Mouse Hepatitis Virus Genomic RNA Research Articles

Identification of a 1.4-kb-Long Sequence Located in the nsp12 and nsp13 Coding Regions of SARS-CoV-2 Genomic RNA That Mediates Efficient Viral RNA Packaging.

The specific packaging of the viral RNA genome into virus particles is an essential step in the replication cycle of coronaviruses (CoVs). Using a single-cycle, replicable severe acute respiratory syndrome CoV-2 (SARS-CoV-2) mutant, we demonstrated the preferential packaging of the SARS-CoV-2 genomic RNA into purified virus particles. Furthermore, based on the sequence of an efficiently packaged defective interfering RNA of SARS-CoV, a closely related CoV, that was generated after serial passages of SARS-CoV in cell culture, we designed a series of replication-competent SARS-CoV-2 minigenome RNAs to identify the specific viral RNA region that is important for SARS-CoV-2 RNA packaging into virus particles. We showed that a 1.4-kb-long sequence, derived from the nsp12 and nsp13 coding regions of the SARS-CoV-2 genomic RNA, is required for the efficient packaging of SARS-CoV-2 minigenome RNA into SARS-CoV-2 particles. In addition, we also showed that the presence of possibly the entire 1.4-kb-long sequence is important for the efficient packaging of SARS-CoV-2 RNA. Our findings highlight the differences between the RNA packaging sequence identified in SARS-CoV-2, a Sarbecovirus, and the packaging signal of mouse hepatitis virus (MHV), an Embecovirus, which is a 95-nt-long sequence located at the nsp15 coding region of MHV genomic RNA. Collectively, our data imply that both the location and the sequence/structural features of the RNA element(s) that drives the selective and efficient packaging of viral genomic RNA are not conserved among the subgenera Embecovirus and Sarbecovirus within the Betacoronavirus genus. IMPORTANCE Elucidating the mechanism of SARS-CoV-2 RNA packaging into virus particles is important for the rational design of antiviral drugs that inhibit this vital step in the replication cycle of CoVs. However, our knowledge about the RNA packaging mechanism in SARS-CoV-2, including the identification of the viral RNA region important for SARS-CoV-2 RNA packaging, is limited, primarily due to the logistical challenges of handing SARS-CoV-2 in biosafety level 3 (BSL3) facilities. Our study, using a single-cycle, replicable SARS-CoV-2 mutant, which can be handled in a BSL2 lab, demonstrated the preferential packaging of full-length SARS-CoV-2 genomic RNA into virus particles and identified a specific 1.4-kb-long RNA region in SARS-CoV-2 genomic RNA that is required for the efficient packaging of SARS-CoV-2 RNA into virus particles. The information generated in our study could be valuable for clarifying the mechanisms of SARS-CoV-2 RNA packaging and for the development of targeted therapeutics against SARS-CoV-2 and other related CoVs.

An Interaction between the Nucleocapsid Protein and a Component of the Replicase-Transcriptase Complex Is Crucial for the Infectivity of Coronavirus Genomic RNA

The coronavirus nucleocapsid (N) protein plays an essential role in virion assembly via interactions with the large, positive-strand RNA viral genome and the carboxy-terminal endodomain of the membrane protein (M). To learn about the functions of N protein domains in the coronavirus mouse hepatitis virus (MHV), we replaced the MHV N gene with its counterpart from the closely related bovine coronavirus (BCoV). The resulting viral mutant was severely defective, even though individual domains of the N protein responsible for N-RNA, N-M, or N-N interactions were completely interchangeable between BCoV and MHV. The lesion in the BCoV N substitution mutant could be compensated for by reverting mutations in the central, serine- and arginine-rich (SR) domain of the N protein. Surprisingly, a second class of reverting mutations were mapped to the amino terminus of a replicase subunit, nonstructural protein 3 (nsp3). A similarly defective MHV N mutant bearing an insertion of the SR region from the severe acute respiratory syndrome coronavirus N protein was rescued by the same two classes of reverting mutations. Our genetic results were corroborated by the demonstration that the expressed amino-terminal segment of nsp3 bound selectively to N protein from infected cells, and this interaction was RNA independent. Moreover, we found a direct correlation between the N-nsp3 interaction and the ability of N protein to stimulate the infectivity of transfected MHV genomic RNA (gRNA). Our results suggest a role for this previously unknown N-nsp3 interaction in the localization of genomic RNA to the replicase complex at an early stage of infection.

Characterization of coronavirus DI RNA packaging.

Studies of defective interfering (DI) RNAs of mouse hepatitis virus (MHV), suggest that a 69 nt-long packaging signal, which is located about 20 kb from the 5'-end of the 31 kb-long MHV genomic RNA, is necessary and sufficient for MHV genomic RNA packaging into MHV particles. We demonstrated that use of a low pH culture medium combined with subsequent ultrafiltration increased MHV infectivity about 60 times over MHV preparations grown in neutral medium. Using this virus concentration procedure, we successfully prepared DI particle-rich MHV preparations. Characterization of virus samples released from the cells infected with DI particle-rich MHV revealed that infectious MHV genomic RNA was not required for packaging of DI RNAs. These data suggested that interaction of the DI packaging signal with an unidentified region(s) of helper virus genomic RNA is unlikely, and therefore unlikely to facilitate the packaging of MHV DI RNA into the MHV virion. Rather, both DI RNA and MHV genomic RNA probably use the packaging signal for RNA packaging.

Murine coronavirus packaging signal confers packaging to nonviral RNA.

Studies of defective interfering (DI) RNAs of the murine coronavirus mouse hepatitis virus (MHV) suggest that a 69-nucleotide-long packaging signal is necessary for MHV genomic RNA packaging into MHV particles. In this study we showed that when RNA transcripts that consisted of a non-MHV sequence and the packaging signal were expressed in MHV-infected cells, they were packaged into MHV particles. Those RNA transcripts that lacked the packaging signal or those containing a mutated packaging signal did not package efficiently. Thus, the presence of the packaging signal was sufficient for RNA packaging into MHV particles.

The 3' untranslated region of coronavirus RNA is required for subgenomic mRNA transcription from a defective interfering RNA.

The 3'-end of mouse hepatitis virus (MHV) genomic RNA contains a recognition sequence (55 nucleotides [nt]) required for minus-strand RNA synthesis. To determine whether the 3'-end sequence is also involved in subgenomic mRNA transcription, we have constructed MHV defective interfering (DI) RNAs which contain a chloramphenicol acetyltransferase (CAT) gene placed behind an intergenic sequence and a 3'-end sequence with various degrees of internal deletions. The DI RNAs were transfected into MHV-infected cells, and CAT activities, which represent subgenomic mRNA transcription from the intergenic site, were determined. The results demonstrated that the deletions of sequence upstream of the 350 nt at the 3'-end, which include the 3'-untranslated region (3'-UTR), of MHV genomic RNA did not affect subgenomic mRNA transcription. However, deletions that reduced the 3'-end sequences to 270 nt or less completely abolished the mRNA transcription despite the fact that all of these clones synthesized minus-strand RNAs. These results indicated that mRNA transcription from an intergenic site in the MHV DI RNA requires most of the 3'-UTR as a cis-acting signal, which likely exerts its effects during plus-strand RNA synthesis. A substitution of the corresponding bovine coronavirus sequence for the MHV sequence within nt 270 to 305 from the 3'-end abrogated the CAT gene expression, suggesting a very rigid sequence requirement in this region. The deletion of a putative pseudoknot structure within the 3'-UTR also abolished the CAT gene expression. These findings suggest that the 3'-UTR may interact with the other RNA regulatory elements to regulate mRNA transcription.

A 5'-proximal RNA sequence of murine coronavirus as a potential initiation site for genomic-length mRNA transcription

Coronavirus transcription is a discontinuous process, involving interactions between a trans-acting leader and the intergenic transcription initiation sequences. A 9-nucleotide (nt) sequence (UUUAUAAAC), which is located immediately downstream of the leader at the 5' terminus of the mouse hepatitis virus (MHV) genomic RNA, contains a sequence resembling the consensus intergenic sequence (UCUAAAC). It has been shown previously that the presence of the 9-nt sequence facilitates leader RNA switching and may enhance subgenomic mRNA transcription. It is unclear how the 9-nt sequence exerts these functions. In this study, we inserted the 9-nt sequence into a defective interfering (DI) RNA reporter system and demonstrated that mRNA transcription could be initiated from the 9-nt sequence almost as efficiently as from the intergenic sequence between genes 6 and 7. Sequence analysis of the mRNAs showed that the 9-nt sequence served as a site of fusion between the leaders and mRNA. The transcription initiation function of the 9-nt sequence could not be substituted by other 5'-terminal sequences. When the entire 5'-terminal sequence, including four copies of the UCUAA sequence plus the 9-nt sequence, was present, transcription could be initiated from any of the UCUAA copies or the 9-nt sequence, resulting in different copy numbers of the UCUAA sequence and the deletion of the 9-nt sequence in some mRNAs. All of these heterogeneous RNA species were also detected from the 5'-terminal region of the viral genomic-length RNA in MHV-infected cells. These results thus suggest tha the heterogeneity of the copy number of UCUAA sequences at the 5' end, the deletion of the 9-nt sequence in viral and DI RNAs, and the leader RNA switching are the results of transcriptional initiation from the 9-nt site. They also show that an mRNA species (mRNA 1) that lacks the 9-nt sequence can be synthesized during MHV infection. Therefore, MHV genomic RNA replication and mRNA 1 transcription may be distinguishable.

A Conserved Motif at the 3′ End of Mouse Hepatitis Virus Genomic RNA Required for Host Protein Binding and Viral RNA Replication

A conserved 11-nucleotide sequence, UGAAUGAAGUU, at the 3′ end of the genomic RNA of coronavirus mouse hepatitis virus was required for host protein binding and viral RNA synthesis. An RNA probe containing this 11-nucleotide sequence bound four cellular proteins with a highly labeled protein of 120 kDa and three minor species with sizes of 103, 81, and 55 kDa. Mutation of the 11-nucleotide motif abolished cellular protein binding. The RNA–protein complexes observed with cytoplasmic extracts from MHV-JHM-infected cells in both RNase protection/gel mobility shift and UV cross-linking assays were indistinguishable from those observed with extracts from uninfected cells. Both negative-strand synthesis and positive-strand replication of viral defective interfering RNAs in the presence of helper virus were affected by mutations that disrupt RNA–protein complex formation, even though the 11 mutated nucleotides were converted to the wild-type sequence, presumably by recombination with helper virus. Kinetic analysis indicated that recombination between DI RNA and helper virus occurred relatively early in the MHV replicative cycle at 5.5 to 7.5 hr postinfection, a time when viral RNA synthesis and replication of positive-strand DI RNA were at barely detectable levels. A DI RNA with a mutation upstream of the protein binding element replicated as efficiently as wild type without undergoing recombination. Thus, the 11-nucleotide conserved host protein binding motif appears to play an important role in viral RNA replication.

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.

Interactions between the cytoplasmic proteins and the intergenic (promoter) sequence of mouse hepatitis virus RNA: correlation with the amounts of subgenomic mRNA transcribed

Previous studies suggested that coronavirus RNA transcription involves interaction between leader RNA and the intergenic (IG) sequences, probably via protein-RNA interactions (X. M. Zhang, C.-L. Liao, and M. M. C. Lai, J. Virol., 68:4738-4746, 1994; X. M. Zhang and M. M. C. Lai, J. Virol., 68:6626-6633, 1994). To determine whether cellular proteins are involved in this process, we performed UV cross-linking experiments using cytoplasmic extracts of uninfected cells and the IG (promoter) sequence between genes 6 and 7 (IG7) and the 5' untranslational region of mouse hepatitis virus genomic RNA. We demonstrated that three different cellular proteins (p70, p48, and p35/38) bound to the promoter sequence of the template RNA. Deletion analyses of the template RNA mapped the binding site of p35/38 at the consensus transcription initiation signal. In contrast, the binding of p70 and p48 was less specific. p35/38 is the same protein as the one previously identified to bind to the complementary strand of the leader RNA; its binding affinity to the leader was approximately 15 times stronger than that to IG7. Site-directed mutagenesis of the IG sequence revealed that mutations in the consensus sequence of IG7 (UCUAAUCUAAAC to UCGAAAC and GCUAAAG), which resulted in reduced subgenomic mRNA transcription, also caused correspondingly reduced levels of p35/38 binding. These results demonstrated that the extent of protein binding to the IG sequences correlated with the amounts of subgenomic mRNAs transcribed from the IG site. These studies suggest that these RNA-binding proteins are involved in coronavirus RNA transcription and may represent transcription factors.

RNA recombination in a coronavirus: recombination between viral genomic RNA and transfected RNA fragments.

Mouse hepatitis virus (MHV), a coronavirus, has been shown to undergo a high frequency of RNA recombination both in tissue culture and in animal infection. So far, RNA recombination has been demonstrated only between genomic RNAs of two coinfecting viruses. To understand the mechanism of RNA recombination and to further explore the potential of RNA recombination, we studied whether recombination could occur between a replicating MHV RNA and transfected RNA fragments. We first used RNA fragments which represented the 5' end of genomic-sense sequences of MHV RNA for transfection. By using polymerase chain reaction amplification with two specific primers, we were able to detect recombinant RNAs which incorporated the transfected fragment into the 5' end of the viral RNA in the infected cells. Surprisingly, even the anti-genomic-sense RNA fragments complementary to the 5' end of MHV genomic RNA could also recombine with the MHV genomic RNAs. This observation suggests that RNA recombination can occur during both positive- and negative-strand RNA synthesis. Furthermore, the recombinant RNAs could be detected in the virion released from the infected cells even after several passages of virus in tissue culture cells, indicating that these recombinant RNAs represented functional virion RNAs. The crossover sites of these recombinants were detected throughout the transfected RNA fragments. However, when an RNA fragment with a nine-nucleotide (CUUUAUAAA) deletion immediately downstream of a pentanucleotide (UCUAA) repeat sequence in the leader RNA was transfected into MHV-infected cells, most of the recombinants between this RNA and the MHV genome contained crossover sites near this pentanucleotide repeat sequence. In contrast, when exogenous RNAs with the intact nine-nucleotide sequence were used in similar experiments, the crossover sites of recombinants in viral genomic RNA could be detected at more-downstream sites. This study demonstrated that recombination can occur between replicating MHV RNAs and RNA fragments which do not replicate, suggesting the potential of RNA recombination for genetic engineering.

Identification and characterization of a coronavirus packaging signal.

Previously, a mouse hepatitis virus (MHV) genomic sequence necessary for defective interfering (DI) RNA packaging into MHV particles (packaging signal) was mapped to within a region of 1,480 nucleotides in the MHV polymerase gene by comparison of two DI RNAs. One of these, DIssF, is 3.6 kb in size and exhibits efficient packaging, whereas the other, DIssE, which is 2.3 kb, does not. For more precise mapping, a series of mutant DIssF RNAs with deletions within this 1,480-nucleotide region were constructed. After transfection of in vitro-synthesized mutant DI RNA in MHV-infected cells, the virus product was passaged several times. The efficiency of DI RNA packaging into MHV virions was then estimated by viral homologous interference activity and by analysis of intracellular virus-specific RNAs and virion RNA. The results indicated that an area of 190 nucleotides was necessary for packaging. A computer-generated secondary structural analysis of the A59 and JHM strains of MHV demonstrated that within this 190-nucleotide region a stable stem-loop of 69 nucleotides was common between the two viruses. A DIssE-derived DI DNA which had these 69 nucleotides inserted into the DIssE sequence demonstrated efficient DI RNA packaging. Site-directed mutagenic analysis showed that of these 69 nucleotides, the minimum sequence of the packaging signal was 61 nucleotides and that destruction of the secondary structure abolished packaging ability. These studies demonstrated that an MHV packaging signal was present within the 61 nucleotides, which are located on MHV genomic RNA 1,381 to 1,441 nucleotides upstream of the 3' end of gene 1.

Interactions between coronavirus nucleocapsid protein and viral RNAs: implications for viral transcription.

The interaction of the mouse hepatitis virus (MHV) nucleocapsid protein (N) and viral RNA was examined. Monoclonal antibody specific for N protein coimmunoprecipitated MHV genomic RNA as well as all six MHV subgenomic mRNAs found in MHV-infected cells. In contrast, monoclonal antibodies to the MHV E2 or E1 envelope glycoproteins, an anti-I-A monoclonal antibody, and serum samples from lupus patients did not immunoprecipitate the MHV mRNAs. Moreover, the anti-N monoclonal antibody did not coimmunoprecipitate vesicular stomatitis virus RNA or host cell RNA under conditions which immunoprecipitated all MHV RNAs. These data suggest a specific interaction between the N protein and the virus-specific mRNAs. Both the membrane-bound and cytosolic small MHV leader-specific RNAs of greater than 65 nucleotides long were immunoprecipitated only by anti-N monoclonal antibody. These data suggest that an N binding site is present within the leader RNA sequences at a site at least 65 nucleotides from the 5' end of genomic RNA and all six subgenomic mRNAs. The larger leader-containing RNAs originating from mRNA 1 and mRNA 6, as well as the MHV negative-stranded RNA, were also immunoprecipitated by the anti-N monoclonal antibody. These data indicate that the MHV N protein is associated with MHV-specific RNAs and RNA intermediates and may play an important functional role during MHV transcription and replication.

Primary structure and translation of a defective interfering rna of murine coronavirus

An intracellular defective-interfering (DI) RNA, DIssE, of mouse hepatitis virus (MHV) obtained after serial high multiplicity passage of the virus was cloned and sequenced. DIssE RNA is composed of three noncontiguous genomic regions, representing the first 864 nucleotides of the Fend, an internal 748 nucleotides of the polymerase gene, and 601 nucleotides from the 3′ end of the parental MHV genome. The DIssE sequence contains one large continuous open reading frame. Two protein products from this open reading frame were identified both by in vitro translation and in DI-infected cells. Sequence comparison of DIssE and the corresponding parts of the parental virus genome revealed that DIssE had three base substitutions within the leader sequence and also a deletion of nine nucleotides located at the junction of the leader and the remaining genomic sequence. The 5′ end of DIssE RNA was heterogeneous with respect to the number of UCUAA repeats within the leader sequence. The parental MHV genomic RNA appears to have extensive and stable secondary structures at the regions where DI RNA rearrangements occurred. These data suggest that MHV DI RNA may have been generated as a result of the discontinuous and nonprocessive manner of MHV RNA synthesis.

Sequence and translation of the murine coronavirus 5'-end genomic RNA reveals the N-terminal structure of the putative RNA polymerase.

A 28-kilodalton protein has been suggested to be the amino-terminal protein cleavage product of the putative coronavirus RNA polymerase (gene A) (M.R. Denison and S. Perlman, Virology 157:565-568, 1987). To elucidate the structure and mechanism of synthesis of this protein, the nucleotide sequence of the 5' 2.0 kilobases of the coronavirus mouse hepatitis virus strain JHM genome was determined. This sequence contains a single, long open reading frame and predicts a highly basic amino-terminal region. Cell-free translation of RNAs transcribed in vitro from DNAs containing gene A sequences in pT7 vectors yielded proteins initiated from the 5'-most optimal initiation codon at position 215 from the 5' end of the genome. The sequence preceding this initiation codon predicts the presence of a stable hairpin loop structure. The presence of an RNA secondary structure at the 5' end of the RNA genome is supported by the observation that gene A sequences were more efficiently translated in vitro when upstream noncoding sequences were removed. By comparing the translation products of virion genomic RNA and in vitro transcribed RNAs, we established that our clones encompassing the 5'-end mouse hepatitis virus genomic RNA encode the 28-kilodalton N-terminal cleavage product of the gene A protein. Possible cleavage sites for this protein are proposed.

In vitro replication of mouse hepatitis virus strain A59.

An in vitro replication system for mouse hepatitis virus (MHV) strain A59 was developed using lysolecithin to produce cell extracts. In extracts of MHV-infected cells, radiolabeled UMP was incorporated at a linear rate for up to 1 h into RNA, which hybridized to MHV-specific cDNA probes and migrated in denaturing formaldehyde-agarose gels to the same position as MHV genomic RNA. The incorporation of [32P]UMP into genome-sized RNA in vitro correlated with the observed increase of [3H]uridine incorporation in MHV-infected cells labeled in vivo. Incorporation of [32P]UMP into genome-sized RNA was inhibited when extracts were incubated with puromycin. The addition to the assay of antiserum to the MHV-A59 nucleocapsid protein N inhibited synthesis of genome-sized RNA by 90% compared with the addition of preimmune serum. In contrast, antiserum to the E1 or E2 glycoproteins did not significantly inhibit RNA replication. In vitro-synthesized RNA banded in cesium chloride gradients as a ribonucleoprotein complex with the characteristic density of MHV nucleocapsids isolated from virions. These experiments suggest that ongoing protein synthesis is necessary for replication of MHV genomic RNA and indicate that the N protein plays an important role in MHV replication.

The 5′-end sequence of the murine coronavirus genome: Implications for multiple fusion sites in leader-primed transcription

The coronavirus leader-primed transcription model proposes that free leader RNA species derived from the 5′-end of the genomic RNA are utilized as a primer for the transcription of subgenomic mRNAs. To elucidate the precise mechanism of leader-priming, we cloned and sequenced the 5′-end of the mouse hepatitis virus genomic RNA. The 5′-terminal sequences are identical to the leader sequences present at the 5′-end of the subgenomic mRNAs. Two possible hairpin loop structures and an AU-rich region around the 3′-end of the leader sequence may provide the termination site for leader RNA synthesis. The comparison of 5′-end genomic sequences and the intergenic start sites for mRNA transcription revealed that there are homologous regions of 7–18 nucleotides at the putative leader/body junction sites. Some intergenic regions contain a mismatching nucleotide within this homologous region. We propose that free leader RNA binds to the intergenic region due to this homology and is cleaved at the mismatching nucleotide before serving as a primer. Thus, the free leader RNA species may be longer than the leader sequences in the subgenomic mRNAs and different mRNAs may have different leader/body junction sites.

Studies on the mechanism of RNA synthesis of a murine coronavirus.

The mechanism of viral RNA replication in mouse hepatitis virus (MHV)-infected cells was studied by oligonucleotide mapping of every mRNA. We discovered that an oligonucleotide, No. 10, was localized at the 5'-end of every mRNA, and was not colinear with the sequences of the virion genomic RNA. This result indicates that all of the mRNAs contain a leader sequence which is joined to the body sequences of the mRNAs. We have also studied the structure of the replicative intermediate (RI) RNA in the MHV-infected cells. This RI RNA consists of a single species corresponding to the MHV genomic RNA. No subgenomic RI structures were detected. Furthermore, the nascent RNA chains in the RI structure contained the leader sequences, suggesting that the leader RNA was not added to the mRNA post- transcriptionally , but rather, it was probably synthesized independently and then used as a primer for the synthesis of mRNAs. We have also shown that the poly (A) sequences in the MHV genome were transcribed from the poly (U) sequences present in the negative-strand template. The RNA polymerases involved in the MHV RNA synthesis were also characterized. The early polymerase synthesizes a single negative-stranded, full-length RNA. The late polymerases could be separated into two activities, one synthesizing positive-stranded genomic RNA, and the other synthesizing genomic as well as subgenomic RNAs. Thus, the replication and transcription functions of MHV could probably be separated. A plausible model of MHV replication is presented.