synthesis to be studied conveniently until at least 10 h postinfection. Seven species of poly(A)-containing viral RNAs were synthesized at early and late times after infection, in nonequal but constant ratios. MHV minus-strand RNA synthesis was first detected at about 3 h after infection and was found exclusively in the viral replicative intermediates and was not detected in 60S single-stranded form in infected cells. Early in the replication cycle, from 45 to 65% of the [3H]uridine pulse-labeled RF core of purified MHV replicative intermediates was in minus-strand RNA. The rate of minus-strand synthesis peaked at 5 to 6 h postinfection and then declined to about 20% of the maximum rate. The addition of cycloheximide before 3 h postinfection prevented viral RNA synthesis, whereas the addition of cycloheximide after viral RNA synthesis had begun resulted in the inhibition of viral RNA synthesis. The synthesis of both genome and subgenomic mRNAs and of viral minus strands required continued protein synthesis, and minus-strand RNA synthesis was three- to fourfold more sensitive to inhibition by cycloheximide than was plus-strand synthesis.     

Homologous RNA recombination allows efficient introduction of site-specific mutations into the genome of coronavirus MHV-A59 via synthetic co-replicating RNAs.

We describe a novel strategy to site-specifically mutagenize the genome of an RNA virus by exploiting homologous RNA recombination between synthetic defective interfering (DI) RNA and the viral RNA. The construction of a full-length cDNA clone, pMIDI, of a DI RNA of coronavirus MHV strain A59 was reported previously (R.G. Van der Most, P.J. Bredenbeek, and W.J.M. Spaan (1991). J. Virol. 65, 3219-3226). RNA transcribed from this construct, is replicated efficiently in MHV-infected cells. Marker mutations introduced in MIDI RNA were replaced by the wild-type residues during replication. More importantly, however, these genetic markers were introduced into viral genome: even in the absence of positive selection MHV recombinants could be isolated. This finding provides new prospects for the study of coronavirus replication using recombinant DNA techniques. As a first application, we describe the rescue of the temperature sensitive mutant MHV Albany-4 using DI-directed mutagenesis. Possibilities and limitations of this strategy are discussed.     

Complete replication of poliovirus in vitro: preinitiation RNA replication complexes require soluble cellular factors for the synthesis of VPg-linked RNA.

Translation of poliovirion RNA in HeLa S10 extracts resulted in the formation of RNA replication complexes which catalyzed the asymmetric replication of poliovirus RNA. Synthesis of poliovirus RNA was detected in unfractionated HeLa S10 translation reactions and in RNA replication complexes isolated from HeLa S10 translation reactions by pulse-labeling with [32P]CTP. The RNA replication complexes formed in vitro contained replicative-intermediate RNA and were enriched in viral protein 3CD and the membrane-associated viral proteins 2C, 2BC, and 3AB. Genome-length poliovirus RNA covalently linked to VPg was synthesized in large amounts by the replication complexes. RNA replication was highly asymmetric, with predominantly positive-polarity RNA products. Both anti-VPg antibody and guanidine HCl inhibited RNA replication and virus formation in the HeLa S10 translation reactions without affecting viral protein synthesis. The inhibition of RNA synthesis by guanidine was reversible. The reversible nature of guanidine inhibition was used to demonstrate the formation of preinitiation RNA replication complexes in reaction mixes containing 2 mM guanidine HCl. Preinitiation complexes sedimented upon centrifugation at 15,000 x g and initiated RNA replication upon their resuspension in reaction mixes lacking guanidine. Initiation of RNA synthesis by preinitiation complexes did not require active protein synthesis or the addition of soluble viral proteins. Initiation of RNA synthesis by preinitiation complexes, however, was absolutely dependent on soluble HeLa cytoplasmic factors. Preinitiation complexes also catalyzed the formation of infectious virus in reaction mixes containing exogenously added capsid proteins. The titer of infectious virus produced in such trans-encapsidation reactions reached 4 x 10(7) PFU/ml. The HeLa S10 translation-RNA replication reactions represent an efficient in vitro system for authentic poliovirus replication, including protein synthesis, polyprotein processing, RNA replication, and virus assembly.