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The coronavirus nucleocapsid (N) protein comprises two RNA-binding domains connected by a central spacer, which contains a serine- and arginine-rich (SR) region. The SR region engages the largest subunit of the viral replicase-transcriptase, nonstructural protein 3 (nsp3), in an interaction that is essential for efficient initiation of infection by genomic RNA. We carried out an extensive genetic analysis of the SR region of the N protein of mouse hepatitis virus in order to more precisely define its role in RNA synthesis. We further examined the N-nsp3 interaction through construction of nsp3 mutants and by creation of an interspecies N protein chimera. Our results indicate a role for the central spacer as an interaction hub of the N molecule that is partially regulated by phosphorylation. These findings are discussed in relation to the recent discovery that nsp3 forms a molecular pore in the double-membrane vesicles that sequester the coronavirus replicase-transcriptase., Graphical abstract Image 1

RNA viruses carry out selective packaging of their genomes in a variety of ways, many involving a genomic packaging signal. The first coronavirus packaging signal was discovered nearly thirty years ago, but how it functions remains incompletely understood. This review addresses the current state of knowledge of coronavirus genome packaging, which has mainly been studied in two prototype species, mouse hepatitis virus and transmissible gastroenteritis virus. Despite the progress that has been made in the mapping and characterization of some packaging signals, there is conflicting evidence as to whether the viral nucleocapsid protein or the membrane protein plays the primary role in packaging signal recognition. The different models for the mechanism of genomic RNA packaging that have been prompted by these competing views are described. Also discussed is the recent exciting discovery that selective coronavirus genome packaging is critical for in vivo evasion of the host innate immune response., Graphical abstract Image 1, Highlights • Selective incorporation of the coronavirus genome into virions is mediated by a cis-acting RNA packaging signal. • Packaging signals vary across different coronavirus genera and lineages. • Different lines of evidence attribute packaging signal recognition to either the nucleocapsid or the membrane protein. • Selective coronavirus genome packaging plays a role in evasion of host innate immunity.

The psychrophilic (cold-loving) fungus Pseudogymnoascus destructans was discovered more than a decade ago to be the pathogen responsible for white-nose syndrome, an emerging disease of North American bats causing unprecedented population declines. The same species of fungus is found in Europe but without associated mortality in bats. We found P. destructans was infected with a mycovirus [named Pseudogymnoascus destructans partitivirus 1 (PdPV-1)]. The virus is bipartite, containing two double-stranded RNA (dsRNA) segments designated as dsRNA1 and dsRNA2. The cDNA sequences revealed that dsRNA1 dsRNA is 1,683 bp in length with an open reading frame (ORF) that encodes 539 amino acids (molecular mass of 62.7 kDa); dsRNA2 dsRNA is 1,524 bp in length with an ORF that encodes 434 amino acids (molecular mass of 46.9 kDa). The dsRNA1 ORF contains motifs representative of RNA-dependent RNA polymerase (RdRp), whereas the dsRNA2 ORF sequence showed homology with the putative capsid proteins (CPs) of mycoviruses. Phylogenetic analyses with PdPV-1 RdRp and CP sequences indicated that both segments constitute the genome of a novel virus in the family Partitiviridae. The purified virions were isometric with an estimated diameter of 33 nm. Reverse transcription PCR (RT-PCR) and sequencing revealed that all US isolates and a subset of Czech Republic isolates of P. destructans were infected with PdPV-1. However, PdPV-1 appears to be not widely dispersed in the fungal genus Pseudogymnoascus, as non-pathogenic fungi P. appendiculatus (1 isolate) and P. roseus (6 isolates) tested negative. P. destructans PdPV-1 could be a valuable tool to investigate fungal biogeography and the host–pathogen interactions in bat WNS.

The coronavirus membrane (M) protein is the central actor in virion morphogenesis. M organizes the components of the viral membrane, and interactions of M with itself and with the nucleocapsid (N) protein drive virus assembly and budding. In order to further define M-M and M-N interactions, we constructed mutants of the model coronavirus mouse hepatitis virus (MHV) in which all or part of the M protein was replaced by its phylogenetically divergent counterpart from severe acute respiratory syndrome coronavirus (SARS-CoV). We were able to obtain viable chimeras containing the entire SARS-CoV M protein as well as mutants with intramolecular substitutions that partitioned M protein at the boundaries between the ectodomain, transmembrane domains, or endodomain. Our results show that the carboxy-terminal domain of N protein, N3, is necessary and sufficient for interaction with M protein. However, despite some previous genetic and biochemical evidence that mapped interactions with N to the carboxy terminus of M, it was not possible to define a short linear region of M protein sufficient for assembly with N. Thus, interactions with N protein likely involve multiple linearly discontiguous regions of the M endodomain. The SARS-CoV M chimera exhibited a conditional growth defect that was partially suppressed by mutations in the envelope (E) protein. Moreover, virions of the M chimera were markedly deficient in spike (S) protein incorporation. These findings suggest that the interactions of M protein with both E and S protein are more complex than previously thought. IMPORTANCE The assembly of coronavirus virions entails concerted interactions among the viral structural proteins and the RNA genome. One strategy to study this process is through construction of interspecies chimeras that preserve or disrupt particular inter- or intramolecular associations. In this work, we replaced the membrane (M) protein of the model coronavirus mouse hepatitis virus with its counterpart from a heterologous coronavirus. The results clarify our understanding of the interaction between the coronavirus M protein and the nucleocapsid protein. At the same time, they reveal unanticipated complexities in the interactions of M with the viral spike and envelope proteins.

Coronaviruses, the largest RNA viruses, have a complex program of RNA synthesis that entails genome replication and transcription of subgenomic mRNAs. RNA synthesis by the prototype coronavirus mouse hepatitis virus (MHV) is carried out by a replicase-transcriptase composed of 16 nonstructural protein (nsp) subunits. Among these, nsp3 is the largest and the first to be inserted into the endoplasmic reticulum. nsp3 comprises multiple structural domains, including two papain-like proteases (PLPs) and a highly conserved ADP-ribose-1″-phosphatase (ADRP) macrodomain. We have previously shown that the ubiquitin-like domain at the amino terminus of nsp3 is essential and participates in a critical interaction with the viral nucleocapsid protein early in infection. In the current study, we exploited atypical expression schemes to uncouple PLP1 from the processing of nsp1 and nsp2 in order to investigate the requirements of nsp3 domains for viral RNA synthesis. In the first strategy, a mutant was created in which replicase polyprotein translation initiated with nsp3, thereby establishing that complete elimination of nsp1 and nsp2 does not abolish MHV viability. In the second strategy, a picornavirus autoprocessing element was used to separate a truncated nsp1 from nsp3. This provided a platform for further dissection of amino-terminal domains of nsp3. From this, we found that catalytic mutation of PLP1 or complete deletion of PLP1 and the adjacent ADRP domain was tolerated by the virus. These results showed that neither the PLP1 domain nor the ADRP domain of nsp3 provides integral activities essential for coronavirus genomic or subgenomic RNA synthesis. IMPORTANCE The largest component of the coronavirus replicase-transcriptase complex, nsp3, contains multiple modules, many of which do not have clearly defined functions in genome replication or transcription. These domains may play direct roles in RNA synthesis, or they may have evolved for other purposes, such as to combat host innate immunity. We initiated a dissection of MHV nsp3 aimed at identifying those activities or structures in this huge molecule that are essential to replicase activity. We found that both PLP1 and ADRP could be entirely deleted, provided that the requirement for proteolytic processing by PLP1 was offset by an alternative mechanism. This demonstrated that neither PLP1 nor ADRP plays an essential role in coronavirus RNA synthesis.

Coronaviruses selectively package genomic RNA into assembled virions, despite the great molar excess of subgenomic RNA species that is present in infected cells. The genomic packaging signal (PS) for the coronavirus mouse hepatitis virus (MHV) was originally identified as an element that conferred packaging capability to defective interfering RNAs. The MHV PS is an RNA structure that maps to the region of the replicase gene encoding the nonstructural protein 15 subunit of the viral replicase-transcriptase complex. To begin to understand the role and mechanism of action of the MHV PS in its native genomic locus, we constructed viral mutants in which this cis -acting element was altered, deleted, or transposed. Our results demonstrated that the PS is pivotal in the selection of viral genomic RNA for incorporation into virions. Mutants in which PS RNA secondary structure was disrupted or entirely ablated packaged large quantities of subgenomic RNAs, in addition to genomic RNA. Moreover, the PS retained its function when displaced to an ectopic site in the genome. Surprisingly, the PS was not essential for MHV viability, nor did its elimination have a severe effect on viral growth. However, the PS was found to provide a distinct selective advantage to MHV. Viruses containing the PS readily outcompeted their otherwise isogenic counterparts lacking the PS.

The prototype coronavirus mouse hepatitis virus (MHV) exhibits highly selective packaging of its genomic positive-stranded RNA into assembled virions, despite the presence in infected cells of a large excess of subgenomic viral mRNAs. One component of this selectivity is the MHV packaging signal (PS), an RNA structure found only in genomic RNA and not in subgenomic RNAs. It was previously shown that a major determinant of PS recognition is the second of the two RNA-binding domains of the viral nucleocapsid (N) protein. We have now found that PS recognition additionally depends upon a segment of the carboxy-terminal tail (domain N3) of the N protein. Since domain N3 is also the region of N protein that interacts with the membrane (M) protein, this finding suggests a mechanism by which selective genome packaging is accomplished, through the coupling of genome encapsidation to virion assembly.

The coronavirus small envelope (E) protein plays a crucial, but poorly defined, role in the assembly of virions. To investigate E protein function, we previously generated E gene point mutants of mouse hepatitis virus (MHV) that were defective in growth and assembled virions with anomalous morphologies. We subsequently constructed an E gene deletion (ΔE) mutant that was only minimally viable. The ΔE virus formed tiny plaques and reached optimal infectious titers many orders of magnitude below those of wild-type virus. We have now characterized highly aberrant viral transcription patterns that developed in some stocks of the ΔE mutant. Extensive analysis of three independent stocks revealed that, in each, a faster-growing virus harboring a genomic duplication had been selected. Remarkably, the net result of each duplication was the creation of a variant version of the membrane protein (M) gene that was situated upstream of the native copy of the M gene. Each different variant M gene encoded an expressed protein (M*) containing a truncated endodomain. Reconstruction of one variant M gene in a ΔE background showed that expression of the M* protein markedly enhanced the growth of the ΔE mutant and that the M* protein was incorporated into assembled virions. These findings suggest that M* proteins were repeatedly selected as surrogates for the E protein and that one role of E is to mediate interactions between transmembrane domains of M protein monomers. Our results provide a demonstration of the capability of coronaviruses to evolve new gene functions through recombination.

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.

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.

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, MΔ2, was constructed. This mutant contained a 2-amino-acid carboxy-terminal truncation of the M protein. Analysis of second-site revertants of MΔ2 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 MΔ2. 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.

The severe acute respiratory syndrome coronavirus (SARS-CoV) is the causative agent of the recent outbreak of severe acute respiratory syndrome. VeroE6 cells, fetal rhesus monkey kidney cells, and human peripheral blood mononuclear cells were the only cells known to be susceptible to SARS-CoV. We developed a multiplex reverse transcription-PCR assay to analyze the susceptibility of cells derived from a variety of tissues and species to SARS-CoV. Additionally, productive infection was determined by titration of cellular supernatants. Cells derived from three species of monkey were susceptible to SARS-CoV. However, the levels of SARS-CoV produced differed by 4 log 10 . Mink lung epithelial cells (Mv1Lu) and R-Mix, a mixed monolayer of human lung-derived cells (A549) and mink lung-derived cells (Mv1Lu), are used by diagnostic laboratories to detect respiratory viruses (e.g., influenza virus); they were also infected with SARS-CoV, indicating that the practices of diagnostic laboratories should be examined to ensure appropriate biosafety precautions. Mv1Lu cells produce little SARS-CoV compared to that produced by VeroE6 cells, which indicates that they are a safer alternative for SARS-CoV diagnostics. Evaluation of cells permissive to other coronaviruses indicated that these cell types are not infected by SARS-CoV, providing additional evidence that SARS-CoV binds an alternative receptor. Analysis of human cells derived from lung, kidney, liver, and intestine led to the discovery that human cell lines were productively infected by SARS-CoV. This study identifies new cell lines that may be used for SARS-CoV diagnostics and/or basic research. Our data and other in vivo studies indicate that SARS-CoV has a wide host range, suggesting that the cellular receptor(s) utilized by SARS-CoV is highly conserved and is expressed by a variety of tissues.

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.

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 (ΔE) was obtained by a targeted RNA recombination method that makes use of a powerful host range-based selection system. The ΔE 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 ΔE 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.

The coronavirus membrane (M) protein is the most abundant virion protein and the key component in viral assembly and morphogenesis. The M protein of mouse hepatitis virus (MHV) is an integral membrane protein with a short ectodomain, three transmembrane segments, and a large carboxy-terminal endodomain facing the interior of the viral envelope. The carboxy terminus of MHV M has previously been shown to be extremely sensitive to mutation, both in a virus-like particle expression system and in the intact virion. We have constructed a mutant, MΔ2, containing a two-amino-acid truncation of the M protein that was previously thought to be lethal. This mutant was isolated by means of targeted RNA recombination with a powerful host range-based selection allowed by the interspecies chimeric virus fMHV (MHV containing the ectodomain of the feline infectious peritonitis virus S protein). Analysis of multiple second-site revertants of the MΔ2 mutant has revealed changes in regions of both the M protein and the nucleocapsid (N) protein that can compensate for the loss of the last two residues of the M protein. Our data thus provide the first genetic evidence for a structural interaction between the carboxy termini of the M and N proteins of MHV. In addition, this work demonstrates the efficacy of targeted recombination with fMHV for the systematic genetic analysis of coronavirus structural protein interactions.

The coronavirus nucleocapsid (N) protein forms a helical ribonucleoprotein with the viral positive-strand RNA genome and binds to the principal constituent of the virion envelope, the membrane (M) protein, to facilitate assembly and budding. Besides these structural roles, N protein associates with a component of the replicase-transcriptase complex, nonstructural protein 3, at a critical early stage of infection. N protein has also been proposed to participate in the replication and selective packaging of genomic RNA and the transcription and translation of subgenomic mRNA. Coronavirus N proteins contain two structurally distinct RNA-binding domains, an unusual characteristic among RNA viruses. To probe the functions of these domains in the N protein of the model coronavirus mouse hepatitis virus (MHV), we constructed mutants in which each RNA-binding domain was replaced by its counterpart from the N protein of severe acute respiratory syndrome coronavirus (SARS-CoV). Mapping of revertants of the resulting chimeric viruses provided evidence for extensive intramolecular interactions between the two RNA-binding domains. Through analysis of viral RNA that was packaged into virions we identified the second of the two RNA-binding domains as a principal determinant of MHV packaging signal recognition. As expected, the interaction of N protein with M protein was not affected in either of the chimeric viruses. Moreover, the SARS-CoV N substitutions did not alter the fidelity of leader-body junction formation during subgenomic mRNA synthesis. These results more clearly delineate the functions of N protein and establish a basis for further exploration of the mechanism of genomic RNA packaging. IMPORTANCE This work describes the interactions of the two RNA-binding domains of the nucleocapsid protein of a model coronavirus, mouse hepatitis virus. The main finding is that the second of the two domains plays an essential role in recognizing the RNA structure that allows the selective packaging of genomic RNA into assembled virions.

The protein encoded by ORF 4 of mouse hepatitis virus (MHV) is not required for growth of some strains in tissue culture cells, but its role in pathogenesis in the murine host has not been defined previously in a controlled manner. MHV strain JHM causes acute and chronic neurological diseases in susceptible strains of rodents. To genetically manipulate the structural proteins of this and other strains of MHV, we have generalized an interspecies-targeted RNA recombination selection that was originally developed for the A59 strain of MHV. Using this approach, a recombinant MHV–JHM was constructed in which gene 4 was genetically inactivated. Virus lacking gene 4 expression replicated in tissue culture cells with similar kinetics to recombinant virus in which gene 4 expression was not disrupted. Both types of viruses exhibited similar virulence when analyzed in a murine model of encephalitis. These results establish a targeted recombination system for inserting mutations into MHV–JHM. Furthermore, the protein encoded by ORF 4 is not essential for growth in tissue culture cells or in the CNS of the infected host.

Viruses with RNA genomes often capture and redirect host cell components to assist in mechanisms particular to RNA-dependent RNA synthesis. The nidoviruses are an order of positive-stranded RNA viruses, comprising coronaviruses and arteriviruses, that employ a unique strategy of discontinuous transcription, producing a series of subgenomic mRNAs linking a 5′ leader to distal portions of the genome. For the prototype coronavirus mouse hepatitis virus (MHV), heterogeneous nuclear ribonucleoprotein (hnRNP) A1 has been shown to be able to bind in vitro to the negative strand of the intergenic sequence, a cis-acting element found in the leader RNA and preceding each downstream ORF in the genome. hnRNP A1 thus has been proposed as a host factor in MHV transcription. To test this hypothesis genetically, we initially constructed MHV mutants with a very high-affinity hnRNP A1 binding site inserted in place of, or adjacent to, an intergenic sequence in the MHV genome. This inserted hnRNP A1 binding site was not able to functionally replace, or enhance transcription from, the intergenic sequence. This finding led us to test more directly the role of hnRNP A1 by analysis of MHV replication and RNA synthesis in a murine cell line that does not express this protein. The cellular absence of hnRNP A1 had no detectable effect on the production of infectious virus, the synthesis of genomic RNA, or the quantity or quality of subgenomic mRNAs. These results strongly suggest that hnRNP A1 is not a required host factor for MHV discontinuous transcription or genome replication.

The coronavirus spike glycoprotein (S) mediates both the attachment of virus to the host cell receptor and membrane fusion. We describe here the characterization of a temperature-sensitive mutant of the coronavirus mouse hepatitis virus A59 (MHV-A59) having multiple S protein-related defects. The most remarkable of these was that the mutant, designated Albany 18 (Alb 18), assembled virions devoid of the S glycoprotein at the nonpermissive temperature. Alb18 also failed to bring about syncytia formation in cells infected at the nonpermissive temperature. Virions of the mutant assembled at the permissive temperature were much more thermolabile than wild type. Moreover, mutant S protein that was incorporated into virions at the permissive temperature showed enhanced pH-dependent thermolability in its ability to bind to the MHV receptor. Alb18 was found to have a single point mutation in S resulting in a change of serine 287 to isoleucine, and it was shown by revertant analysis that this was the lesion responsible for the phenotype of the mutant.

Expression studies have shown that the coronavirus small envelope protein E and the much more abundant membrane glycoprotein M are both necessary and sufficient for the assembly of virus-like particles in cells. As a step toward understanding the function of the mouse hepatitis virus (MHV) E protein, we carried out clustered charged-to-alanine mutagenesis on the E gene and incorporated the resulting mutations into the MHV genome by targeted recombination. Of the four possible clustered charged-to-alanine E gene mutants, one was apparently lethal and one had a wild-type phenotype. The two other mutants were partially temperature sensitive, forming small plaques at the nonpermissive temperature. Revertant analyses of these two mutants demonstrated that the created mutations were responsible for the temperature-sensitive phenotype of each and provided support for possible interactions among E protein monomers. Both temperature-sensitive mutants were also found to be markedly thermolabile when grown at the permissive temperature, suggesting that there was a flaw in their assembly. Most significantly, when virions of one of the mutants were examined by electron microscopy, they were found to have strikingly aberrant morphology in comparison to the wild type: most mutant virions had pinched and elongated shapes that were rarely seen among wild-type virions. These results demonstrate an important, probably essential, role for the E protein in coronavirus morphogenesis.

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.

The coronavirus nucleocapsid protein (N) plays an essential structural role in virions through a network of interactions with positive-strand viral genomic RNA, the envelope membrane protein (M), and other N molecules. Additionally, N protein participates in at least one stage of the complex mechanism of coronavirus RNA synthesis. We previously uncovered an unanticipated interaction between N and the largest subunit of the viral replicase-transcriptase complex, nonstructural protein 3 (nsp3). This was found through analysis of revertants of a severely defective mutant of murine hepatitis virus (MHV) in which the N gene was replaced with that of its close relative, bovine coronavirus (BCoV). In the work reported here, we constructed BCoV chimeras and other mutants of MHV nsp3 and obtained complementary genetic evidence for its association with N protein. We found that the N-nsp3 interaction maps to the amino-terminal ubiquitin-like domain of nsp3, which is essential for the virus. The interaction does not require the adjacent acidic domain of nsp3, which is dispensable. In addition, we demonstrated a complete correspondence between N-nsp3 genetic interactions and the ability of N protein to enhance the infectivity of transfected coronavirus genomic RNA. The latter function of N was shown to depend on both of the RNA-binding domains of N, as well as on the serine- and arginine-rich central region of N, which binds nsp3. Our results support a model in which the N-nsp3 interaction serves to tether the genome to the newly translated replicase-transcriptase complex at a very early stage of infection.

Coronavirus spike (S) protein assembles into virions via its carboxy-terminus, which is composed of a transmembrane domain and an endodomain. Here, the carboxy-terminal charge-rich motif in the endodomain was verified to be critical for the specificity of S assembly into mouse hepatitis virus (MHV). Recombinant MHVs exhibited a range of abilities to accommodate the homologous S endodomains from the betacoronaviruses bovine coronavirus and human SARS-associated coronavirus, the alphacoronavirus porcine transmissible gastroenteritis virus (TGEV), and the gammacoronavirus avian infectious bronchitis virus respectively. Interestingly, in TGEV endodomain chimeras the reverting mutations resulted in stronger S incorporation into virions, and a net gain of negatively charged residues in the charge-rich motif accounted for the improvement. Additionally, MHV S assembly could also be rescued by the acidic carboxy-terminal domain of the nucleocapsid protein. These results indicate an important role for negatively charged endodomain residues in the incorporation of MHV S protein into assembled virions.

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.

The type I interferon (IFN) response plays an essential role in the control of in vivo infection by the coronavirus mouse hepatitis virus (MHV). However, in vitro , most strains of MHV are largely resistant to the action of this cytokine, suggesting that MHV encodes one or more functions that antagonize or evade the IFN system. A particular strain of MHV, MHV-S, exhibited orders-of-magnitude higher sensitivity to IFN than prototype strain MHV-A59. Through construction of interstrain chimeric recombinants, the basis for the enhanced IFN sensitivity of MHV-S was found to map entirely to the region downstream of the spike gene, at the 3′ end of the genome. Sequence analysis revealed that the major difference between the two strains in this region is the absence of gene 5a from MHV-S. Creation of a gene 5a knockout mutant of MHV-A59 demonstrated that a major component of IFN resistance maps to gene 5a. Conversely, insertion of gene 5a, or its homologs from related group 2 coronaviruses, at an upstream genomic position in an MHV-A59/S chimera restored IFN resistance. This is the first demonstration of a coronavirus gene product that can protect that same virus from the antiviral state induced by IFN. Neither protein kinase R, which phosphorylates eukaryotic initiation factor 2, nor oligoadenylate synthetase, which activates RNase L, was differentially activated in IFN-treated cells infected with MHV-A59 or MHV-S. Thus, the major IFN-induced antiviral activities that are specifically inhibited by MHV, and possibly by other coronaviruses, remain to be identified.

The coronavirus nucleocapsid protein (N), together with the large, positive-strand RNA viral genome, forms a helically symmetric nucleocapsid. This ribonucleoprotein structure becomes packaged into virions through association with the carboxy-terminal endodomain of the membrane protein (M), which is the principal constituent of the virion envelope. Previous work with the prototype coronavirus mouse hepatitis virus (MHV) has shown that a major determinant of the N-M interaction maps to the carboxy-terminal domain 3 of the N protein. To explore other domain interactions of the MHV N protein, we expressed a series of segments of the MHV N protein as fusions with green fluorescent protein (GFP) during the course of viral infection. We found that two of these GFP-N-domain fusion proteins were selectively packaged into virions as the result of tight binding to the N protein in the viral nucleocapsid, in a manner that did not involve association with either M protein or RNA. The nature of each type of binding was further explored through genetic analysis. Our results defined two strongly interacting regions of the N protein. One is the same domain 3 that is critical for M protein recognition during assembly. The other is domain N1b, which corresponds to the N-terminal domain that has been structurally characterized in detail for two other coronaviruses, infectious bronchitis virus and the severe acute respiratory syndrome coronavirus.

Targeted RNA recombination has proven to be a powerful tool for the genetic engineering of the coronavirus genome, particularly in its 3' part. Here we describe procedures for the generation of recombinant and mutant mouse hepatitis virus and feline infectious peritonitis virus. Key to the two-step method is the efficient selection of recombinant viruses based on host cell switching. The first step consists of the preparation---using this selection principle--of an interspecies chimeric coronavirus. In this virus the ectodomain of the spike glycoprotein is replaced by that of a coronavirus with a different species tropism. In the second step this chimeric virus is used as the recipient for recombination with synthetic donor RNA carrying the original spike gene. Recombinant viruses are then isolated on the basis of their regained natural (e.g., murine or feline) cell tropism. Additional mutations created in the donor RNA can be co-incorporated into the recombinant virus in order to generate mutant viruses.

To obtain information about the structure and evolution of the nucleocapsid (N) protein of the coronavirus mouse hepatitis virus (MHV), we determined the entire nucleotide sequences of the N genes of MHV-A59, MHV-3, MHV-S, and MHV-1 from cDNA clones. At the nucleotide level, the N gene sequences of these viral strains, and that of MHV-JHM, were more than 92% conserved overall. Even higher nucleotide sequence identity was found in the 3′ untranslated regions (3′ UTRs) of the five strains, which may reflect the role of the 3′ UTR in negative-strand RNA synthesis. All five N genes were found to encode markedly basic proteins of 454 or 455 residues having at least 94% sequence identity in pairwise comparisons. However, amino acid sequence divergences were found to be clustered in two short segments of N, putative spacer regions that, together, constituted only 11% of the molecule. Thus, the data suggest that the MHV N protein is composed of three highly conserved structural domains connected to each other by regions that have much less constraint on their amino acid sequences. The first two conserved domains contain most of the excess of basic amino acid residues; by contrast, the carboxy-terminal domain is acidic. Finally, we noted that four of the five N genes contain an internal open reading frame that potentially encodes a protein of 207 amino acids having a large proportion of basic and hydrophobic residues.

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 (ΔE) 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 ΔE 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.

Triaryl pyrazoline {[5-(4-chloro-phenyl)-3-thiophen-2-yl-4,5-dihydro-pyrazol-1-yl]-phenyl-methanone} inhibits flavivirus infection in cell culture. The inhibitor was identified through high-throughput screening of a compound library using a luciferase-expressing West Nile (WN) virus infection assay. The compound inhibited an epidemic strain of WN virus without detectable cytotoxicity (a 50% effective concentration of 28 μM and a compound concentration of ≥300 μM required to reduce 50% cell viability). Besides WN virus, the compound also inhibited other flaviviruses (dengue, yellow fever, and St. Louis encephalitis viruses), an alphavirus (Western equine encephalitis virus), a coronavirus (mouse hepatitis virus), and a rhabdovirus (vesicular stomatitis virus). However, the compound did not suppress an orthomyxovirus (influenza virus) or a retrovirus (human immunodeficiency virus type 1). Mode-of-action analyses in WN virus showed that the compound did not inhibit viral entry or virion assembly but specifically suppressed viral RNA synthesis. To examine the mechanism of inhibition of dengue virus, we developed two replicon systems for dengue type 1 virus: (i) a stable cell line that harbored replicons containing a luciferase reporter and a neomycin phosphotransferase selection marker and (ii) a luciferase-expressing replicon that could differentiate between viral translation and RNA replication. Analyses of the compound in the dengue type 1 virus replicon systems showed that it weakly suppressed viral translation but significantly inhibited viral RNA synthesis. Overall, the results demonstrate that triaryl pyrazoline exerts a broad spectrum of antiflavivirus activity through potent inhibition of viral RNA replication. This novel inhibitor could be developed for potential treatment of flavivirus infection.

A number of approaches have been taken to elucidate the network of interactions, among the canonical structural proteins S, M, E, and N, and the genomic RNA, that lead to assembly of virions. The earliest efforts employed the fractionation and reassociation of components of purified virions. These studies were followed by molecular genetic and co-immunoprecipitation analyses of expressed proteins or proteins from virus-infected cells. More recently, reverse-genetic techniques have become available. This chapter will briefly review the current understanding of CoV assembly, highlighting some recent results from our laboratory in the context of work that has been done by numerous other groups in this field. From a large body of work extending over two decades, the main principle that has emerged is that M is the central organizer of CoV assembly. The M protein (~25 kDa)

Coronaviruses are large, enveloped RNA viruses of both medical and veterinary importance. Interest in this viral family has intensified in the past few years as a result of the identification of a newly emerged coronavirus as the causative agent of severe acute respiratory syndrome (SARS). At the molecular level, coronaviruses employ a variety of unusual strategies to accomplish a complex program of gene expression. Coronavirus replication entails ribosome frameshifting during genome translation, the synthesis of both genomic and multiple subgenomic RNA species, and the assembly of progeny virions by a pathway that is unique among enveloped RNA viruses. Progress in the investigation of these processes has been enhanced by the development of reverse genetic systems, an advance that was heretofore obstructed by the enormous size of the coronavirus genome. This review summarizes both classical and contemporary discoveries in the study of the molecular biology of these infectious agents, with particular emphasis on the nature and recognition of viral receptors, viral RNA synthesis, and the molecular interactions governing virion assembly.

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.

The 3′ untranslated region (3′ UTR) of the genome of the severe acute respiratory syndrome coronavirus can functionally replace its counterpart in the prototype group 2 coronavirus mouse hepatitis virus (MHV). By contrast, the 3′ UTRs of representative group 1 or group 3 coronaviruses cannot operate as substitutes for the MHV 3′ UTR.

The large size of the Coronavirus genome has made reverse genetics difficult. Targeted recombination, a technique developed by Masters and colleagues, has facilitated the introduction of mutations into the Coronavirus genome (Kuo et al., 2000). Previous work by Skinner and Siddell showed that MHV-JHM ORF 4 encodes a 15 kDa protein composed of 139 amino acids. This protein is relatively rich in threonines and includes a hydrophobic region. The N-terminus contains a potential membrane-anchoring region and the C-terminus has a possible RNA binding region (Skinner and Siddell, 1985). MHV-S, a natural variant, does not encode a functional ORF 4 suggesting that the ORF 4 product was not necessary for growth in tissue culture cells or animals. Additionally this strain contained a deletion within ORF 5a (Yokomori and Lai, 1991). Lack of mRNA 4 synthesis most likely resulted from a point mutation in the intergenic sequence (UCUAAAC to UUUAAAC). In this study, targeted recombination was used to genetically disrupt ORF 4. This recombinant virus was then analyzed in a murine model of encephalitis.

The subgenomic mRNAs of the coronavirus mouse hepatitis virus (MHV) are composed of a leader sequence, identical to the 5′ 70 nucleotides of the genome, joined at distant downstream sites to a stretch of sequence that is identical to the 3′ end of the genome. The points of fusion occur at intergenic sequences (IGSs), loci on the genome that contain a tract of sequence homologous to the 3′ end of the leader RNA. We have constructed a mutant of MHV-A59 containing an extra IGS inserted into the genome immediately downstream of the 3′-most gene, that encoding the nucleocapsid (N) protein. We show that in cells infected with the mutant, there is synthesis of an additional leader-containing subgenomic RNA of the predicted size. Our study demonstrates that (i) an IGS can be a sufficient cis -acting element to dictate MHV transcription, (ii) the relative efficiency of an IGS must be influenced by factors other than the nucleotides immediately adjacent to the 5′AAUCUAAAC3′ core consensus sequence or its position relative to the 3′ end of the genome, (iii) a downstream IGS can exert a polar attenuating effect on upstream IGSs, and (iv) unknown factors prevent the insertion of large exogenous elements between the N gene and the 3′ untranslated region of MHV. These results confirm and extend conclusions previously derived from the analysis of defective interfering RNAs.

Publisher Summary The capped and polyadenylated genomes of coronaviruses, spanning some 27 to 31 kb, are the largest of all RNA virus genomes, including those of the segmented RNA viruses. This chapter presents the reverse genetics of the largest RNA viruses. Just as all other positive-sense RNA viruses (retroviruses excluded), coronavirus genomic RNA is infectious when transfected into the cells of a permissive host. Therefore, in principle, the most direct way to perform reverse genetics on a coronavirus ought to involve the construction of a full-length genomic complementary DNA (cDNA) clone from which infectious RNA could be transcribed in vitro . The method––targeted recombination––is less direct and more laborious, and so far it has been applied exclusively to site-directed mutagenesis of mouse hepatitis virus (MHV). Thus, at least for structural gene mutations that are not expected to be severely deleterious, targeted recombination may remain the less complicated alternative for the creation of MHV mutants. The chapter discusses targeted RNA recombination, such as development of system, genetic analysis of coronavirus structural proteins, genetic analysis of coronavirus RNA synthesis, and limitations of targeted recombination.

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.

The genome of the coronavirus mouse hepatitis virus (MHV) contains genes which have been shown to be nonessential for viral replication and which could, in principle, be used as sites for the introduction of foreign sequences. We have inserted heterologous genetic material into gene 4 of MHV in order (i) to test the applicability of targeted RNA recombination for site-directed mutagenesis of the MHV genome upstream of the N gene; (ii) to develop further genetic tools for mutagenesis of structural genes other than N; and (iii) to examine the feasibility of using MHV as an expression vector. A DI-like donor RNA vector containing the MHV S gene and all genes distal to S was constructed. Initially, a derivative of this was used to insert a 19-nucleotide tag into the start of ORF 4a of MHV-A59 using the N gene deletion mutant Alb4 as the recipient virus. Subsequently, the entire gene for the green fluorescent protein (GFP) was inserted in place of gene 4. This heterologous gene was shown to be expressed by recombinant viruses but not at levels sufficient to allow detection of fluorescence of viral plaques. Northern blot analysis of transcripts of GFP recombinants showed the expected displacement of the mobility, relative to those of wild-type, of all subgenomic mRNAs larger than mRNA5. An unexpected result of the Northern analysis was the observation that GFP recombinants also produced an RNA species the same size as that of wild-type mRNA4. RT-PCR analysis of the 5’ end of this species revealed that it was actually a collection of mRNAs originating from a cluster of 10 different sites, none of which possessed a canonical intergenic sequence. The finding of these aberrant mRNAs, all of nearly the same size as wild-type mRNA4, suggests that long range structure of the MHV genome can sometimes be the sole determinant of the site of initiation of transcription.

The 3’ untranslated regions (3’ UTRs) of coronaviruses contain the signals necessary for negative strand RNA synthesis and may also harbor elements essential for positive strand replication and subgenomic RNA transcription. The 3’ UTRs of mouse hepatitis virus (MHV) and bovine Coronavirus (BCV) are more than 30% divergent. In an effort to learn what parts of these regions might be functionally interchangeable, we attempted to replace the 3’ UTR of MHV with its BCV counterpart by targeted RNA recombination. Initially, we tried to substitute the 3’ 267 nucleotides (nt) of the 301 nt MHV 3’ UTR with the corresponding region of the BCV 3’ UTR. This exchange did not yield viable recombinant viruses, and the donor DI RNA was shown to be unable to replicate with MHV as a helper virus. Subsequent analysis revealed that the entire BCV 3’ UTR could be inserted into the MHV genome in place of the entire MHV 3’ UTR. It resulted that the failure of the initial attempted substitution was due to the inadvertent disruption of an essential conserved bulged stem-loop secondary structure in the MHV and BCV 3’ UTRs immediately downstream of the N gene stop codon.

A set of viruses in which various segments of the nucleocapsid (N) gene of MHV have been substituted with the corresponding segments of bovine coronavirus (BCV) by targeted recombination were analyzed for their biologic properties. Histology for organ pathology and plaque assay for viral titer analysis following intracerebral (IC) inoculation were studied. One chimeric virus (A1b85), in which only a small segment of the N gene was replaced, exhibited a phenotype similar to wild type MHV-A59. However, three of the chimeric viruses (A1b 106, A1b 112 and A1b 100) produced acute encephalitis and demyelination but without hepatitis following IC inoculation. Intravenous (IV) and intrahepatic (IH) inoculations were able to restore the ability of these viruses to produce hepatitis. The common denominator of the three chimeric viruses with a different phenotype is a region between aa 306 and aa 386 in which 17 amino acids (aa) differences exist between the two strains. Thus this region may contain determinants which enable the virus to exit the brain and reach the blood stream.

MHV-A59 produces acute encephalitis, acute hepatitis and chronic demyelination in infected mice. MHV-2 produces only hepatitis and mild meningitis but without encephalitis or demyelination. We have previously studied a set of recombinant viruses between these two strains. The common denominator of viruses that produced encephalitis was a membrane (M) gene derived from MHV-A59. Thus to study the potential contribution of the M gene to acute encephalitis, chimeric viruses were produced in which the M gene of MHV-A59 was substituted with the M gene of MHV-2 by targeted recombination. A control virus was produced in which the M gene of A59 was recombined back into an A59 background. Viruses were then analyzed for their biologic properties and compared with the phenotypes of MHV-A59 and MHV-2 by histopathology and plaque assays for viral titers in organs following intracerebral (IC) inoculation. All three chimeric viruses had a phenotype similar to MHV-A59. Thus, the replacement of the M gene of MHV-A59 with that of MHV-2 is insufficient to produce a phenotype that lacks encephalitis similar to MHV-2.

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.

We have inserted heterologous genetic material into the nonessential gene 4 of the coronavirus mouse hepatitis virus (MHV) in order to test the applicability of targeted RNA recombination for site-directed mutagenesis of the MHV genome upstream of the nucleocapsid (N) gene and to develop further genetic tools for site-directed mutagenesis of structural genes other than N. Initially, a 19-nucleotide tag was inserted into the start of gene 4a of MHV strain A59 with the N gene deletion mutant Alb4 as the recipient virus. In further work, the entire gene for the green fluorescent protein (GFP) was inserted in place of gene 4, creating the currently largest known RNA virus. The expression of GFP was demonstrated by Western blot analysis of infected cell lysates; however, the level of GFP expression was not sufficient to allow detection of fluorescence of viral plaques. Northern blot analysis of transcripts of GFP recombinants showed the expected alteration of the pattern of the nested MHV subgenomic mRNAs. Surprisingly, though, GFP recombinants also produced an RNA species that was the same size as wild-type mRNA4. Analysis of the 5' end of this species revealed that it was actually a collection of mRNAs originating from 10 different genomic fusion sites, none possessing a canonical intergenic sequence. The finding of these aberrant mRNAs suggests that long-range RNA or the ribonucleoprotein structure of the MHV genome can sometimes be the sole determinant of the site of initiation of transcription.

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.

Our laboratory has described a method for 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. By using a thermolabile N protein mutant of MHV as the recipient virus and synthetic RNA7 (the mRNA for the nucleocapsid protein N) as the donor, engineered recombinant viruses were selected as heat-stable progeny resulting from cotransfection. We have recently reported an optimization of 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 recombinants can often be directly identified without employing a thermal selection. We present here a progress report on our use of this system to map MHV mutants and to construct N gene mutants which include (1) a mutant in which the internal open reading frame within the N gene (the I gene) has been disrupted, and (2) a series of recombinants in which portions of the MHV N gene have been replaced by the homologous regions from the N gene of bovine coronavirus. We also report on some mutants we have not been able to construct.

The nucleocapsid (N) proteins of coronaviruses, as with most enveloped viruses, have received less attention than the surface glycoproteins and generally have been perceived to be of lesser concern. Interest in this class of proteins, however, has been stimulated in recent years by such diverse developments as the recognition of the importance of RNA-protein interactions, most notably in the study of spliceosomes and related ribonucleoproteins, as well as the finding in numerous viral systems that internal virion proteins can be major determinants of the cellular immune response.

Coronaviruses, like virtually all single-stranded RNA viruses, contain a nucleocapsid (N) protein in close structural association with their genomes.1 Knowledge of the structure and functions of N proteins would be fundamental to an understanding of the viral life cycle and pathogenesis. We have undertaken a study of the N protein of mouse hepatitis virus (MHV) in an attempt to learn the roles of this protein in viral assembly, transcription and translation. Specifically, we would like to examine: (i) the nature of the interaction between N protein and the genome RNA; (ii) the nature of the interactions between adjacent and more distant pairs of N monomers in the helically symmetric viral nucleocapsid; and (iii) the location and purpose of the phosphoserine residue(s) known to occur in the N molecule.2,3 To date, we have pursued three approaches to these questions: (i) a comparison of the sequences of the N proteins of different, independently isolated strains of MHV; (ii) the examination of an N protein mutant of MHV-A59, Albany-4; and (iii) the in vitro expression and functional assay of N protein and of engineered mutants of N protein. Our results suggest that the MHV N protein has three structural domains, at least two of which may be functionally distinct.

The effects of a subsaturating, long treatment (24 h) dose of a highly purified cloned subspecies of human leukocyte interferon (IFN-alpha A) on vesicular stomatitis virus (VSV) primary macromolecular synthesis in tsG41-infected human amnion U cells were examined. IFN-alpha A, under these conditions, was found to inhibit primary VSV protein synthesis ten-fold while producing no detectable effect on the amount or integrity of primary viral message transcripts. There was no selective reduction by IFN-alpha A of the VSV G or M proteins.

The nucleotide sequence of the 3′ end of the genome of Chandipura (CHP) virus, including the complete sequences of the nucleocapsid (N) and phosphoprotein (NS) genes was determined, principally from cloned cDNAs of the N and NS mRNAs. The NS mRNA of CHIP virus is 908 bases in length and encodes a protein of 293 amino acids. Comparison of the CHP virus NS protein sequence with those of vesicular stomatitis virus of the New Jersey serotype (VSV (NJ)) and of the Indiana serotype (VSV (IND)) revealed homologies of only 23 and 21%, respectively, with no consecutive stretches of more than four amino acids identical among the three sequences. As with the two VSV serotypes, the highest homology between the NS proteins of CHIP and VSV was in a 20-amino acid region near the carboxy termini of the proteins. Of the potential phosphorylation sites, there are eight conserved serine or threonine residues among the three sequences. Despite the dissimilarity among primary sequences of the NS proteins, their overall structure, as assessed by amino acid composition and by the relative hydropathicities of the sequences, has been conserved throughout evolution. The N mRNA of CHP virus is 1291 bases long and encodes a protein of 422 amino acids. In contrast to the NS protein, the CHIP N protein is at least 50% homologous to the N proteins of each of the VSV serotypes. We have identified a region near the center of these N protein sequences which is conserved among members of both the rhabdovirus and paramyxovirus families. This extent of conservation of the N protein sequences underscores the high rate of mutability of the NS protein sequences among the vesiculoviruses.