Expanded subgenomic mRNA transcriptome and coding capacity of a nidovirus.
ABSTRACT: Members of the order Nidovirales express their structural protein ORFs from a nested set of 3' subgenomic mRNAs (sg mRNAs), and for most of these ORFs, a single genomic transcription regulatory sequence (TRS) was identified. Nine TRSs were previously reported for the arterivirus Simian hemorrhagic fever virus (SHFV). In the present study, which was facilitated by next-generation sequencing, 96 SHFV body TRSs were identified that were functional in both infected MA104 cells and macaque macrophages. The abundance of sg mRNAs produced from individual TRSs was consistent over time in the two different cell types. Most of the TRSs are located in the genomic 3' region, but some are in the 5' ORF1a/1b region and provide alternative sources of nonstructural proteins. Multiple functional TRSs were identified for the majority of the SHFV 3' ORFs, and four previously identified TRSs were found not to be the predominant ones used. A third of the TRSs generated sg mRNAs with variant leader-body junction sequences. Sg mRNAs encoding E', GP2, or ORF5a as their 5' ORF as well as sg mRNAs encoding six previously unreported alternative frame ORFs or 14 previously unreported C-terminal ORFs of known proteins were also identified. Mutation of the start codon of two C-terminal ORFs in an infectious clone reduced virus yield. Mass spectrometry detected one previously unreported protein and suggested translation of some of the C-terminal ORFs. The results reveal the complexity of the transcriptional regulatory mechanism and expanded coding capacity for SHFV, which may also be characteristic of other nidoviruses.
Project description:The 3' end of the simian hemorrhagic fever virus (SHFV) single-stranded RNA genome was cloned and sequenced. Adjacent to the 3' poly(A) tract, we identified a 76-nucleotide noncoding region preceded by two overlapping reading frames (ORFs). The ultimate 3' ORF of the viral genome encodes the capsid protein, and the penultimate ORF encodes the smallest SHFV envelope protein. These two ORFs overlap each other by 26 nucleotides. Northern (RNA) blot hybridization analyses of cytoplasmic RNA extracts from SHFV-infected MA-104 cells with gene-specific probes revealed the presence of full-length genomic RNA as well as six subgenomic SHFV-specific mRNA species. The subgenomic mRNAs are 3' coterminal. In its virion morphology and size, genome structure and length, and replication strategy, SHFV is most similar to lactate dehydrogenase-elevating virus, equine arteritis virus, and porcine reproductive and respiratory syndrome virus.
Project description:The simian hemorrhagic fever virus (SHFV) genome differs from those of other members of the family Arterivirus in encoding two adjacent sets of four minor structural protein open reading frames (ORFs). A stable, full-length, infectious SHFV-LVR cDNA clone was constructed. Virus produced from this clone had replication characteristics similar to those of the parental virus. A subgenomic mRNA was identified for the SHFV ORF previously identified as 2b. As an initial means of analyzing the functional relevance of each of the SHFV minor structural proteins, a set of mutant infectious clones was generated, each with the start codon of one minor structural protein ORF mutated. Different phenotypes were observed for each ortholog of the pairs of minor glycoproteins and all of the eight minor structural proteins were required for the production of infectious extracellular virus indicating that the duplicated sets of SHFV minor structural proteins are not functionally redundant.
Project description:Coronavirus (CoV) transcription includes a discontinuous mechanism during the synthesis of sub-genome-length minus-strand RNAs leading to a collection of mRNAs in which the 5' terminal leader sequence is fused to contiguous genome sequences. It has been previously shown that transcription-regulating sequences (TRSs) preceding each gene regulate transcription. Base pairing between the leader TRS (TRS-L) and the complement of the body TRS (cTRS-B) in the nascent RNA is a determinant factor during CoV transcription. In fact, in transmissible gastroenteritis CoV, a good correlation has been observed between subgenomic mRNA (sg mRNA) levels and the free energy (DeltaG) of TRS-L and cTRS-B duplex formation. The only exception was sg mRNA N, the most abundant sg mRNA during viral infection in spite of its minimum DeltaG associated with duplex formation. We postulated that additional factors should regulate transcription of sg mRNA N. In this report, we have described a novel transcription regulation mechanism operating in CoV by which a 9-nucleotide (nt) sequence located 449 nt upstream of the N gene TRS core sequence (CS-N) interacts with a complementary sequence just upstream of CS-N, specifically increasing the accumulation of sg mRNA N. Alteration of this complementarity in mutant replicon genomes showed a correlation between the predicted stability of the base pairing between 9-nt sequences and the accumulation of sg mRNA N. This interaction is exclusively conserved in group 1a CoVs, the only CoV subgroup in which the N gene is not the most 3' gene in the viral genome. This is the first time that a long-distance RNA-RNA interaction regulating transcriptional activity specifically enhancing the transcription of one gene has been described to occur in CoVs.
Project description:SHFV is a member of a new virus family which includes the genus arterivirus. We have cloned and sequenced 6,314 nt from the 3' end of the SHFV genome. This sequence encompasses nine complete ORFs which is three additional ORFs as compared to the other arteriviruses. We have numbered these ORFs 2a, 2b, 3, 4, 5, 6, 7, 8 and 9. At the 5' end of this sequence is a partial ORF (ORF 1b) of 1590 nt and at the 3' end is a poly(A) tract preceded by a 76 nt noncoding region. The coding capacity for each of the SHFV ORFs as well as the potential mass, pI and number of N-linked glycosylation sites for each of the encoded peptides was determined.
Project description:Arteriviruses are enveloped positive-strand RNA viruses for which the attachment proteins and cellular receptors have remained largely controversial. Arterivirus particles contain at least eight envelope proteins, an unusually large number among RNA viruses. These appear to segregate into three groups: major structural components (major glycoprotein GP5 and membrane protein [M]), minor glycoproteins (GP2a, GP3, and GP4), and small hydrophobic proteins (E and the recently discovered ORF5a protein). Biochemical studies previously suggested that the GP5-M heterodimer of porcine reproductive and respiratory syndrome virus (PRRSV) interacts with porcine sialoadhesin (pSn) in porcine alveolar macrophages (PAM). However, another study proposed that minor protein GP4, along with GP2a, interacts with CD163, another reported cellular receptor for PRRSV. In this study, we provide genetic evidence that the minor envelope proteins are the major determinant of arterivirus entry into cultured cells. A PRRSV infectious cDNA clone was equipped with open reading frames (ORFs) encoding minor envelope and E proteins of equine arteritis virus (EAV), the only known arterivirus displaying a broad tropism in cultured cells. Although PRRSV and EAV are only distantly related and utilize diversified transcription-regulating sequences (TRSs), a viable chimeric progeny virus was rescued. Strikingly, this chimeric virus (vAPRRS-EAV2ab34) acquired the broad in vitro cell tropism of EAV, demonstrating that the minor envelope proteins play a critical role as viral attachment proteins. We believe that chimeric arteriviruses of this kind will be a powerful tool for further dissection of the arterivirus replicative cycle, including virus entry, subgenomic RNA synthesis, and virion assembly.
Project description:To express its structural proteins, the arterivirus Equine arteritis virus (EAV) produces a nested set of six subgenomic (sg) RNA species. These RNA molecules are generated by a mechanism of discontinuous transcription, during which a common leader sequence, representing the 5' end of the genomic RNA, is attached to the bodies of the sg RNAs. The connection between the leader and body parts of an mRNA is formed by a short, conserved sequence element termed the transcription-regulating sequence (TRS), which is present at the 3' end of the leader as well as upstream of each of the structural protein genes. With the exception of RNA3, only one body TRS was previously assumed to be used to join the leader and body of each EAV sg RNA. Here we show that for the synthesis of two other sg RNAs, RNA4 and RNA5, alternative leader-body junction sites that differ substantially in transcriptional activity are used. By site-directed mutagenesis of an EAV infectious cDNA clone, the alternative TRSs used to generate RNA3, -4, and -5 were inactivated, which strongly influenced the corresponding RNA levels and the production of infectious progeny virus. The relative amounts of RNA produced from alternative TRSs differed significantly and corresponded to the relative infectivities of the virus mutants. This strongly suggested that the structural proteins that are expressed from these RNAs are limiting factors during the viral life cycle and that the discontinuous step in sg RNA synthesis is crucial for the regulation of their expression. On the basis of a theoretical analysis of the predicted RNA structure of the 3' end of the EAV genome, we propose that the local secondary RNA structure of the body TRS regions is an important factor in the regulation of the discontinuous step in EAV sg mRNA synthesis.
Project description:The N-terminal region of simian hemorrhagic fever virus (SHFV) nonstructural polyprotein 1a is predicted to encode three papain-like proteases (PLP1?, PLP1?, and PLP1?). Catalytic residues and cleavage sites for each of the SHFV PLP1s were predicted by alignment of the SHFV PLP1 region sequences with each other as well as with those of other arteriviruses, and the predicted catalytic residues were shown to be proximal by homology modeling of the SHFV nsp1s on porcine respiratory and reproductive syndrome virus (PRRSV) nsp1 crystal structures. The functionality of the predicted catalytic Cys residues and cleavage sites was tested by analysis of the autoproteolytic products generated in in vitro transcription/translation reactions done with wild-type or mutant SHFV nsp1 constructs. Cleavage sites were also analyzed by mass spectroscopy analysis of selected immunoprecipitated cleavage products. The data showed that each of the three SHFV PLP1s is an active protease. Cys63 was identified as the catalytic Cys of SHFV PLP1? and is adjacent to an Ala instead of the canonical Tyr observed in other arterivirus PLP1s. SHFV PLP1? is able to cleave at both downstream and upstream nsp1 junction sites. Although intermediate precursor polyproteins as well as alternative products generated by each of the SHFV PLP1s cleaving at sites within the N-terminal region of nsp1? were produced in the in vitro reactions, Western blotting of SHFV-infected, MA104 cell lysates with SHFV nsp1 protein-specific antibodies detected only the three mature nsp1 proteins.SHFV is unique among arteriviruses in having three N-terminal papain-like protease 1 (PLP1) domains. Other arteriviruses encode one or two active PLP1s. This is the first functional study of the SHFV PLP1s. Analysis of the products of in vitro autoprocessing of an N-terminal SHFV nonstructural 1a polypeptide fragment showed that each of the three SHFV PLP1s is active, and the predicted catalytic Cys residues and cleavage sites for each PLP1 were confirmed by testing mutant constructs. Several unique features of the SHFV PLP1s were discovered. The SHFV PLP1? catalytic Cys63 is unique among arterivirus PLP1s in being adjacent to an Ala instead of a Trp. Other arterivirus PLP1s cleave only in cis at a single downstream site, but SHFV PLP1? can cleave at both the downstream nsp1?-nsp2 and upstream nsp1?-nsp1? junctions. The three mature nsp1 proteins were produced both in the in vitro reactions and in infected cells.
Project description:The arterivirus family (order Nidovirales) of single-stranded, positive-sense RNA viruses includes porcine respiratory and reproductive syndrome virus and equine arteritis virus (EAV). Their replicative enzymes are translated from their genomic RNA, while their seven structural proteins are encoded by a set of small, partially overlapping genes in the genomic 3'-proximal region. The latter are expressed via synthesis of a set of subgenomic mRNAs that, in general, are functionally monocistronic (except for a bicistronic mRNA encoding the E and GP2 proteins). ORF5, which encodes the major glycoprotein GP5, has been used extensively for phylogenetic analyses. However, an in-depth computational analysis now reveals the arterivirus-wide conservation of an additional AUG-initiated ORF, here termed ORF5a, that overlaps the 5' end of ORF5. The pattern of substitutions across sequence alignments indicated that ORF5a is subject to functional constraints at the amino acid level, while an analysis of substitutions at synonymous sites in ORF5 revealed a greatly reduced frequency of substitution in the portion of ORF5 that is overlapped by ORF5a. The 43-64 aa ORF5a protein and GP5 are probably expressed from the same subgenomic mRNA, via a translation initiation mechanism involving leaky ribosomal scanning. Inactivation of ORF5a expression by reverse genetics yielded a severely crippled EAV mutant, which displayed lower titres and a tiny plaque phenotype. These defects, which could be partially complemented in ORF5a-expressing cells, indicate that the novel protein, which may be the eighth structural protein of arteriviruses, is expressed and important for arterivirus infection.
Project description:Arteri-, corona-, toro- and roniviruses are evolutionarily related positive-strand RNA viruses, united in the order Nidovirales. The best studied nidoviruses, the corona- and arteriviruses, employ a unique transcription mechanism, which involves discontinuous RNA synthesis, a process resembling similarity-assisted copy-choice RNA recombination. During infection, multiple subgenomic (sg) mRNAs are transcribed from a mirror set of sg negative-strand RNA templates. The sg mRNAs all possess a short 5' common leader sequence, derived from the 5' end of the genomic RNA. The joining of the non-contiguous 'leader' and 'body' sequences presumably occurs during minus-strand synthesis. To study whether toroviruses use a similar transcription mechanism, we characterized the 5' termini of the genome and the four sg mRNAs of Berne virus (BEV). We show that BEV mRNAs 3-5 lack a leader sequence. Surprisingly, however, RNA 2 does contain a leader, identical to the 5'-terminal 18 residues of the genome. Apparently, BEV combines discontinuous and non-discontinuous RNA synthesis to produce its sg mRNAs. Our findings have important implications for the understanding of the mechanism and evolution of nidovirus transcription.
Project description:Porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus that emerged in the late 1980s in both Europe and North America as the causative agent of porcine reproductive and respiratory syndrome (PRRS), now the most important disease of swine worldwide. Despite extensive characterization of PRRSV proteins by direct analysis and comparison with other arteriviruses, determinants of virulence, pathogenesis and protective immune recognition remain poorly understood. Thus, we hypothesized that additional ORFs are present in the PRRSV genome that may contribute to its biological properties, and so we screened highly purified virions of strain VR2332, the prototype type 2 PRRSV, for evidence of novel polypeptides. A 51 aa polypeptide was discovered that is encoded by an alternative ORF of the subgenomic mRNA encoding the major envelope glycoprotein, GP5, and which is incorporated into virions. The protein, referred to as ORF5a protein, is expressed in infected cells, and pigs infected with PRRSV express anti-ORF5a protein antibodies. A similar ORF is present as an alternative reading frame in all PRRSV subgenomic RNA5 genes and in all other arteriviruses, suggesting that this ORF5a protein plays a significant role in arterivirology. Its discovery also provides a new potential target for immunological and pharmacological intervention in PRRS.