Retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier

Coronaviruses generally have a narrow host range, infecting one or just a few species. Using targeted RNA recombination, we constructed a mutant of the coronavirus mouse hepatitis virus (MHV) in which the ectodomain of the spike glycoprotein (S) was replaced with the highly divergent ectodomain of the S protein of feline infectious peritonitis virus. The resulting chimeric virus, designated fMHV, acquired the ability to infect feline cells and simultaneously lost the ability to infect murine cells in tissue culture. This reciprocal switch of species specificity strongly supports the notion that coronavirus host cell range is determined primarily at the level of interactions between the S protein and the virus receptor. The isolation of fMHV allowed the localization of the region responsible for S protein incorporation into virions to the carboxy-terminal 64 of the 1,324 residues of this protein. This establishes a basis for further definition of elements involved in virion assembly. In addition, fMHV is potentially the ideal recipient virus for carrying out reverse genetics of MHV by targeted RNA recombination, since it presents the possibility of selecting recombinants, no matter how defective, that have regained the ability to replicate in murine cells.     

A major determinant for membrane protein interaction localizes to the carboxy-terminal domain of the mouse coronavirus nucleocapsid protein

The two major constituents of coronavirus virions are the membrane (M) and nucleocapsid (N) proteins. The M protein is anchored in the viral envelope by three transmembrane segments flanked by a short amino-terminal ectodomain and a large carboxy-terminal endodomain. The M endodomain interacts with the viral nucleocapsid, which consists of the positive-strand RNA genome helically encapsidated by N protein monomers. In previous work with the coronavirus mouse hepatitis virus (MHV), a highly defective M protein mutant, MDelta2, was constructed. This mutant contained a 2-amino-acid carboxy-terminal truncation of the M protein. Analysis of second-site revertants of MDelta2 revealed mutations in the carboxy-terminal region of the N protein that compensated for the defect in the M protein. To seek further genetic evidence corroborating this interaction, we generated a comprehensive set of clustered charged-to-alanine mutants in the carboxy-terminal domain 3 of N protein. One of these mutants, CCA4, had a highly defective phenotype similar to that of MDelta2. Transfer of the CCA4 mutation into a partially diploid MHV genome showed that CCA4 was a loss-of-function mutation rather than a dominant-negative mutation. Analysis of multiple second-site revertants of CCA4 revealed mutations in both the M protein and the N protein that could compensate for the original lesion in N. These data more precisely define the region of the N protein that interacts with the M protein. Further, we found that fusion of domain 3 of the N protein to the carboxy terminus of a heterologous protein caused it to be incorporated into MHV virions.     

Coronavirus Particle Assembly: Primary Structure Requirements of the Membrane Protein

Coronavirus-like particles morphologically similar to normal virions are assembled when genes encoding the viral membrane proteins M and E are coexpressed in eukaryotic cells. Using this envelope assembly assay, we have studied the primary sequence requirements for particle formation of the mouse hepatitis virus (MHV) M protein, the major protein of the coronavirion membrane. Our results show that each of the different domains of the protein is important. Mutations (deletions, insertions, point mutations) in the luminal domain, the transmembrane domains, the amphiphilic domain, or the carboxy-terminal domain had effects on the assembly of M into enveloped particles. Strikingly, the extreme carboxy-terminal residue is crucial. Deletion of this single residue abolished particle assembly almost completely; most substitutions were strongly inhibitory. Site-directed mutations in the carboxy terminus of M were also incorporated into the MHV genome by targeted recombination. The results supported a critical role for this domain of M in viral assembly, although the M carboxy terminus was more tolerant of alteration in the complete virion than in virus-like particles, likely because of the stabilization of virions by additional intermolecular interactions. Interestingly, glycosylation of M appeared not essential for assembly. Mutations in the luminal domain that abolished the normal O glycosylation of the protein or created an N-glycosylated form had no effect. Mutant M proteins unable to form virus-like particles were found to inhibit the budding of assembly-competent M in a concentration-dependent manner. However, assembly-competent M was able to rescue assembly-incompetent M when the latter was present in low amounts. These observations support the existence of interactions between M molecules that are thought to be the driving force in coronavirus envelope assembly.     

Optimization of targeted RNA recombination and mapping of a novel nucleocapsid gene mutation in the coronavirus mouse hepatitis virus.

We have recently described a method of introducing site-specific mutations into the genome of the coronavirus mouse hepatitis virus (MHV) by RNA recombination between cotransfected genomic RNA and a synthetic subgenomic mRNA (C. A. Koetzner, M. M. Parker, C. S. Ricard, L. S. Sturman, and P. S. Masters, J. Virol. 66:1841-1848, 1992). By using a thermolabile N protein mutant of MHV (Alb4) as the recipient virus and synthetic RNA7 (the mRNA for the nucleocapsid protein N) as the donor, we selected engineered recombinant viruses as heat-stable progeny resulting from cotransfection. We have now been able to greatly increase the efficiency of targeted recombination in this process by using a synthetic defective interfering (DI) RNA in place of RNA7. The frequency of recombination is sufficiently high that, with Alb4 as the recipient, recombinants can be directly identified without using thermal selection. The synthetic DI RNA has been used to demonstrate that the lesion in another temperature-sensitive and thermolabile MHV mutant, Alb1, maps to the N gene. Sequencing of the Alb1 N gene revealed two closely linked point mutations that fall in a region of the N molecule previously noted as being the most highly conserved region among all of the coronavirus N proteins. Analysis of revertants of the Alb1 mutant revealed that one of the two mutations is critical for the temperature-sensitive phenotype; the second mutation is phenotypically silent.     

Switching species tropism: an effective way to manipulate the feline coronavirus genome

Feline infectious peritonitis virus (FIPV), a coronavirus, is the causative agent of an invariably lethal infection in cats. Like other coronaviruses, FIPV contains an extremely large positive-strand RNA genome of ca. 30 kb. We describe here the development and use of a reverse genetics strategy for FIPV based on targeted RNA recombination that is analogous to what has been described for the mouse hepatitis virus (MHV) (L. Kuo et al., J. Virol. 74:1393-1406, 2000). In this two-step process, we first constructed by targeted recombination a mutant of FIPV, designated mFIPV, in which the ectodomain of the spike glycoprotein was replaced by that of MHV. This switch allowed for the selection of the recombinant virus in murine cells: mFIPV grows to high titers in these cells but has lost the ability to grow in feline cells. In a second, reverse process, mFIPV was used as the recipient, and the reintroduction of the FIPV spike now allowed for selection of candidate recombinants by their regained ability to grow in feline cells. In this fashion, we reconstructed a wild-type recombinant virus (r-wtFIPV) and generated a directed mutant FIPV in which the initiation codon of the nonstructural gene 7b had been disrupted (FIPV Delta 7b). The r-wtFIPV was indistinguishable from its parental virus FIPV 79-1146 not only for its growth characteristics in tissue culture but also in cats, exhibiting a highly lethal phenotype. FIPV Delta 7b had lost the expression of its 7b gene but grew unimpaired in cell culture, confirming that the 7b glycoprotein is not required in vitro. We establish the second targeted RNA recombination system for coronaviruses and provide a powerful tool for the genetic engineering of the FIPV genome.     

The small envelope protein E is not essential for murine coronavirus replication

The importance of the small envelope (E) protein in the assembly of coronaviruses has been demonstrated in several studies. While its precise function is not clearly defined, E is a pivotal player in the morphogenesis of the virion envelope. Expression of the E protein alone results in its incorporation into vesicles that are released from cells, and the coexpression of the E protein with the membrane protein M leads to the assembly of coronavirus-like particles. We have previously generated E gene mutants of mouse hepatitis virus (MHV) that had marked defects in viral growth and produced virions that were aberrantly assembled in comparison to wild-type virions. We have now been able to obtain a viable MHV mutant in which the entire E gene, as well as the nonessential upstream genes 4 and 5a, has been deleted. This mutant (Delta E) was obtained by a targeted RNA recombination method that makes use of a powerful host range-based selection system. The Delta E mutant produces tiny plaques with an unusual morphology compared to plaques formed by wild-type MHV. Despite its low growth rate and low infectious titer, the Delta E mutant is genetically stable, showing no detectable phenotypic changes after several passages. The properties of this mutant provide further support for the importance of E protein in MHV replication, but surprisingly, they also show that E protein is not essential.     

Construction of murine coronavirus mutants containing interspecies chimeric nucleocapsid proteins.

Targeted RNA recombination was used to construct mouse hepatitis virus (MHV) mutants containing chimeric nucleocapsid (N) protein genes in which segments of the bovine coronavirus N gene were substituted in place of their corresponding MHV sequences. This defined portions of the two N proteins that, despite evolutionary divergence, have remained functionally equivalent. These regions included most of the centrally located RNA-binding domain and two putative spacers that link the three domains of the N protein. By contrast, the amino terminus of N, the acidic carboxy-terminal domain, and a serine- and arginine-rich segment of the central domain could not be transferred from bovine coronavirus to MHV, presumably because these parts of the molecule participate in protein-protein interactions that are specific for each virus (or, possibly, each host). Our results demonstrate that targeted recombination can be used to make extensive substitutions in the coronavirus genome and can generate recombinants that could not otherwise be made between two viruses separated by a species barrier. The implications of these findings for N protein structure and function as well as for coronavirus RNA recombination are discussed.     

Importance of the penultimate positive charge in mouse hepatitis coronavirus A59 membrane protein

The coronavirus membrane (M) protein carboxy tail interacts with the nucleocapsid during virus assembly. Previous studies demonstrated that the two terminal residues are important, and the charged residue (R227) in the penultimate position in the mouse hepatitis coronavirus (MHV) A59 M protein was suggested to participate in intermolecular interactions with negative charges in the nucleocapsid (N) protein. To determine the significance of the positive charge at position 227, we substituted the arginine with lysine (K), aspartic acid (D), glutamic acid (E), or alanine (A) and studied these by reverse genetics in the context of a MHV full-length infectious clone. Viruses with wild-type phenotype were readily recovered with the K or A substitutions. In contrast, negative-charge substitutions were not tolerated as well. In all recovered R227D viruses the negative charge was replaced with heterologous residues resulting from apparent template switching during negative-strand synthesis of subgenomic RNA 7. An additional second-site compensatory V202I substitution was present in some viruses. Recovered R227E viruses had second-site changes within the M protein carboxy tail that were partially compensatory. Significantly, most of the second site changes in the R227E mutant viruses were previously shown to compensate for the removal of negative charges in the N protein. Our results strongly indicate that a positive charge is not absolutely required. It is clear that other regions within the tail must also be involved in helping mediate interactions between the M protein and the nucleocapsid.     

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.     

Repair and mutagenesis of the genome of a deletion mutant of the coronavirus mouse hepatitis virus by targeted RNA recombination.

The genetic characterization of a nucleocapsid (N) protein mutant of the coronavirus mouse hepatitis virus (MHV) is described. The mutant, Albany 4 (Alb4), is both temperature sensitive and thermolabile. Analysis of the progeny of a mixed infection showed that the defective Alb4 allele is recessive to wild type, and its gene product is diffusible. The N protein of Alb4 was found to be smaller than its wild-type counterpart, and sequence analysis of the Alb4 N gene revealed that it contains an internal deletion of 87 nucleotides, producing an in-frame deletion of 29 amino acids. All of these properties of Alb4 made it ideal for use as a recipient in a targeted RNA recombination experiment in which the deletion in Alb4 was repaired by recombination with synthetic RNA7, the smallest MHV subgenomic mRNA. Progeny from a cotransfection of Alb4 genomic RNA and synthetic RNA7 were selected for thermal stability. Polymerase chain reaction analysis of candidate recombinants showed that they had regained the material that is deleted in the Alb4 mutant. They also had acquired a five-nucleotide insertion in the 3' untranslated region, which had been incorporated into the synthetic RNA7 as a molecular tag. The presence of the tag was directly verified, as well, by sequencing the genomic RNA of purified recombinant viruses. This provided a clear genetic proof that the Alb4 phenotype was due to the observed deletion in the N gene. In addition, these results demonstrated that it is possible to obtain stable, independently replicating progeny from recombination between coronavirus genomic RNA and a tailored, synthetic RNA species.     

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.     

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.     

RNA recombination of murine coronaviruses: recombination between fusion-positive mouse hepatitis virus A59 and fusion-negative mouse hepatitis virus 2.

It has previously been shown that the murine coronavirus mouse hepatitis virus (MHV) undergoes RNA recombination at a relatively high frequency in both tissue culture and infected animals. Thus far, all of the recombination sites had been localized at the 5' half of the RNA genome. We have now performed a cross between MHV-2, a fusion-negative murine coronavirus, and a temperature-sensitive mutant of the A59 strain of MHV, which is fusion positive at the permissive temperature. By selecting fusion-positive viruses at the nonpermissive temperature, we isolated several recombinants containing multiple crossovers in a single genome. Some of the recombinants became fusion negative during the plaque purification. The fusion ability of the recombinants parallels the presence or absence of the A59 genomic sequences encoding peplomers. Several of the recombinants have crossovers within 3' end genes which encode viral structural proteins, N and E1. These recombination sites were not specifically selected with the selection markers used. This finding, together with results of previous recombination studies, indicates that RNA recombination can occur almost anywhere from the 5' end to the 3' end along the entire genome. The data also show that the replacement of A59 genetic sequences at the 5' end of gene C, which encodes the peplomer protein, with the fusion-negative MHV-2 sequences do not affect the fusion ability of the recombinant viruses. Thus, the crucial determinant for the fusion-inducing capability appears to reside in the more carboxyl portion of the peplomer protein.     

The internal open reading frame within the nucleocapsid gene of mouse hepatitis virus encodes a structural protein that is not essential for viral replication.

The coronavirus mouse hepatitis virus (MHV) contains a large open reading frame embedded entirely within the 5' half of its nucleocapsid (N) gene. This internal gene (designated I) is in the +1 reading frame with respect to the N gene, and it encodes a mostly hydrophobic 23-kDa polypeptide. We have found that this protein is expressed in MHV-infected cells and that it is a previously unrecognized structural protein of the virion. To analyze the potential biological importance of the I gene, we disrupted its expression by site-directed mutagenesis using targeted RNA recombination. The start codon for I was replaced by a threonine codon, and a stop codon was introduced at a short interval downstream. Both alterations created silent changes in the N reading frame. In vitro translation studies showed that these mutations completely abolished synthesis of I protein, and immunological analysis of infected cell lysates confirmed this conclusion. The MHV I mutant was viable and grew to high titer. However, the I mutant had a reduced plaque size in comparison with its isogenic wild-type counterpart, suggesting that expression of I confers some minor growth advantage to the virus. The engineered mutations were stable during the course of experimental infection in mice, and the I mutant showed no significant differences from wild type in its ability to replicate in the brains or livers of infected animals. These results demonstrate that I protein is not essential for the replication of MHV either in tissue culture or in its natural host.     

Localization and membrane topology of coronavirus nonstructural protein 4: involvement of the early secretory pathway in replication

The coronavirus nonstructural proteins (nsp's) derived from the replicase polyproteins collectively constitute the viral replication complexes, which are anchored to double-membrane vesicles. Little is known about the biogenesis of these complexes, the membrane anchoring of which is probably mediated by nsp3, nsp4, and nsp6, as they contain several putative transmembrane domains. As a first step to getting more insight into the formation of the coronavirus replication complex, the membrane topology, processing, and subcellular localization of nsp4 of the mouse hepatitis virus (MHV) and severe acute respiratory syndrome-associated coronavirus (SARS-CoV) were elucidated in this study. Both nsp4 proteins became N glycosylated, while their amino and carboxy termini were localized to the cytoplasm. These observations imply nsp4 to assemble in the membrane as a tetraspanning transmembrane protein with a Nendo/Cendo topology. The amino terminus of SARS-CoV nsp4, but not that of MHV nsp4, was shown to be (partially) processed by signal peptidase. nsp4 localized to the endoplasmic reticulum (ER) when expressed alone but was recruited to the replication complexes in infected cells. nsp4 present in these complexes did not colocalize with markers of the ER or Golgi apparatus, while the susceptibility of its sugars to endoglycosidase H indicated that the protein had also not traveled trough the latter compartment. The important role of the early secretory pathway in formation of the replication complexes was also demonstrated by the inhibition of coronaviral replication when the ER export machinery was blocked by use of the kinase inhibitor H89 or by expression of a mutant, Sar1[H79G].     

Demyelination determinants map to the spike glycoprotein gene of coronavirus mouse hepatitis virus

Demyelination is the pathologic hallmark of the human immune-mediated neurologic disease multiple sclerosis, which may be triggered or exacerbated by viral infections. Several experimental animal models have been developed to study the mechanism of virus-induced demyelination, including coronavirus mouse hepatitis virus (MHV) infection in mice. The envelope spike (S) glycoprotein of MHV contains determinants of properties essential for virus-host interactions. However, the molecular determinants of MHV-induced demyelination are still unknown. To investigate the mechanism of MHV-induced demyelination, we examined whether the S gene of MHV contains determinants of demyelination and whether demyelination is linked to viral persistence. Using targeted RNA recombination, we replaced the S gene of a demyelinating virus (MHV-A59) with the S gene of a closely related, nondemyelinating virus (MHV-2). Recombinant viruses containing an S gene derived from MHV-2 in an MHV-A59 background (Penn98-1 and Penn98-2) exhibited a persistence-positive, demyelination-negative phenotype. Thus, determinants of demyelination map to the S gene of MHV. Furthermore, viral persistence is insufficient to induce demyelination, although it may be a prerequisite for the development of demyelination.     

Genetic analysis of determinants for spike glycoprotein assembly into murine coronavirus virions: distinct roles for charge-rich and cysteine-rich regions of the endodomain

The coronavirus spike protein (S) forms the distinctive virion surface structures that are characteristic of this viral family, appearing in negatively stained electron microscopy as stems capped with spherical bulbs. These structures are essential for the initiation of infection through attachment of the virus to cellular receptors followed by fusion to host cell membranes. The S protein can also mediate the formation of syncytia in infected cells. The S protein is a type I transmembrane protein that is very large compared to other viral fusion proteins, and all except a short carboxy-terminal segment of the S molecule constitutes the ectodomain. For the prototype coronavirus mouse hepatitis virus (MHV), it has previously been established that S protein assembly into virions is specified by the carboxy-terminal segment, which comprises the transmembrane domain and the endodomain. We have genetically dissected these domains in the MHV S protein to localize the determinants of S incorporation into virions. Our results establish that assembly competence maps to the endodomain of S, which was shown to be sufficient to target a heterologous integral membrane protein for incorporation into MHV virions. In particular, mutational analysis indicated a major role for the charge-rich carboxy-terminal region of the endodomain. Additionally, we found that the adjacent cysteine-rich region of the endodomain is critical for fusion of infected cells, confirming results previously obtained with S protein expression systems.