Brenda G. Hogue

Sequence analysis of the bovine coronavirus nucleocapsid and matrix protein genes

Abstract The 3′ end of the 20-kb genome of the Mebus strain of bovine enteric coronavirus (BCV) was copied into cDNA and cloned into the PstI site of the pUC9 vector. Four clones from the 3′ end of the genome were sequenced either completely or in part to determine the sequence of the first 2451 bases. Within this sequence were identified, in order, a 3′-noncoding region of 291 bases, the gene for a 448-amino acid nucleocapsid protein (N) having a molecular weight of 49,379, and the gene for a 230-amino acid matrix protein (M) having a molecular weight of 26,376. A third large open reading frame is contained entirely within the N gene sequence but is positioned in a different reading frame; it potentially encodes a polypeptide of 207 amino acids having a molecular weight of 23,057. A higher degree of amino acid sequence homology was found between the M proteins of BCV and MHV (87%) than between the N proteins (70%). For the M proteins of BCV and MHV, notable differences were found at the amino terminus, the most probable site of O-glycosylation, where the sequence is N-Met-Ser-Ser-Val-Thr-Thr for BCV and N-Met-Ser-Ser-Thr-Thr for MHV. BCV apparently uses two of its six potential O-glycosylation sites.

Host Protein Interactions with the 3′ End of Bovine Coronavirus RNA and the Requirement of the Poly(A) Tail for Coronavirus Defective Genome Replication

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

Mouse Hepatitis Coronavirus A59 Nucleocapsid Protein Is a Type I Interferon Antagonist

ABSTRACT The recent emergence of several new coronaviruses, including the etiological cause of severe acute respiratory syndrome, has significantly increased the importance of understanding virus-host cell interactions of this virus family. We used mouse hepatitis virus (MHV) A59 as a model to gain insight into how coronaviruses affect the type I alpha/beta interferon (IFN) system. We demonstrate that MHV is resistant to type I IFN. Protein kinase R (PKR) and the alpha subunit of eukaryotic translation initiation factor are not phosphorylated in infected cells. The RNase L activity associated with 2′,5′-oligoadenylate synthetase is not activated or is blocked, since cellular RNA is not degraded. These results are consistent with lack of protein translation shutoff early following infection. We used a well-established recombinant vaccinia virus (VV)-based expression system that lacks the viral IFN antagonist E3L to screen viral genes for their ability to rescue the IFN sensitivity of the mutant. The nucleocapsid (N) gene rescued VVΔE3L from IFN sensitivity. N gene expression prevents cellular RNA degradation and partially rescues the dramatic translation shutoff characteristic of the VVΔE3L virus. However, it does not prevent PKR phosphorylation. The results indicate that the MHV N protein is a type I IFN antagonist that likely plays a role in circumventing the innate immune response.

Structural proteins of human respiratory coronavirus OC43

Abstract The human respiratory coronavirus OC43 was grown on a human rectal tumor cell line and was isotopically labeled with amino acids, glucosamine, and orthophosphate to analyze virion structural proteins. Four major protein species were resolved by electrophoresis and many of their properties were deduced from digestion studies using proteolytic enzymes. The four proteins are: (1) A 190 kDa protein, the presumed peplomeric protein, that was glycosylated and proteolytically cleavable by trypsin into subunits of 110 and 90 kDa. The subunits each represent a different amino acid sequence on the basis of peptide mapping; (2) a 130 kDa protein that was glycosylated and behaved as a disulfide-linked dimer of 65 kDa molecules. It is the apparent virion hemagglutinin on the basis of digestion studies with trypsin, bromelain and pronase; (3) a 55 kDa nucleocapsid protein that was phosphorylated; (4) a 26 kDa matrix protein that was glycosylated. The 190, 130, 55 and 26 kDa species can therefore be designated P, H, N and M, respectively. They exist in molar ratios of 4:1: 33 : 33, and are calculated to be present at the rate of 88, 22, 726, and 726 molecules per virion, respectively.

The 9-kDa hydrophobic protein encoded at the 3' end of the porcine transmissible gastroenteritis coronavirus genome is membrane-associated.

Abstract The open reading frame potentially encoding a 78 amino acid, 9101 Da hydrophobic protein (HP) and, mapping at the 3′ end of the porcine transmissible gastroenteritis coronavirus (TGEV) genome, was shown to be expressed during virus replication. The cloned HP gene was placed in a plasmid under control of the T7 RNA polymerase promoter and in vitro translation of transcripts generated in vitro yielded a 9.1-kDa protein that was immunoprecipitable with porcine hyperimmune anti-TGEV serum. Antiserum raised in rabbits against a 31 amino acid synthetic polypeptide that represented the central hydrophilic region of HP specifically immunoprecipitated HP from TGEV-infected cells. HP was further shown to become associated with microsomal membranes during synthesis in vitro and was found to be closely associated with the endoplasmic reticulum and cell surface membranes in infected cells. The intracellular location of HP suggests that it may play a role in the membrane association of replication complexes or in virion assembly.

The Coronavirus Hemagglutinin Esterase Glycoprotein

Early reviews on Coronavirus structure (Siddell et al., 1983a,b; Sturman and Holmes, 1985) described coronaviruses as having three major structural proteins: a large-surface (or peplomer) glycoprotein of around 200 kDa, a phos-phorylated nucleocapsid protein of around 50 kDa, and a glycosylated, multi-spanning membrane protein of around 30 kDa. This description was based primarily on studies of the prototypic avian infectious bronchitis virus (IBV) and the highly studied mouse hepatitis virus (MHV), strain A59. Although IBV was shown early on to have a weak hemagglutinating property, detection of the hemagglutinating activity required that the virus first be treated with phospho-lipase C or concentrated by centrifugation in sucrose gradients (Bingham et al., 1975). Not all strains of IBV demonstrated hemagglutination, however, and hemagglutinating activity by IBV, as well as by the porcine transmissible gastroenteritis virus (TGEV) (Noda et al., 1987, 1988) was probably a cryptic property of the spike protein (Cavanagh and Davis, 1986). The nature of this hemagglutinating activity is not well understood.

Identification of Nucleocapsid Binding Sites within Coronavirus-Defective Genomes

Abstract The coronavirus nucleocapsid (N) protein is a major structural component of virions that associates with the genomic RNA to form a helical nucleocapsid. N appears to be a multifunctional protein since data also suggest that the protein may be involved in viral RNA replication and translation. All of these functions presumably involve interactions between N and viral RNAs. As a step toward understanding how N interacts with viral RNAs, we mapped high-efficiency N-binding sites within BCV- and MHV-defective genomes. Both in vivo and in vitro assays were used to study binding of BCV and MHV N proteins to viral and nonviral RNAs. N–viral RNA complexes were detected in bovine coronavirus (BCV)-infected cells and in cells transiently expressing the N protein. Filter binding was used to map N-binding sites within Drep, a BCV-defective genome that is replicated and packaged in the presence of helper virus. One high-efficiency N-binding site was identified between nucleotides 1441 and 1875 at the 3′ end of the N ORF within Drep. For comparative purposes N-binding sites were also mapped for the mouse hepatitis coronavirus (MHV)-defective interfering (DI) RNA MIDI-C. Binding efficiencies similar to those for Drep were measured for RNA transcripts of a region encompassing the MHV packaging signal (nts 3949–4524), as well as a region at the 3′ end of the MHV N ORF (nts 4837–5197) within MIDI-C. Binding to the full-length MIDI-C transcript (∼5500 nts) and to an ∼1-kb transcript from the gene 1a region (nts 935–1986) of MIDI-C that excluded the packaging signal were both significantly higher than that measured for the smaller transcripts. This is the first identification of N-binding sequences for BCV. It is also the first report to demonstrate that N interacts in vitro with sequences other than the packaging signal and leader within the MHV genome. The data clearly demonstrate that N binds coronavirus RNAs more efficiently than nonviral RNAs. The results have implications with regard to the multifunctional role of N.

Role of the Coronavirus E Viroporin Protein Transmembrane Domain in Virus Assembly

ABSTRACT Coronavirus envelope (E) proteins are small (∼75- to 110-amino-acid) membrane proteins that have a short hydrophilic amino terminus, a relatively long hydrophobic membrane domain, and a long hydrophilic carboxy-terminal domain. The protein is a minor virion structural component that plays an important, not fully understood role in virus production. It was recently demonstrated that the protein forms ion channels. We investigated the importance of the hydrophobic domain of the mouse hepatitis coronavirus (MHV) A59 E protein. Alanine scanning insertion mutagenesis was used to examine the effect of disruption of the domain on virus production in the context of the virus genome by using a MHV A59 infectious clone. Mutant viruses exhibited smaller plaque phenotypes, and virus production was significantly crippled. Analysis of recovered viruses suggested that the structure of the presumed α-helical structure and positioning of polar hydrophilic residues within the predicted transmembrane domain are important for virus production. Generation of viruses with restored wild-type helical pitch resulted in increased virus production, but some exhibited decreased virus release. Viruses with the restored helical pitch were more sensitive to treatment with the ion channel inhibitor hexamethylene amiloride than were the more crippled parental viruses with the single alanine insertions, suggesting that disruption of the transmembrane domain affects the functional activity of the protein. Overall the results indicate that the transmembrane domain plays a crucial role during biogenesis of virions.

Identification of a Bovine Coronavirus Packaging Signal

ABSTRACT A region of the bovine coronavirus (BCV) genome that functions as a packaging signal has been cloned. The 291-nucleotide clone shares 72% homology with the region of mouse hepatitis coronavirus (MHV) gene1b that contains the packaging signal. RNA transcripts were packaged into both BCV and MHV virions when the cloned region was appended to a noncoronavirus RNA. This is the first identification of a BCV packaging signal. The data demonstrate that the BCV genome contains a sequence that is conserved at both the sequence and functional levels, thus broadening our insight into coronavirus packaging.

The amino-terminal signal peptide on the porcine transmissible gastroenteritis coronavirus matrix protein is not an absolute requirement for membrane translocation and glycosylation

Abstract cDNA clones mapping within the first 2601 bases of the 3′ end of the porcine transmissible gastroenteritis coronavirus (TGEV) genome were sequenced by the method of Maxam and Gilbert and an open reading frame yielding a protein having properties of the matrix (M or E1) protein was identified. It is positioned at the 5′ side of the nucleocapsid (N) gene from which it is separated by an intergenic stretch of 12 bases. The deduced M protein comprises 262 amino acids, has a molecular weight of 29,544, is moderately hydrophobic, and has a net charge of +7 at neutral pH. Thirty-four percent of its amino acid sequence is homologous with the M protein of the bovine coronavirus (BCV), 32% with that of the mouse hepatitis coronavirus (MHV), and 19% with that of the avian infectious bronchitis coronavirus (IBV). Judging from alignment with the BCV, MHV, and IBV M proteins, the amino terminus of the TGEV M protein extends 54 amino acids from the virion envelope which compares with only 28 for BCV, 26 for MHV, and 21 for IBV. Eleven of the sixteen amino-terminal amino acids are hydrophobic and the positions of charged amino acids around this sequence suggest that the first 16 amino acids comprise a potentially cleavable signal peptide for membrane insertion. A similar sequence is not found in the M proteins of BCV, MHV, or IBV. When mRNA from infected cells, or RNA prepared by in vitro transcription of the reconstructed M gene, was translated in vitro in the presence of microsomes, the M protein became translocated and glycosylated. When a protein without the amino-terminal signal peptide was made by translating a truncated version of the M gene transcript, some translocation and glycosylation also occurred suggesting that the amino-terminal signal peptide on the TGEV M protein is not an absolute requirement for membrane translocation. Interestingly, the amino-terminal peptide did not appear to be cleaved during in vitro translation in the presence of microsomes suggesting that a step in virion assembly may be required for proper exposure of the cleavage site to the signal peptidase.