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Category: Viruses and Viral Pathogenesis
Nidovirus Genome Organization and Expression Mechanisms, Page 1 of 2
< Previous page | Next page > /docserver/preview/fulltext/10.1128/9781555815790/9781555814557_Chap03-1.gif /docserver/preview/fulltext/10.1128/9781555815790/9781555814557_Chap03-2.gifAbstract:
This chapter covers the genome organization and expression mechanisms of the nidoviruses. Following infection of a susceptible cell by a nidovirus and uncoating of the RNA genome, the first step in a successful replication cycle is the production of the replicase proteins. The nidovirus genomic RNA (gRNA) initially acts as a eukaryotic mRNA for the translation of the replicase proteins. Following synthesis of the replicase proteins, the positive-sense gRNA is copied into negative-sense counterparts which act as templates for the synthesis of new gRNAs. In addition to a negative-sense gRNA, nidoviruses produce a series of negative-sense counterparts of the subgenomic mRNAs (sg mRNAs). The synthesis of both full-length and subgenome-length negative-sense RNAs is initiated at the 3’ end of the gRNA. Synthesis of negative-sense RNAs may terminate at different points along the gRNA template, yielding subgenome-length minus-strand RNAs. Attenuation of minus-strand RNA synthesis occurs at sequences, known as transcription regulatory sequences (TRSs), which are well conserved in a virus type but differ between groups, genera, and families of viruses. The negative-sense sgRNAs act as templates for the synthesis of the positive-sense sgRNAs, which are usually generated in a large excess compared to their negative-sense counterparts. The mechanism for the synthesis of nidovirus sgRNAs is called discontinuous extension of minus-strand RNA.
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Comparison of the genomic organizations of selected nidoviruses. A representative virus from each coronavirus group is included. The genomes are drawn to scale to emphasize that the first two-thirds of most nidovirus genomes consist of the replicase genes, ORF1a and ORF1b. The —1 frameshift site (RFS) for each virus is indicated. The drawings are derived from complete genome sequences of the representative viruses: porcine TGEV, MHV, SARS-CoV, avian IBV, bovine torovirus (BToV), EAV, and GAV. All the nonreplicase nonstructural protein genes are indicated with open boxes. The structural genes are those encoding S, M, E, N, HE, and GP proteins; I is an internal ORF identified in the N protein genes of some group 2 coronaviruses. S is shown as a double-shaded box, with the GP116/GP64 region of GAV having some structural similarities to an S gene. E is indicated as a dark gray box, M as a black box, and N as light gray box. The 5’ end of the GAV GP116/GP64 is colored black, as this part of the gene product is predicted to have triple membrane-spanning motifs that are reminiscent of the M protein. The arterivirus GP5 gene has a function equivalent to that of the S gene in coronaviruses and toroviruses.
Schematic diagram representing the replication cycle of a nidovirus following infection of a susceptible cell. The diagram has five numbered regions that represent the topics discussed in this chapter: ( 1 ) gRNA, released from a virus particle that has infected the cell, to highlight that it initially acts as an mRNA for the translation of the replicase proteins; ( 2 ) programmed —1 frameshifting event, common to all nidoviruses, for the translation of the replicase polyproteins, pp1a and pp1ab, in differing amounts; ( 3 ) another common feature of nidoviruses, proteolytic cleavage of the replicase polyproteins by virus-encoded proteinases; ( 4 ) another feature of nidoviruses, the generation of sg mRNAs (mainly polycistronic but functionally monocistronic) for the expression of the other virus-derived proteins; ( 5 ) expression strategies used for the translation of the sg mRNAs into virus proteins. Only the sg mRNAs encoding the structural proteins are shown. The rest of the diagram represents the interaction of the virus proteins for assembly and release of virus particles. ERGIC, endoplasmic reticulum-Golgi intermediate compartment.
RNA structural elements in the 3’ UTR of the avian coronavirus IBV. The IBV 3’ UTR contains three predicted RNA structures, a stem-loop associated with a pseudoknot structure and a second stem-loop structure, s2m. Similar stem-loop and associated pseudoknot RNA structures have been identified in the 3’ UTRs of other coronaviruses. The s2m structure in the IBV 3’ UTR also occurs in the 3’ UTRs of various astroviruses and a human rhino-virus as well as in 3’ UTR of TCoV, another group 3 coronavirus related to IBV. Neither the s2m structure nor its composite sequence was identified in the 3’ UTR of any other non-group 3 coronavirus, until the isolation of SARS-CoV. The numbers represent the IBV genomic nucleotide positions.
Sequence of the IBV ribosome frameshifting site. The top drawing represents the IBV gRNA, as shown in Fig. 1 and 3. The sequence representing the junction of ORF1a and ORF1b, corresponding to the RFS site, is expanded below the gRNA drawing. The numbers represent the IBV genomic nucleotide positions. The positions of the heptameric UUUAAC slip site sequences (in bold) are shown; the nucleotides forming the stem and loop structures of the pseudoknot (shown in Fig. 6 ) are underlined. The amino acid sequences corresponding to ORF1a, -1b, and -1ab are shown below the IBV genomic nucleotide sequence. ORF1a terminates at nucleotide 12382. There is no initiation codon for ORF1b, but a contiguous amino acid-encoding sequence starts at nucleotide 12342 and terminates at nucleotide 20417. The —1 frameshift site, highlighted in bold, takes place within the coding context of the slip site in which an asparagine residue, encoded by ORF1a, is replaced by a lysine residue, encoded by ORF1b, due to the ribosome moving the RNA back one nucleotide (shown in Fig. 5 ). As a result of the —1 frameshift event, ORF1a is extended by the ORF1b coding sequence and terminates as an ORF1ab fusion protein at nucleotide 20415. Translation termination codons are marked with asterisks.
Schematic diagram representing the IBV —1 frameshift event for the synthesis of pp1ab. The top part shows the progression of a ribosome along the IBV gRNA over the UUUAAAC slip site. Shown are the positions of the aminoacyl-tRNAs decoding the ORF1a codons, elongation of the polypeptide chain, and decoding of the next codon, resulting in the synthesis of pp1a. The lower part represents a —1 frameshift event as proposed by the simultaneous-slippage model, in which the ribosome-bound aminoacyl-tRNAs are proposed to slip simultaneously one nucleotide to a —1 frame position from their initial frame. Frameshifting can only occur when the anticodons of the two aminoacyl-tRNAs associated with the ribosome and mRNA can still form two base pairs with the RNA in the shifted —1 frame, indicated by the first and second anticodon-codon base pairings, but with disruption of base pairing at the third position. Following the —1 slippage event, aminoacyl-tRNAAsn dissociates from the ribosome complex, before decoding the mRNA, allowing the next aminoacyl-tRNA, aminoacyl-tRNALys, to move into the aminoacyl site of the ribosome, in which there is full complementarity between the anticodon of the tRNA and codon on the mRNA in the ORF1b frame. The mRNA is then decoded at this position, allowing elongation and movement of the ribosome to the next codon. The IBV replicase gene is now decoded in the ORF1b frame, resulting in translation of pp1ab.
Comparison of the nidovirus replicase gene frameshifting sites. (A) Independent alignment of the gRNA sequences, from the slip site, over the pseudoknot sequences of various coronavirus, torovirus, arterivirus, and ronivirus sequences. Nucleotides common within the grouped sequences are highlighted in black. The RFS slip site is underlined. (B) Phylogenetic groupings of all the aligned nidovirus sequences from panel A to demonstrate that as well as falling within their genus groupings, the coronavirus-derived sequences also fall within their groups, indicating that the viruses are related through their RFSs. FIPV, feline infectious peritonitis virus; PEDV, porcine epidemic diarrhea virus; BToV, bovine torovirus; YHV, yellow head virus.
Schematic diagram indicating the pausing of ribosomes by a pseudoknot structure. The top part represents ribosomes encountering a stem-loop structure on an mRNA being decoded. The ribosomes may be slowed down by such a structure, but they are able to melt the structure and decode the mRNA. The lower part represents ribosomes encountering a pseudoknot structure with an upstream slip site. Provided the slip site and pseudoknot are separated by no more than 5 to 7 nucleotides, the pausing effect of the pseudoknot can cause a —1 frameshift as illustrated in Fig. 6 . The predicted RNA structure of the IBV pseudoknot is shown as an example to highlight the nucleotides forming the slip site and the stem and loop structures.
Minimal lengths of nidovirus terminal 5’ and 3’ regions involved in replication
Coronavirus sg mRNAs expressing more than one product