Identification of sequences in Brome mosaic virus replicase protein 1a that mediate association with endoplasmic reticulum membranes.

RNA replication of all positive-strand RNA viruses is closely associated with intracellular membranes. Brome mosaic virus (BMV) RNA replication occurs on the perinuclear region of the endoplasmic reticulum (ER), both in its natural plant host and in the yeast Saccharomyces cerevisiae. The only viral component in the BMV RNA replication complex that localizes independently to the ER is 1a, a multifunctional protein with an N-terminal RNA capping domain and a C-terminal helicase-like domain. The other viral replication components, the RNA polymerase-like protein 2a and the RNA template, depend on 1a for recruitment to the ER. We show here that, in membrane extracts, 1a is fully susceptible to proteolytic digestion in the absence of detergent and thus, a finding consistent with its roles in RNA replication, is wholly or predominantly on the cytoplasmic face of the ER with no detectable lumenal protrusions. Nevertheless, 1a association with membranes is resistant to high-salt and high-pH treatments that release most peripheral membrane proteins. Membrane flotation gradient analysis of 1a deletion variants and 1a segments fused to green fluorescent protein (GFP) showed that sequences in the N-terminal RNA capping module of 1a mediate membrane association. In particular, a region C-terminal to the core methyltransferase homology was sufficient for high-affinity ER membrane association. Confocal immunofluorescence microscopy showed that even though these determinants mediate ER localization, they fail to localize GFP to the narrow region of the perinuclear ER, where full-length 1a normally resides. Instead, they mediate a more globular or convoluted distribution of ER markers. Thus, additional sequences in 1a that are distinct from the primary membrane association determinants contribute to 1a's normal subcellular distribution, possibly through effects on 1a conformation, orientation, or multimerization on the membrane.

Helicase and capping enzyme active site mutations in brome mosaic virus protein 1a cause defects in template recruitment, negative-strand RNA synthesis, and viral RNA capping.

Brome mosaic virus (BMV) encodes two RNA replication proteins: 1a, which contains RNA capping and helicase-like domains, and 2a, which is related to polymerases. BMV 1a and 2a can direct virus-specific RNA replication in the yeast Saccharomyces cerevisiae, which reproduces the known features of BMV replication in plant cells. We constructed single amino acid point mutations at the predicted capping and helicase active sites of 1a and analyzed their effects on BMV RNA3 replication in yeast. The helicase mutants showed no function in any assays used: they were strongly defective in template recruitment for RNA replication, as measured by 1a-induced stabilization of RNA3, and they synthesized no detectable negative-strand or subgenomic RNA. Capping domain mutants divided into two groups. The first exhibited increased template recruitment but nevertheless allowed only low levels of negative-strand and subgenomic mRNA synthesis. The second was strongly defective in template recruitment, made very low levels of negative strands, and made no detectable subgenomes. To distinguish between RNA synthesis and capping defects, we deleted chromosomal gene XRN1, encoding the major exonuclease that degrades uncapped mRNAs. XRN1 deletion suppressed the second but not the first group of capping mutants, allowing synthesis and accumulation of large amounts of uncapped subgenomic mRNAs, thus providing direct evidence for the importance of the viral RNA capping function. The helicase and capping enzyme mutants showed no complementation. Instead, at high levels of expression, a helicase mutant dominantly interfered with the function of the wild-type protein. These results are discussed in relation to the interconnected functions required for different steps of positive-strand RNA virus replication.

An infectious arterivirus cDNA clone: identification of a replicase point mutation that abolishes discontinuous mRNA transcription.

Equine arteritis virus (EAV) is a positive-strand RNA virus that uses a discontinuous transcription mechanism to generate a nested set of six subgenomic mRNAs from which its structural genes are expressed. A stable bacterial plasmid (pEAV030) containing a full-length cDNA copy of the 12.7-kb EAV genome was constructed. After removal of a single point mutation in the replicase gene, RNA transcripts generated in vitro from pEAV030 were shown to be infectious upon electroporation into BHK-21 cells. A genetic marker mutation was introduced at the cDNA level and recovered from the genome of the progeny virus. The potential of pEAV030 as a tool to express foreign genes was demonstrated by the efficient expression of the chloramphenicol acetyltransferase (CAT) reporter gene from two different subgenomic mRNAs. The point mutation that initially rendered the full-length clone noninfectious was found to result in a particularly intriguing phenotype: RNA carrying this mutation can replicate efficiently but does not produce the subgenomic mRNAs required for structural protein expression. To our knowledge, this mutant provides the first evidence that the requirements for arterivirus genome replication and discontinuous mRNA synthesis are, at least partially, different and that these processes may be separated experimentally.

Equine arteritis virus subgenomic mRNA synthesis: analysis of leader-body junctions and replicative-form RNAs.

In addition to the genomic RNA, a 3' coterminal nested set of six subgenomic mRNAs is produced in equine arteritis virus (EAV)-infected cells. The seven viral RNAs are also 5' coterminal, since they all contain a 206-nucleotide common leader sequence which is identical to the 5' end of the genome. A conserved penta-nucleotide sequence motif, 5' UCAAC 3', was shown to be present at the junctions between the leader and body sequences in each of the mRNAs. In addition, two alternative junction sites were detected for mRNA 3. Seven replicative-form (RF) RNAs (RFs I to VII), corresponding to the genomic RNA and each of the subgenomic EAV mRNAs, could be prepared from lysates of infected cells. The minus-strand RNA contents of these RF RNAs were analyzed by using an RNase protection assay with an RNA probe containing the mRNA 2 leader-body junction. It was established that RF II contained a negative-stranded copy of mRNA 2, including a complementary leader sequence. The presence of subgenomic minus-strand RNA in RFs is indicative of a function as a transcription template during the production of EAV subgenomic mRNAs.

Equine arteritis virus subgenomic RNA transcription: UV inactivation and translation inhibition studies.

The expression of the genetic information of equine arteritis virus (EAV), an arterivirus, involves the synthesis of six subgenomic (sg) mRNAs. These are 5' and 3' coterminal since they are composed of a leader and a body sequence, which are identical to the 5' and 3' ends of the genome, respectively. Previously, it has been suggested that cis-splicing of a genome-length precursor RNA is involved in their synthesis. This was reevaluated in a comparative analysis of the sg RNA synthesis of EAV, the coronavirus mouse hepatitis virus (MHV), and the alphavirus Sindbis virus. UV transcription mapping showed that the majority of the EAV sg RNAs made at later stages of infection is not derived from a genome-length precursor. However, complete independence of sg RNA synthesis from that of genomic RNA was never observed during the course of infection. The possibility that this resulted from UV irradiation-induced effects on the synthesis of the viral replicase was investigated by inhibiting translation using cycloheximide. For EAV, ongoing protein synthesis was found to be more important for the synthesis of sg RNA than for that of genomic RNA. In general, MHV transcription was extremely sensitive to translation inhibition, whereas EAV genomic RNA synthesis became independent of de novo protein synthesis late in infection.

Processing and evolution of the N-terminal region of the arterivirus replicase ORF1a protein: identification of two papainlike cysteine proteases.

Two adjacent papainlike cysteine protease (PCP) domains, PCP alpha and PCP beta, were identified in the N-terminal region of the open reading frame 1a replicase proteins of the arteriviruses porcine reproductive and respiratory syndrome virus and lactate dehydrogenase-elevating virus. The replicase of the related virus equine arteritis virus contains only one active PCP in the corresponding region. Sequence comparison revealed that the equine arteritis virus PCP alpha counterpart probably was inactivated by loss of its catalytic Cys residue. For both porcine reproductive and respiratory syndrome virus and lactate dehydrogenase-elevating virus, the generation of two processing products, nsp1 alpha and nsp1 beta, was demonstrated by in vitro translation. Site-directed mutagenesis and sequence comparison were used to identify the putative active-site residues of the PCP alpha and PCP beta protease domains and to show that they mediate the nsp1 alpha/1 beta and nsp1 beta/2 cleavages, respectively.

Responsiveness of the increase in C-fos mRNA levels depends on the inducer and the cell's past.

In this paper we show that in C3H10T1/2 mouse fibroblasts, the inducibility of c-fos mRNA by heat shock or serum addition is strongly dependent on the cell's past. Four hours after a heat shock, a time point where the induced c-fos mRNA has disappeared, c-fos mRNA could not be induced again by a second heat shock. Four hours after serum addition, by which c-fos was induced, a second serum addition also failed to induce c-fos mRNA again. When, however, serum was added 4 hours after heat shock or heat shock was given 4 hours after serum addition, levels of c-fos mRNA could be enhanced again. The induction by serum of c-fos mRNA levels in thermotolerant cells might be related to their increased stimulation of DNA synthesis as compared to control cells.

Characterization of defective interfering RNAs of Berne virus.

Defective interfering (DI) RNAs of Berne virus (BEV) were generated by serial undiluted passaging of the virus in embryonic mule skin cells. Two DI RNAs of 1.0 and 1.4 kb (designated DI1000 and DI1400) were characterized in more detail. Isokinetic sucrose gradient analysis showed that these DI RNAs are specifically packaged into particles with smaller S values than standard virions. Both DI RNAs were cloned and sequenced. Three genomic cDNA clones were identified using probes complementary to the 5' end of a DI RNA, which are thought to be derived from the 5'-terminal region of the BEV genome. A non-translated region of about 700 nt and the 5' end of the putative BEV replicase gene were identified in the consensus nucleotide sequence. Both DI RNAs were shown to contain sequences from the 5' and 3' ends of the BEV genome. A conserved sequence motif, which has been postulated to be involved in sub-genomic RNA transcription, was also identified just downstream of the extreme 5' ends of DI1000 and DI1400.

Equine arteritis virus is not a togavirus but belongs to the coronaviruslike superfamily.

The nucleotide sequence of the genome of equine arteritis virus (EAV) was determined from a set of overlapping cDNA clones and was found to contain eight open reading frames (ORFs). ORFs 2 through 7 are expressed from six 3'-coterminal subgenomic mRNAs, which are transcribed from the 3'-terminal quarter of the viral genome. A number of these ORFs are predicted to encode structural EAV proteins. The organization and expression of the 3' part of the EAV genome are remarkably similar to those of coronaviruses and toroviruses. The 5'-terminal three-quarters of the genome contain the putative EAV polymerase gene, which also shares a number of features with the corresponding gene of corona- and toroviruses. The gene contains two large ORFs, ORF1a and ORF1b, with an overlap region of 19 nucleotides. The presence of a "shifty" heptanucleotide sequence in this region and a downstream RNA pseudoknot structure indicate that ORF1b is probably expressed by ribosomal frameshifting. The frameshift-directing potential of the ORF1a/ORF1b overlap region was demonstrated by using a reporter gene. Moreover, the predicted ORF1b product was found to contain four domains which have been identified in the same relative positions in coronavirus and torovirus ORF1b products. The sequences of the EAV and coronavirus ORF1a proteins were found to be much more diverged. The EAV ORF1a product contains a putative trypsinlike serine protease motif. Our data indicate that EAV, presently considered a togavirus, is evolutionarily related to viruses from the coronaviruslike superfamily.

Another triple-spanning envelope protein among intracellularly budding RNA viruses: the torovirus E protein.

The nucleotide sequence of the Berne virus envelope (E) protein gene was determined and its 26.5K translation product was identified by in vitro transcription and translation. Computer analysis of the protein sequence revealed the characteristics of a class III membrane protein lacking a cleaved signal sequence but containing three successive transmembrane alpha-helices in the N-terminal half, much the same as the coronavirus membrane (M) protein. The disposition of the E protein in the membrane was studied by in vitro translation in the presence of microsomes and by subsequent proteinase K digestion. Only small portions of either end of the polypeptide were found to be exposed on opposite sides of the vesicle membranes. Experiments with a hybrid E protein (EM) containing the C-terminal tail of a coronavirus M protein, to which an anti-peptide serum was available, showed that this C-terminus was present at the cytoplasmic side of the membrane, which is another similarity to the coronavirus M protein. Immunofluorescence experiments indicated that the EM protein, expressed by a recombinant vaccinia virus, accumulated in intracellular membranes, predominantly those of the endoplasmic reticulum. The common features of the torovirus E and the coronavirus M protein support our hypothesis that an evolutionary relationship exists between these groups of intracellularly budding viruses.

Comparison of the genome organization of toro- and coronaviruses: evidence for two nonhomologous RNA recombination events during Berne virus evolution.

Recently, toroviruses and coronaviruses have been found to be ancestrally related by divergence of their polymerase and envelope proteins from common ancestors. In addition, their genome organization and expression strategy, which involves the synthesis of a 3'-coterminal nested set of mRNAs, are comparable. Nucleotide sequence analysis of the genome of the torovirus prototype, Berne virus (BEV), has now revealed the results of two independent nonhomologous RNA recombinations during torovirus evolution. Berne virus open reading frame (ORF) 4 encodes a protein with significant sequence similarity (30-35% identical residues) to a part of the hemagglutinin esterase proteins of coronaviruses and influenza virus C. The sequence of the C-terminal part of the predicted BEV polymerase ORF1a product contains 31-36% identical amino acids when compared with the sequence of a nonstructural 30/32K coronavirus protein. The cluster of coronaviruses which contains this nonstructural gene expresses it not as a part of their polymerase, but by synthesizing an additional subgenomic mRNA.

Primary structure and post-translational processing of the Berne virus peplomer protein.

The nucleotide sequence of the peplomer (P) protein gene of Berne virus (BEV), the torovirus prototype, was determined. The gene encodes an apoprotein of 1581 amino acids with an Mr of about 178K. The open reading frame was cloned behind the T7 RNA polymerase promoter and its translation product was identified as the BEV P protein precursor by in vivo expression and immunoprecipitation. The deduced amino acid sequence contains a number of domains which are typical for type I membrane glycoproteins: an N-terminal signal sequence, a putative C-terminal transmembrane anchor, and a cytoplasmic tail. Eighteen potential N-glycosylation sites, two heptad repeat domains, and a possible "trypsin-like" cleavage site were identified. The mature P protein consists of two subunits and their electrophoretic mobility upon endoglycosidase F treatment strongly suggests that the predicted cleavage site is functional in vivo. The heptad repeat domains are probably involved in the generation of an intra-chain coiled-coil secondary structure; similar inter-chain interactions can play a role in P protein oligomerization. Using a sucrose gradient assay the P protein was indeed shown to form dimers. The intra- and inter-chain coiled-coil interactions may stabilize the elongated BEV peplomers.

The carboxyl-terminal part of the putative Berne virus polymerase is expressed by ribosomal frameshifting and contains sequence motifs which indicate that toro- and coronaviruses are evolutionarily related.

Sequence analysis of the 3' part (8 kb) of the polymerase gene of the torovirus prototype Berne virus (BEV) revealed that this area contains at least two open reading frames (provisionally designated ORF1a and ORF1b) which overlap by 12 nucleotides. The complete sequence of ORF1b (6873 nucleotides) was determined. Like the coronaviruses, BEV was shown to express its ORF1b by ribosomal frameshifting during translation of the genomic RNA. The predicted tertiary RNA structure (a pseudoknot) in the toro- and coronaviral frameshift-directing region is similar. Analysis of the amino acid sequence of the predicted BEV ORF1b translation product revealed homology with the ORF1b product of coronaviruses. Four conserved domains were identified: the putative polymerase domain, an area containing conserved cysteine and histidine residues, a putative helicase motif, and a domain which seems to be unique for toro- and coronaviruses. The data on the 3' part of the polymerase gene of BEV supplement previously observed similarities between toro- and coronaviruses at the level of genome organization and expression. The two virus families are more closely related to each other than to other families of positive-stranded RNA viruses.

The polymerase gene of corona- and toroviruses: evidence for an evolutionary relationship.

In this paper we demonstrate that the organization of the polymerase gene of toroviruses and coronaviruses is similar. The polymerase gene of both virus families consists of at least two large ORFs (1a and 1b). Four domains of conserved amino acid sequences have been identified in nearly identical positions in the 3' ORF of the pol gene of toroviruses and coronaviruses. The most 3' conserved domain which is still unique for these viruses encodes a 33-kDA protein in MHV-A59, which is cleaved from a precursor protein. Expression of ORF1b of the pol gene of both virus families occurs by ribosomal frameshifting. A predicted stem-loop structure and pseudoknot are conserved in the ORF1a/ORF1b overlap of toro- and coronaviruses. On the basis of these results we postulate that toro- and coronaviruses are ancestrally more related to each other than to other families of positive stranded RNA viruses.

Identification and primary structure of the gene encoding the Berne virus nucleocapsid protein.

The nucleotide sequence of the nucleocapsid (N) protein gene of Berne virus (BEV; proposed family Toroviridae) was determined from two independent clones of a cDNA library. From the deduced amino acid sequence a basic protein of 18.3K was predicted. In vitro transcription and translation, followed by immunoprecipitation, were used to identify the gene. The identification was confirmed by metabolic labelling, using the knowledge that cysteine residues are absent from the amino acid sequence of the N protein. Smaller N-related polypeptides encountered in BEV-infected cell lysates were shown to be probable products of aberrant translation, due to initiation on AUG codons further downstream in the N protein gene.