The influence of downstream protein-coding sequence on internal ribosome entry on hepatitis C virus and other flavivirus RNAs.

Some studies suggest that the hepatitis C virus (HCV) internal ribosome entry site (IRES) requires downstream 5' viral polyprotein-coding sequence for efficient initiation of translation, but the role of this RNA sequence in internal ribosome entry remains unresolved. We confirmed that the inclusion of viral sequence downstream of the AUG initiator codon increased IRES-dependent translation of a reporter RNA encoding secretory alkaline phosphatase, but found that efficient translation of chloramphenicol acetyl transferase (CAT) required no viral sequence downstream of the initiator codon. However, deletion of an adenosine-rich domain near the 5' end of the CAT sequence, or the insertion of a small stable hairpin structure (deltaG = -18 kcal/mol) between the HCV IRES and CAT sequences (hpCAT) substantially reduced IRES-mediated translation. Although translation could be restored to both mutants by the inclusion of 14 nt of the polyprotein-coding sequence downstream of the AUG codon, a mutational analysis of the inserted protein-coding sequence demonstrated no requirement for either a specific nucleotide or amino acid-coding sequence to restore efficient IRES-mediated translation to hpCAT. Similar results were obtained with the structurally and phylogenetically related IRES elements of classical swine fever virus and GB virus B. We conclude that there is no absolute requirement for viral protein-coding sequence with this class of IRES elements, but that there is a requirement for an absence of stable RNA structure immediately downstream of the AUG initiator codon. Stable RNA structure immediately downstream of the initiator codon inhibits internal initiation of translation but, in the case of hpCAT, did not reduce the capacity of the RNA to bind to purified 40S ribosome subunits. Thus, stable RNA structure within the 5' proximal protein-coding sequence does not alter the capacity of the IRES to form initial contacts with the 40S subunit, but appears instead to prevent the formation of subsequent interactions between the 40S subunit and viral RNA in the vicinity of the initiator codon that are essential for efficient internal ribosome entry.

Internal entry of ribosomes is directed by the 5' noncoding region of classical swine fever virus and is dependent on the presence of an RNA pseudoknot upstream of the initiation codon.

Bicistronic RNAs containing the 373-nucleotide-long 5' nontranslated region (NTR) of the classical swine fever virus (CSFV) genome as intercistronic spacer were used to show the presence of an internal ribosome entry site (IRES) in the 5' end of the CSFV genome. By coexpression of the poliovirus 2A protease it was demonstrated that the CSFV 5' NTR-driven translation is independent of the presence of functional eukaryotic initiation factor eIF-4F. Deletion analysis indicated that the 5' border of the IRES is located between nucleotides 28 and 66. The role of a proposed pseudoknot structure at the 3' end of the CSFV 5' NTR in IRES-mediated translation was investigated by site-directed mutagenesis. Mutant RNAs that had lost the ability to base pair in stem II of the pseudoknot were translationally inactive. Translation to wild-type levels could be restored through the introduction of compensatory complementary base changes that repaired base pairing in stem II. In addition, we showed that the AUG codon, which is located 7 nucleotides upstream of the polyprotein initiation site and is conserved in pestiviruses, could not be used to initiate translation. Also, an AUG codon introduced downstream of the polyprotein initiation site was not recognized as an initiation site by ribosomes. These data suggest that after internal entry on the CSFV 5' NTR, ribosomal scanning for the initiation codon is limited to a small region.

The influence of AUG codons in the hepatitis C virus 5' nontranslated region on translation and mapping of the translation initiation window.

The initiation of translation of hepatitis C virus (HCV) is cap-independent and mediated by an internal ribosome entry site (IRES) that is located in the 5' nontranslated region (5' NTR) of the viral genome. This 5' NTR is relatively long and folds into a complex structure involving multiple hairpins and a pseudoknot. Within the sequence encompassing the IRES there are several AUG triplets. Some of these AUG codons are conserved between HCV genotypes and the related pestiviruses. In this study the 5 AUG codons (positions 13, 32, 85, 96, and 215) that are present in the 5' NTR of the HCV H-strain have been mutagenized to determine their influence on HCV cap-independent translation. The effect of these mutations on the expression of a chloramphenicol acetyl transferase (CAT) gene was tested in vaccinia virus. vTF7-3 infected Hep2 cells transfected with plasmids for the expression of a monocistronic HCV 5' NTR-CAT mRNA. Mutating the AUG codons at positions 13, 32, and 215 does not have a significant effect on CAT expression, inactivating the AUG codons at either position 85 or position 96 severely impaired IRES function. To determine whether ribosomes scan the RNA to select the initiation site, AUG codons were inserted up- and downstream of the authentic HCV polyprotein translation initiation codon (position 342). Analysis of these mutants has revealed that the ribosome is unable to use an AUG codon that is placed either 7 nucleotides upstream or 8 nucleotides downstream of the inactivated AUG at position 342. These results indicate that when scanning is involved in the recognition of the translation initiating AUG, it is limited to a narrow region between nucleotides 335 and 350.

Sindbis virus expression vectors: packaging of RNA replicons by using defective helper RNAs.

Since the recovery of infectious RNA transcripts from full-length cDNA clones, alphavirus genome RNAs have been engineered to allow expression of heterologous RNAs and proteins. The highest levels of expression of heterologous products are achieved when the viral structural genes are replaced by the heterologous coding sequences. Such recombinant RNAs are self-replicating (replicons) and can be introduced into cells as naked RNA, but they require trans complementation to be packaged and released from cells as infectious virion particles. In this report, we describe a series of defective Sindbis virus helper RNAs which can be used for packaging Sindbis virus RNA replicons. The defective helper RNAs contain the cis-acting sequences required for replication as well as the subgenomic RNA promoter which drives expression of the structural protein genes. In cells cotransfected with both the replicon and defective helper RNAs, viral nonstructural proteins translated from the replicon RNA allow replication and transcription of the defective helper RNA to produce the virion structural proteins. A series of defective helper RNAs were compared for the ability to package the replicon RNA as well as for the ability to be replicated and packaged. One defective helper RNA not only packaged the replicon but also was itself encapsidated and would be useful under conditions in which extensive amplification is advantageous. Other defective helper RNAs were able to package the replicon efficiently but were packaged very poorly themselves. These helpers should be useful for applications in which expression of the viral structural proteins or virus spread is not desired.

A domain at the 3' end of the polymerase gene is essential for encapsidation of coronavirus defective interfering RNAs.

Two murine hepatitis virus strain A59 defective interfering (DI) RNAs were generated by undiluted virus passages. The DI RNAs were encapsidated efficiently. The smallest DI particle, DI-a, contained a 5.5-kb RNA consisting of the following three noncontiguous regions from the MHV-A59 genome, which were joined in frame: the 5'-terminal 3.9 kb, a 798-nucleotide fragment from the 3' end of the polymerase gene, and the 3'-terminal 805 nucleotides. A full-length cDNA clone of the DI-a genome was constructed and cloned downstream of the bacteriophage T7 promoter. Transcripts derived from this clone, pMIDI, were used for transfection of MHV-A59-infected cells and found to be amplified and packaged. Deletion analysis of pMIDI allowed us to identify a 650-nucleotide region derived from the 3' end of the second open reading frame of the polymerase gene that was required for efficient encapsidation.

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.

All subgenomic mRNAs of equine arteritis virus contain a common leader sequence.

During the replication of equine arteritis virus (EAV) six subgenomic mRNAs are synthesized. We present evidence that the viral mRNAs form a 3'-coterminal nested set and contain a common leader sequence of 208 nucleotides which is encoded by the 5'-end of the genome. The leader is joined to the bodies of mRNA 5 and 6 at positions defined by the sequence 5' UCAAC 3'. The part of the leader sequence flanking the UCAAC motif is very similar to the 5'-splice site of the Tetrahymena pre-rRNA. A possible internal guide sequence has been identified 43 nucleotides downstream of the leader sequence on the genome. Hybridization analysis shows that all EAV intracellular RNAs contain the leader sequence. These data imply that the viral subgenomic mRNAs are composed of leader and body sequences which are non-contiguous on the genome.

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

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

Identification and stability of a 30-kDa nonstructural protein encoded by mRNA 2 of mouse hepatitis virus in infected cells.

A bacterial expression vector encoding a fusion protein containing almost the entire first open reading frame (ORF1) of mRNA 2 of MHV-A59 has been constructed. The purified fusion protein was used to raise antibodies to the protein encoded by mRNA 2 ORF1. Specificity of the antibodies was verified by immunoprecipitation of the in vitro translation product of ORF1, which was reconstructed downstream of a T7 promoter. In vivo the antiserum reacted specifically with a 30-kDa protein synthesized in MHV-A59- and MHV-JHM-infected cells. This 30-kDa protein could not be identified in purified virions and is therefore a nonstructural viral protein. The expression pattern of this 30-kDa nonstructural viral protein in infected cells was shown to be identical to that of the viral structural proteins. However, in comparison to the nucleocapsid protein pulse-chase studies revealed a relative short half life for this 30-kDa protein in vivo.

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.

Molecular cloning of the gene encoding the putative polymerase of mouse hepatitis coronavirus, strain A59.

Complementary DNA (cDNA) libraries were constructed representing the genome RNA of the coronavirus mouse hepatitis virus, strain A59 (MHV-A59). From these libraries clones were selected to form a linear map across the entire gene A, the putative viral polymerase gene. This gene is approximately 23 kb in length, considerably larger than earlier estimates. Sequence analysis of the 5' terminal region of the genome indicates the presence of the 66-nucleotide leader that is found on all mRNAs. Secondary structure analysis of the 5' terminal region suggests that transcription of leader terminates in the region of nucleotide 66. The sequence of the first 2000 nucleotides is very similar to that reported for the closely related JHM strain of MHV and potentially encodes p28, a basic protein thought to be a component of the viral polymerase (L. Soe, C. K. Shieh, S. Baker, M. F. Chang, and M. M. C. Lai, 1987, J. Virol., 61, 3968-3976). Gene A contains two of the consensus sequences found in intergenic regions. One is adjacent to the 5' leader sequence and the other is upstream from the initiation codon for translation of gene B.

Differential premature termination of transcription as a proposed mechanism for the regulation of coronavirus gene expression.

We propose that the different subgenomic mRNA levels of coronaviruses are controlled through differential premature termination of transcription, and are modulated by the relative strength of transcriptional initiation/blockage events. We present the complete set of sequences covering the leader encoding and intergenic regions of the MHV-A59 strain. A computer-assisted analysis of the two now complete sets of these sequences of strain IBV-M42 and MHV-A59 shows that, in contrast to the previous theory, differences amongst stabilities of intermolecular base-pairings between the leader and the intergenic regions are not sufficient to determine the mRNA gradients in both MHV and IBV infected cells. Neither can the accessibility of the interacting regions on the leader and the negative stranded genome, as revealed by secondary structure analysis, explain the mRNA levels. The nested gene organisation itself, on the other hand, could be responsible for observed mRNA levels gradually increasing with gene order. Relatively slow new initiation events at intergenic regions are proposed to block elongation of passing transcripts which, via temporary pausing, can cause premature termination of transcription. This effects longer transcripts more than shorter ones.

Sequence of mouse hepatitis virus A59 mRNA 2: indications for RNA recombination between coronaviruses and influenza C virus.

The nucleotide sequence of the unique region of coronavirus MHV-A59 mRNA 2 has been determined. Two open reading frames (ORF) are predicted: ORF1 potentially encodes a protein of 261 amino acids; its amino acid sequence contains elements which indicate nucleotide binding properties. ORF2 predicts a 413 amino acids protein; it lacks a translation initiation codon and is therefore probably a pseudogene. The amino acid sequence of ORF2 shares 30% homology with the HA1 hemagglutinin sequence of influenza C virus. A short stretch of nucleotides immediately upstream of ORF2 shares 83% homology with the MHC class I nucleotide sequences. We discuss the possibility that both similarities are the result of recombinations and present a model for the acquisition and the subsequent inactivation of ORF2; the model applies also to MHV-A59-related coronaviruses in which we expect ORF2 to be still functional.

Primary structure of the glycoprotein E2 of coronavirus MHV-A59 and identification of the trypsin cleavage site.

The nucleotide sequence of the peplomer (E2) gene of MHV-A59 was determined from a set of overlapping cDNA clones. The E2 gene encodes a protein of 1324 amino acids including a hydrophobic signal peptide. A second large hydrophobic domain is found near the COOH terminus and probably represents the membrane anchor. Twenty glycosylation sites are predicted. Cleavage of the E2 protein results in two different 90K species, 90A and 90B (L.S. Sturman, C. S. Ricard, and K. V. Holmes (1985) J. Virol. 56, 904-911), and activates cell fusion. Protein sequencing of the trypsin-generated N-terminus revealed the position of the cleavage site. 90A and 90B could be identified as the C-terminal and the N-terminal parts, respectively. Amino acid sequence comparison of the A59 and JHM E2 proteins showed extensive homology and revealed a stretch of 89 amino acids in the 90B region of the A59 E2 protein that is absent in JHM.