A bulged stem-loop structure in the 3' untranslated region of the genome of the coronavirus mouse hepatitis virus is essential for replication.

The 3' untranslated region (UTR) of the positive-sense RNA genome of the coronavirus mouse hepatitis virus (MHV) contains sequences that are necessary for the synthesis of negative-strand viral RNA as well as sequences that may be crucial for both genomic and subgenomic positive-strand RNA synthesis. We have found that the entire 3' UTR of MHV could be replaced by the 3' UTR of bovine coronavirus (BCV), which diverges overall by 31% in nucleotide sequence. This exchange between two viruses that are separated by a species barrier was carried out by targeted RNA recombination. Our results define regions of the two 3' UTRs that are functionally equivalent despite having substantial sequence substitutions, deletions, or insertions with respect to each other. More significantly, our attempts to generate an unallowed substitution of a particular portion of the BCV 3' UTR for the corresponding region of the MHV 3' UTR led to the discovery of a bulged stem-loop RNA secondary structure, adjacent to the stop codon of the nucleocapsid gene, that is essential for MHV viral RNA replication.     

Host protein interactions with the 3' end of bovine coronavirus RNA and the requirement of the poly(A) tail for coronavirus defective genome replication

RNA viruses have 5' and 3' untranslated regions (UTRs) that contain specific signals for RNA synthesis. The coronavirus genome is capped at the 5' end and has a 3' UTR that consists of 300 to 500 nucleotides (nt) plus a poly(A) tail. To further our understanding of coronavirus replication, we have begun to examine the involvement of host factors in this process for two group II viruses, bovine coronavirus (BCV) and mouse hepatitis coronavirus (MHV). Specific host protein interactions with the BCV 3' UTR [287 nt plus poly(A) tail] were identified using gel mobility shift assays. Competition with the MHV 3' UTR [301 nt plus poly(A) tail] suggests that the interactions are conserved for the two viruses. Proteins with molecular masses of 99, 95, and 73 kDa were detected in UV cross-linking experiments. Less heavily labeled proteins were also detected in the ranges of 40 to 50 and 30 kDa. The poly(A) tail was required for binding of the 73-kDa protein. Immunoprecipitation of UV-cross-linked proteins identified the 73-kDa protein as the cytoplasmic poly(A)-binding protein (PABP). Replication of the defective genomes BCV Drep and MHV MIDI-C, along with several mutants, was used to determine the importance of the poly(A) tail. Defective genomes with shortened poly(A) tails consisting of 5 or 10 A residues were replicated after transfection into helper virus-infected cells. BCV Drep RNA that lacked a poly(A) tail did not replicate, whereas replication of MHV MIDI-C RNA with a deleted tail was detected after several virus passages. All mutants exhibited delayed kinetics of replication. Detectable extension or addition of the poly(A) tail to the mutants correlated with the appearance of these RNAs in the replication assay. RNAs with shortened poly(A) tails exhibited less in vitro PABP binding, suggesting that decreased interactions with the protein may affect RNA replication. The data strongly indicate that the poly(A) tail is an important cis-acting signal for coronavirus replication.     

An optimal cis-replication stem-loop IV in the 5' untranslated region of the mouse coronavirus genome extends 16 nucleotides into open reading frame 1

The 288-nucleotide (nt) 3' untranslated region (UTR) in the genome of the bovine coronavirus (BCoV) and 339-nt 3' UTR in the severe acute respiratory syndrome (SARS) coronavirus (SCoV) can each replace the 301-nt 3' UTR in the mouse hepatitis coronavirus (MHV) for virus replication, thus demonstrating common 3' cis-replication signals. Here, we show that replacing the 209-nt MHV 5' UTR with the ～63%-sequence-identical 210-nt BCoV 5' UTR by reverse genetics does not yield viable virus, suggesting 5' end signals are more stringent or possibly are not strictly 5' UTR confined. To identify potential smaller, 5'-common signals, each of three stem-loop (SL) signaling domains and one inter-stem-loop domain from the BCoV 5' UTR was tested by replacing its counterpart in the MHV genome. The SLI/II domain (nucleotides 1 to 84) and SLIII domain (nucleotides 85 to 141) each immediately enabled near-wild-type (wt) MHV-like progeny, thus behaving similarly to comparable 5'-proximal regions of the SCoV 5' UTR as shown by others. The inter-stem-loop domain (nt 142 to 173 between SLs III and IV) enabled small plaques only after genetic adaptation. The SLIV domain (nt 174 to 210) required a 16-nt extension into BCoV open reading frame 1 (ORF1) for apparent stabilization of a longer BCoV SLIV (nt 174 to 226) and optimal virus replication. Surprisingly, pleiomorphic SLIV structures, including a terminal loop deletion, were found among debilitated progeny from intra-SLIV chimeras. The results show the inter-stem-loop domain to be a potential novel species-specific cis-replication element and that cis-acting SLIV in the viral genome extends into ORF1 in a manner that stabilizes its lower stem and is thus not 5' UTR confined.     

Characterization of the RNA components of a putative molecular switch in the 3' untranslated region of the murine coronavirus genome

RNA virus genomes contain cis-acting sequence and structural elements that participate in viral replication. We previously identified a bulged stem-loop secondary structure at the upstream end of the 3' untranslated region (3' UTR) of the genome of the coronavirus mouse hepatitis virus (MHV). This element, beginning immediately downstream of the nucleocapsid gene stop codon, was shown to be essential for virus replication. Other investigators discovered an adjacent downstream pseudoknot in the 3' UTR of the closely related bovine coronavirus (BCoV). This pseudoknot was also shown to be essential for replication, and it has a conserved counterpart in every group 1 and group 2 coronavirus. In MHV and BCoV, the bulged stem-loop and pseudoknot are, in part, mutually exclusive, because of the overlap of the last segment of the stem-loop and stem 1 of the pseudoknot. This led us to hypothesize that they form a molecular switch, possibly regulating a transition occurring during viral RNA synthesis. We have now performed an extensive genetic analysis of the two components of this proposed switch. Our results define essential and nonessential components of these structures and establish the limits to which essential parts of each element can be destabilized prior to loss of function. Most notably, we have confirmed the interrelationship of the two putative switch elements. Additionally, we have identified a pseudoknot loop insertion mutation that appears to point to a genetic interaction between the pseudoknot and a distant region of the genome.     

A hypervariable region within the 3' cis-acting element of the murine coronavirus genome is nonessential for RNA synthesis but affects pathogenesis

The 3' cis-acting element for mouse hepatitis virus (MHV) RNA synthesis resides entirely within the 301-nucleotide 3' untranslated region (3' UTR) of the viral genome and consists of three regions. Encompassing the upstream end of the 3' UTR are a bulged stem-loop and an overlapping RNA pseudoknot, both of which are essential to MHV and common to all group 2 coronaviruses. At the downstream end of the genome is the minimal signal for initiation of negative-strand RNA synthesis. Between these two ends is a hypervariable region (HVR) that is only poorly conserved between MHV and other group 2 coronaviruses. Paradoxically, buried within the HVR is an octanucleotide motif (oct), 5'-GGAAGAGC-3', which is almost universally conserved in coronaviruses and is therefore assumed to have a critical biological function. We conducted an extensive mutational analysis of the HVR. Surprisingly, this region tolerated numerous deletions, rearrangements, and point mutations. Most striking, a mutant deleted of the entire HVR was only minimally impaired in tissue culture relative to the wild type. By contrast, the HVR deletion mutant was highly attenuated in mice, causing no signs of clinical disease and minimal weight loss compared to wild-type virus. Correspondingly, replication of the HVR deletion mutant in the brains of mice was greatly reduced compared to that of the wild type. Our results show that neither the HVR nor oct is essential for the basic mechanism of MHV RNA synthesis in tissue culture. However, the HVR appears to play a significant role in viral pathogenesis.     

Genetic evidence of a long-range RNA-RNA interaction between the genomic 5' untranslated region and the nonstructural protein 1 coding region in murine and bovine coronaviruses

Higher-order RNA structures in the 5' untranslated regions (UTRs) of the mouse hepatitis coronavirus (MHV) and bovine coronavirus (BCoV), separate species in the betacoronavirus genus, appear to be largely conserved despite an ～36% nucleotide sequence divergence. In a previous study, each of three 5'-end-proximal cis-acting stem-loop domains in the BCoV genome, I/II, III, and IV, yielded near-wild-type (wt) MHV phenotypes when used by reverse genetics to replace its counterpart in the MHV genome. Replacement with the BCoV 32-nucleotide (nt) inter-stem-loop fourth domain between stem-loops III and IV, however, required blind cell passaging for virus recovery. Here, we describe suppressor mutations within the transplanted BCoV 32-nt domain that along with appearance of potential base pairings identify an RNA-RNA interaction between this domain and a 32-nt region ～200 nt downstream within the nonstructural protein 1 (Nsp1)-coding region. Mfold and phylogenetic covariation patterns among similarly grouped betacoronaviruses support this interaction, as does cotransplantation of the BCoV 5' UTR and its downstream base-pairing domain. Interestingly, cotransplantation of the BCoV 5' UTR and BCoV Nsp1 coding region directly yielded an MHV wt-like phenotype, which demonstrates a cognate interaction between these two BCoV regions, which in the MHV genome act in a fully interspecies-compliant manner. Surprisingly, the 30-nt inter-stem-loop domain in the MHV genome can be deleted and viral progeny, although debilitated, are still produced. These results together identify a previously undescribed long-range RNA-RNA interaction between the 5' UTR and Nsp1 coding region in MHV-like and BCoV-like betacoronaviruses that is cis acting for viral fitness but is not absolutely required for viral replication in cell culture.     

Stem-loop IV in the 5' untranslated region is a cis-acting element in bovine coronavirus defective interfering RNA replication

The 210-nucleotide (nt) 5' untranslated region (UTR) in the positive-strand bovine coronavirus (BCoV) genome is predicted to contain four higher-order structures identified as stem-loops I to IV, which may function as cis-acting elements in genomic RNA replication. Here, we describe evidence that stem-loop IV, a bulged stem-loop mapping at nt 186 through 215, (i) is phylogenetically conserved among group 2 coronaviruses and may have a homolog in groups 1 and 3, (ii) exists as a higher-order structure on the basis of enzyme probing, (iii) is required as a higher-order element for replication of a BCoV defective interfering (DI) RNA in the positive but not the negative strand, and (iv) as a higher-order structure in wild-type (wt) and mutant molecules that replicate, specifically binds six cellular proteins in the molecular mass range of 25 to 58 kDa as determined by electrophoretic mobility shift and UV cross-linking assays; binding to viral proteins was not detected. Interestingly, the predicted stem-loop IV homolog in the severe acute respiratory syndrome (SARS) coronavirus appears to be group 1-like in that it is in part duplicated with a group 1-like conserved loop sequence and is not group 2-like, as would be expected by the SARS coronavirus group 2-like 3' UTR structure. These results together indicate that stem-loop IV in the BCoV 5' UTR is a cis-acting element for DI RNA replication and that it might function through interactions with cellular proteins. It is postulated that stem-loop IV functions similarly in the virus genome.     

Exceptional flexibility in the sequence requirements for coronavirus small envelope protein function

The small envelope protein (E) plays a role of central importance in the assembly of coronaviruses. This was initially established by studies demonstrating that cellular expression of only E protein and the membrane protein (M) was necessary and sufficient for the generation and release of virus-like particles. To investigate the role of E protein in the whole virus, we previously generated E gene mutants of mouse hepatitis virus (MHV) that were defective in viral growth and produced aberrantly assembled virions. Surprisingly, however, we were also able to isolate a viable MHV mutant (DeltaE) in which the entire E gene, as well as the nonessential upstream genes 4 and 5a, were deleted. We have now constructed an E knockout mutant that confirms that the highly defective phenotype of the DeltaE mutant is due to loss of the E gene. Additionally, we have created substitution mutants in which the MHV E gene was replaced by heterologous E genes from viruses spanning all three groups of the coronavirus family. Group 2 and 3 E proteins were readily exchangeable for that of MHV. However, the E protein of a group 1 coronavirus, transmissible gastroenteritis virus, became functional in MHV only after acquisition of particular mutations. Our results show that proteins encompassing a remarkably diverse range of primary amino acid sequences can provide E protein function in MHV. These findings suggest that E protein facilitates viral assembly in a manner that does not require E protein to make sequence-specific contacts with M protein.     

Identification of a coronavirus hemagglutinin-esterase with a substrate specificity different from those of influenza C virus and bovine coronavirus

We have characterized the hemagglutinin-esterase (HE) of puffinosis virus (PV), a coronavirus closely related to mouse hepatitis virus (MHV). Analysis of the cloned gene revealed approximately 85% sequence identity to HE proteins of MHV and approximately 60% identity to the corresponding esterase of bovine coronavirus. The HE protein exhibited acetylesterase activity with synthetic substrates p-nitrophenyl acetate, alpha-naphthyl acetate, and 4-methylumbelliferyl acetate. In contrast to other viral esterases, no activity was detectable with natural substrates containing 9-O-acetylated sialic acids. Furthermore, PV esterase was unable to remove influenza C virus receptors from human erythrocytes, indicating a substrate specificity different from HEs of influenza C virus and bovine coronavirus. Solid-phase binding assays revealed that purified PV was unable to bind to sialic acid-containing glycoconjugates like bovine submaxillary mucin, mouse alpha1 macroglobulin or bovine brain extract. Because of the close relationship to MHV, possible implications on the substrate specificity of MHV esterases are suggested.     

Maintenance of pluripotency in mouse embryonic stem cells persistently infected with murine coronavirus.

A persistently coronavirus-infected embryonic stem (ES) cell line A3/MHV was established by infecting an ES cell line, A3-1, with mouse hepatitis virus type-2. Although almost all A3/MHV cells were found infected, both A3/MHV and A3-1 cells expressed comparable levels of cell surface differentiation markers. In addition, A3/MHV cells retained the ability to form embryoid bodies. These results suggest that persistent coronavirus infection does not affect the differentiation of ES cells.     

Antagonism of the interferon-induced OAS-RNase L pathway by murine coronavirus ns2 protein is required for virus replication and liver pathology

Many viruses induce hepatitis in humans, highlighting the need to understand the underlying mechanisms of virus-induced liver pathology. The murine coronavirus, mouse hepatitis virus (MHV), causes acute hepatitis in its natural host and provides a useful model for understanding virus interaction with liver cells. The MHV accessory protein, ns2, antagonizes the type I interferon response and promotes hepatitis. We show that ns2 has 2',5'-phosphodiesterase activity, which blocks the interferon inducible 2',5'-oligoadenylate synthetase (OAS)-RNase L pathway to facilitate hepatitis development. Ns2 cleaves 2',5'-oligoadenylate, the product of OAS, to prevent activation of the cellular endoribonuclease RNase L and consequently block viral RNA degradation. An ns2 mutant virus was unable to replicate in the liver or induce hepatitis in wild-type mice, but was highly pathogenic in RNase L deficient mice. Thus, RNase L is a critical cellular factor for protection against viral infection of the liver and the resulting hepatitis.     

A conserved RNA pseudoknot in a putative molecular switch domain of the 3'-untranslated region of coronaviruses is only marginally stable

The 3'-untranslated region (UTR) of the group 2 coronavirus mouse hepatitis virus (MHV) genome contains a predicted bulged stem-loop (designated P0ab), a conserved cis-acting pseudoknot (PK), and a more distal stem-loop (designated P2). Base-pairing to create the pseudoknot-forming stem (P1(pk)) is mutually exclusive with formation of stem P0a at the base of the bulged stem-loop; as a result, the two structures cannot be present simultaneously. Herein, we use thermodynamic methods to evaluate the ability of individual subdomains of the 3' UTR to adopt a pseudoknotted conformation. We find that an RNA capable of forming only the predicted PK (58 nt; 3' nucleotides 241-185) adopts the P2 stem-loop with little evidence for P1(pk) pairing in 0.1 M KCl and the absence of Mg(2+); as Mg(2+) or 1 M KCl is added, a new thermal unfolding transition is induced and assignable to P1(pk) pairing. The P1(pk) helix is only marginally stable, ΔG(25) ≈ 1.2 ± 0.3 kcal/mol (5.0 mM Mg(2+), 100 mM K(+)), and unfolded at 37°C. Similar findings characterize an RNA 5' extended through the P0b helix only (89 nt; 294-185). In contrast, an RNA capable of forming either the P0a helix or the pseudoknot (97 nt; 301-185) forms no P1(pk) helix. Thermal unfolding simulations are fully consistent with these experimental findings. These data reveal that the PK forms weakly and only when the competing double-hairpin structure cannot form; in the UTR RNA, the double hairpin is the predominant conformer under all solution conditions.     

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.     

The primary structure and expression of the second open reading frame of the polymerase gene of the coronavirus MHV-A59; a highly conserved polymerase is expressed by an efficient ribosomal frameshifting mechanism.

Sequence analysis of a substantial part of the polymerase gene of the murine coronavirus MHV-A59 revealed the 3' end of an open reading frame (ORF1a) overlapping with a large ORF (ORF1b; 2733 amino acids) which covers the 3' half of the polymerase gene. The expression of ORF1b occurs by a ribosomal frameshifting mechanism since the ORF1a/ORF1b overlapping nucleotide sequence is capable of inducing ribosomal frameshifting in vitro as well as in vivo. A stem-loop structure and a pseudoknot are predicted in the nucleotide sequence involved in ribosomal frameshifting. Comparison of the predicted amino acid sequence of MHV ORF1b with the amino acid sequence deduced from the corresponding gene of the avian coronavirus IBV demonstrated that in contrast to the other viral genes this ORF is extremely conserved. Detailed analysis of the predicted amino acid sequence revealed sequence elements which are conserved in many DNA and RNA polymerases.     

Characterization of the coronavirus M protein and nucleocapsid interaction in infected cells

Coronavirus contains three envelope proteins, M, E and S, and a nucleocapsid, which consists of genomic RNA and N protein, within the viral envelope. We studied the macromolecular interactions involved in coronavirus assembly in cells infected with a murine coronavirus, mouse hepatitis virus (MHV). Coimmunoprecipitation analyses demonstrated an interaction between N protein and M protein in infected cells. Pulse-labeling experiments showed that newly synthesized, unglycosylated M protein interacted with N protein in a pre-Golgi compartment, which is part of the MHV budding site. Coimmunoprecipitation analyses further revealed that M protein interacted with only genomic-length MHV mRNA, mRNA 1, while N protein interacted with all MHV mRNAs. These data indicated that M protein interacted with the nucleocapsid, consisting of N protein and mRNA 1, in infected cells. The M protein-nucleocapsid interaction occurred in the absence of S and E proteins. Intracellular M protein-N protein interaction was maintained after removal of viral RNAs by RNase treatment. However, the M protein-N protein interaction did not occur in cells coexpressing M protein and N protein alone. These data indicated that while the M protein-N protein interaction, which is independent of viral RNA, occurred in the M protein-nucleocapsid complex, some MHV function(s) was necessary for the initiation of M protein-nucleocapsid interaction. The M protein-nucleocapsid interaction, which occurred near or at the MHV budding site, most probably represented the process of specific packaging of the MHV genome into MHV particles.