Showing papers on "Subgenomic mRNA published in 2005"

Coronavirus genome structure and replication

Abstract: In addition to the SARS coronavirus (treated separately elsewhere in this volume), the complete genome sequences of six species in the coronavirus genus of the coronavirus family [avian infectious bronchitis virus-Beaudette strain (IBV-Beaudette), bovine coronavirus-ENT strain (BCoV-ENT), human coronavirus-229E strain (HCoV-229E), murine hepatitis virus-A59 strain (MHV-A59), porcine transmissible gastroenteritis-Purdue 115 strain (TGEV-Purdue 115), and porcine epidemic diarrhea virus-CV777 strain (PEDV-CV777)] have now been reported. Their lengths range from 27,317 nt for HCoV-229E to 31,357 nt for the murine hepatitis virus-A59, establishing the coronavirus genome as the largest known among RNA viruses. The basic organization of the coronavirus genome is shared with other members of the Nidovirus order (the torovirus genus, also in the family Coronaviridae, and members of the family Arteriviridae) in that the nonstructural proteins involved in proteolytic processing, genome replication, and subgenomic mRNA synthesis (transcription) (an estimated 14–16 end products for coronaviruses) are encoded within the 5′-proximal two-thirds of the genome on gene 1 and the (mostly) structural proteins are encoded within the 3′-proximal one-third of the genome (8–9 genes for coronaviruses). Genes for the major structural proteins in all coronaviruses occur in the 5′ to 3′ order as S, E, M, and N. The precise strategy used by coronaviruses for genome replication is not yet known, but many features have been established. This chapter focuses on some of the known features and presents some current questions regarding genome replication strategy, the cis-acting elements necessary for genome replication [as inferred from defective interfering (DI) RNA molecules], the minimum sequence requirements for autonomous replication of an RNA replicon, and the importance of gene order in genome replication.

Identification of Novel Subgenomic RNAs and Noncanonical Transcription Initiation Signals of Severe Acute Respiratory Syndrome Coronavirus

Abstract: The expression of the genomic information of severe acute respiratory syndrome coronavirus (SARS CoV) involves synthesis of a nested set of subgenomic RNAs (sgRNAs) by discontinuous transcription. In SARS CoV-infected cells, 10 sgRNAs, including 2 novel ones, were identified, which were predicted to be functional in the expression of 12 open reading frames located in the 3' one-third of the genome. Surprisingly, one new sgRNA could lead to production of a truncated spike protein. Sequence analysis of the leader-body fusion sites of each sgRNA showed that the junction sequences and the corresponding transcription-regulatory sequence (TRS) are unique for each species of sgRNA and are consistent after virus passages. For the two novel sgRNAs, each used a variant of the TRS that has one nucleotide mismatch in the conserved hexanucleotide core (ACGAAC) in the TRS. Coexistence of both plus and minus strands of SARS CoV sgRNAs and evidence for derivation of the sgRNA core sequence from the body core sequence favor the model of discontinuous transcription during minus-strand synthesis. Moreover, one rare species of sgRNA has the junction sequence AAA, indicating that its transcription could result from a noncanonical transcription signal. Taken together, these results provide more insight into the molecular mechanisms of genome expression and subgenomic transcription of SARS CoV.

Characterization of Striped jack nervous necrosis virus subgenomic RNA3 and biological activities of its encoded protein B2.

Abstract: Striped jack nervous necrosis virus (SJNNV), which infects fish, is the type species of the genus Betanodavirus. This virus has a bipartite genome of positive-strand RNAs, designated RNAs 1 and 2. A small RNA (ca. 0.4 kb) has been detected from SJNNV-infected cells, which was newly synthesized and corresponded to the 3'-terminal region of RNA1. Rapid amplification of cDNA ends analysis showed that the 5' end of this small RNA (designated RNA3) initiated at nt 2730 of the corresponding RNA1 sequence and contained a 5' cap structure. Substitution of the first nucleotide of the subgenomic RNA sequence within RNA1 selectively inhibited production of the positive-strand RNA3 but not of the negative-strand RNA3, which suggests that RNA3 may be synthesized via a premature termination model. The single RNA3-encoded protein (designated protein B2) was expressed in Escherichia coli, purified and used to immunize a rabbit to obtain an anti-protein B2 polyclonal antibody. An immunological test showed that the antigen was specifically detected in the central nervous system and retina of infected striped jack larvae (Pseudocaranx dentex), and in the cytoplasm of infected cultured E-11 cells. These results indicate that SJNNV produces subgenomic RNA3 from RNA1 and synthesizes protein B2 during virus multiplication, as reported for alphanodaviruses. In addition, an Agrobacterium co-infiltration assay established in transgenic plants that express green fluorescent protein showed that SJNNV protein B2 has a potent RNA silencing-suppression activity, as discovered for the protein B2 of insect-infecting alphanodaviruses.

Coronavirus Transcription: A Perspective

Abstract: At the VIth International Symposium on Corona and Related Viruses held in Quebec, Canada in 1994 we presented a new model for coronavirus transcription to explain how subgenome-length minus strands, which are used as templates for the synthesis of subgenomic mRNAs, might arise by a process involving discontinuous RNA synthesis. The old model explaining subgenomic mRNA synthesis, which was called leader-primed transcription, was based on erroneous evidence that only genome-length negative strands were present in replicative intermediates. To explain the discovery of subgenome-length minus strands, a related model, called the replicon model, was proposed: The subgenomic mRNAs would be produced initially by leader-primed transcription and then replicated into minus-strand templates that would in turn be transcribed into subgenomic mRNAs. We review the experimental evidence that led us to formulate a third model proposing that the discontinuous event in coronavirus RNA synthesis occurs during minus strand synthesis. With our model the genome is copied both continuously to produce minus-strand templates for genome RNA synthesis and discontinuously to produce minus-strand templates for subgenomic mRNA synthesis, and the subgenomic mRNAs do not function as templates for minus strand synthesis, only the genome does.

Selective Replication of Coronavirus Genomes That Express Nucleocapsid Protein

Abstract: The coronavirus nucleocapsid (N) protein is a structural protein that forms a ribonucleoprotein complex with genomic RNA. In addition to its structural role, it has been described as an RNA-binding protein that might be involved in coronavirus RNA synthesis. Here, we report a reverse genetic approach to elucidate the role of N in coronavirus replication and transcription. We found that human coronavirus 229E (HCoV-229E) vector RNAs that lack the N gene were greatly impaired in their ability to replicate, whereas the transcription of subgenomic mRNA from these vectors was easily detectable. In contrast, vector RNAs encoding a functional N protein were able to carry out both replication and transcription. Furthermore, modification of the transcription signal required for the synthesis of N protein mRNAs in the HCoV-229E genome resulted in the selective replication of genomes that are able to express the N protein. This genetic evidence leads us to conclude that at least one coronavirus structural protein, the N protein, is involved in coronavirus replication.

Replication and packaging of Norwalk virus RNA in cultured mammalian cells

Abstract: Human noroviruses, the most common cause of nonbacterial gastroenteritis, are characterized by high infectivity rate, low infectious dose, and unusually high stability outside the host. However, human norovirus research is hindered by the lack of a cell culture system and a small animal model of infection. Norwalk virus (NV) is the prototype strain of human noroviruses. We report here replication of NV viral RNA and its packaging into virus particles in mammalian cells by intracellular expression of native forms of NV viral RNA devoid of extraneous nucleotide sequences derived from the expression vector by the use of replication-deficient vaccinia virus MVA encoding the bacteriophage T7 RNA polymerase (MVA/T7). Expressed genomic RNA was found to replicate; NV subgenomic RNA was transcribed from genomic RNA by use of NV nonstructural proteins expressed from genomic RNA and was subsequently translated into NV capsid protein VP1. Viral genomic RNA was packaged into virus particles generated in mammalian cells. The cesium chloride (CsCl) density gradient profile of virus particles containing genomic RNA was similar to that of NV purified from stool. These observations indicate that the NV cDNA constructed here is a biologically infectious clone, and that mammalian cells have the ability to replicate NV genomic RNA. This work establishes a mammalian cell-based system for analysis of human norovirus replication and, thus, makes it feasible to investigate antiviral agents in mammalian cells.

Inhibitors of Respiratory Syncytial Virus Replication Target Cotranscriptional mRNA Guanylylation by Viral RNA-Dependent RNA Polymerase

Abstract: Respiratory syncytial virus (RSV) is a major cause of respiratory illness in infants, immunocompromised patients, and the elderly. New antiviral agents would be important tools in the treatment of acute RSV disease. RSV encodes its own RNA-dependent RNA polymerase that is responsible for the synthesis of both genomic RNA and subgenomic mRNAs. The viral polymerase also cotranscriptionally caps and polyadenylates the RSV mRNAs at their 5' and 3' ends, respectively. We have previously reported the discovery of the first nonnucleoside transcriptase inhibitor of RSV polymerase through high-throughput screening. Here we report the design of inhibitors that have improved potency both in vitro and in antiviral assays and that also exhibit activity in a mouse model of RSV infection. We have isolated virus with reduced susceptibility to this class of inhibitors. The mutations conferring resistance mapped to a novel motif within the RSV L gene, which encodes the catalytic subunit of RSV polymerase. This motif is distinct from the catalytic region of the L protein and bears some similarity to the nucleotide binding domain within nucleoside diphosphate kinases. These findings lead to the hypothesis that this class of inhibitors may block synthesis of RSV mRNAs by inhibiting guanylylation of viral transcripts. We show that short transcripts produced in the presence of inhibitor in vitro do not contain a 5' cap but, instead, are triphosphorylated, confirming this hypothesis. These inhibitors constitute useful tools for elucidating the molecular mechanism of RSV capping and represent valid leads for the development of novel anti-RSV therapeutics.

Role of Nucleotides Immediately Flanking the Transcription-Regulating Sequence Core in Coronavirus Subgenomic mRNA Synthesis

Abstract: The generation of subgenomic mRNAs in coronavirus involves a discontinuous mechanism of transcription by which the common leader sequence, derived from the genome 5' terminus, is fused to the 5' end of the mRNA coding sequence (body). Transcription-regulating sequences (TRSs) precede each gene and include a conserved core sequence (CS) surrounded by relatively variable sequences (5' TRS and 3' TRS). Regulation of transcription in coronaviruses has been studied by reverse-genetics analysis of the sequences immediately flanking a unique CS in the Transmissible gastroenteritis virus genome (CS-S2), located inside the S gene, that does not lead to detectable amounts of the corresponding mRNA, in spite of its canonical sequence. The transcriptional inactivity of CS-S2 was genome position independent. The presence of a canonical CS was not sufficient to drive transcription, but subgenomic synthesis requires a minimum base pairing between the leader TRS (TRS-L) and the complement of the body TRS (cTRS-B) provided by the CS and its adjacent nucleotides. A good correlation was observed between the free energy of TRS-L and cTRS-B duplex formation and the levels of subgenomic mRNA S2, demonstrating that base pairing between the leader and body beyond the CS is a determinant regulation factor in coronavirus transcription. In TRS mutants with increasing complementarity between TRS-L and cTRS-B, a tendency to reach a plateau in DeltaG values was observed, suggesting that a more precise definition of the TRS limits might be proposed, specifically that it consists of the central CS and around 4 nucleotides flanking 5' and 3' the CS. Sequences downstream of the CS exert a stronger influence on the template-switching decision according to a model of polymerase strand transfer and template switching during minus-strand synthesis.

Gene 5 of the Avian Coronavirus Infectious Bronchitis Virus Is Not Essential for Replication

Abstract: The avian coronavirus Infectious bronchitis virus (IBV), like other coronaviruses, expresses several small nonstructural (ns) proteins in addition to those from gene 1 (replicase) and the structural proteins. These coronavirus ns genes differ both in number and in amino acid similarity between the coronavirus groups but show some concordance within a group or subgroup. The functions and requirements of the small ns gene products remain to be elucidated. With the advent of reverse genetics for coronaviruses, the first steps in elucidating their role can be investigated. We have used our reverse genetics system for IBV (R. Casais, V. Thiel, S. G. Siddell, D. Cavanagh, and P. Britton, J. Virol. 75:12359-12369, 2001) to investigate the requirement of IBV gene 5 for replication in vivo, in ovo, and ex vivo. We produced a series of recombinant viruses, with an isogenic background, in which complete expression of gene 5 products was prevented by the inactivation of gene 5 following scrambling of the transcription-associated sequence, thereby preventing the expression of IBV subgenomic mRNA 5, or scrambling either separately or together of the translation initiation codons for the two gene 5 products. As all of the recombinant viruses replicated very similarly to the wild-type virus, Beau-R, we conclude that the IBV gene 5 products are not essential for IBV replication per se and that they are accessory proteins.

Dual Role of the Lymphocytic Choriomeningitis Virus Intergenic Region in Transcription Termination and Virus Propagation

Abstract: Each genome segment of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV), encodes two genes in ambisense orientation, separated by an intergenic region (IGR). The 3' ends of subgenomic viral mRNAs have been mapped to a stem-loop structure within the IGR, suggesting structure-dependent transcription termination. We have studied the role of the LCMV IGR by using a minigenome (MG) rescue system based on RNA analogues of the short genome segment. An ambisense MG coding for chloramphenicol acetyltransferase (CAT) and green fluorescent protein reporter genes instead of the nucleoprotein and glycoprotein open reading frames, respectively, served as a template for synthesis of full-length anti-MG (aMG) replicate and subgenomic size mRNA for reporter gene expression. An analogous MG without IGR was amplified by the virus polymerase with equal efficiency, but subgenomic mRNA was undetectable. Reporter gene expression from IGR-deficient aMG CAT-sense RNA of genomic length was approximately 5-fold less efficient than that from subgenomic CAT mRNA derived from an IGR-containing MG, but at least 100-fold more efficient than that from a T7 RNA polymerase transcript with the same sequence. Therefore, in the absence of IGR-mediated transcription termination, a fraction of full-length aMG RNA appears to behave as bona fide mRNA. Unexpectedly, MGs without IGR were dramatically impaired in their ability to passage reporter gene activity via infectious virus-like particles. These data suggest that the LCMV IGR serves individual functions in transcription termination for enhanced gene expression and in the virus assembly and/or budding, which are required for the efficient propagation of LCMV infectivity.

Potent inhibitors of subgenomic hepatitis C virus RNA replication through optimization of indole-N-acetamide allosteric inhibitors of the viral NS5B polymerase.

Abstract: Infections caused by hepatitis C virus (HCV) are a significant world health problem for which novel therapies are in urgent demand. Compounds that block replication of subgenomic HCV RNA in liver cells are of interest because of their demonstrated antiviral effect in the clinic. In followup to our recent report that indole-N-acetamides (e.g., 1) are potent allosteric inhibitors of the HCV NS5B polymerase enzyme, we describe here their optimization as cell-based inhibitors. The crystal structure of 1 bound to NS5B was a guide in the design of a two-dimensional compound array that highlighted that formally zwitterionic inhibitors have strong intracellular potency and that pregnane X receptor (PXR) activation (an undesired off-target activity) is linked to a structural feature of the inhibitor. Optimized analogues devoid of PXR activation (e.g., 55, EC(50) = 127 nM) retain strong cell-based efficacy under high serum conditions and show acceptable pharmacokinetics parameters in rat and dog.

Development and preliminary optimization of indole-N-acetamide inhibitors of hepatitis C virus NS5B polymerase.

Abstract: Allosteric inhibition of the hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase enzyme has recently emerged as a viable strategy toward blocking replication of viral RNA in cell-based systems. We report here a novel class of allosteric inhibitor of NS5B that shows potent affinity for the NS5B enzyme and effective inhibition of subgenomic HCV RNA replication in HUH-7 cells. Inhibitors from this class have promising characteristics for further development as anti-HCV agents.

Rapid Communication A novel, mouse mammary tumor virus encoded protein with Rev-like properties

Abstract: We have identified a novel, multiple spliced, subgenomic mRNA species in MMTV producing cells of different origin containing an open reading frame encoding a 39-kDa Rev-like protein, Rem (regulator of expression of MMTV). An EGFP–Rem fusion protein is shown to be predominantly in the nucleolus. Further leptomycin B inhibits the nuclear export of nonspliced MMTV transcripts, implicating Rem in nuclear export by the Crm1 pathway in MMTV. Rem is thus reminiscent of the Rec protein from the related endogenous human retrovirus, HERV-K.

Discontinuous Subgenomic RNA Synthesis in Arteriviruses Is Guided by an RNA Hairpin Structure Located in the Genomic Leader Region

Abstract: Nidoviruses produce an extensive 3'-coterminal nested set of subgenomic (sg) mRNAs, which are used to express structural proteins and sometimes accessory proteins. In arteriviruses and coronaviruses, these mRNAs contain a common 5' leader sequence, derived from the genomic 5' end. The joining of the leader sequence to different segments derived from the 3'-proximal part of the genome (mRNA bodies) presumably involves a unique mechanism of discontinuous minus-strand RNA synthesis in which base pairing between sense and antisense transcription-regulating sequences (TRSs) plays an essential role. The leader TRS is present in the loop of a hairpin structure that functions in sg mRNA synthesis. In this study, the minimal sequences in the 5'-proximal region of the Equine arteritis virus genome that are required for sg RNA synthesis were delimited through mutagenesis. A full-length cDNA clone was engineered in which this domain was duplicated, allowing us to make mutations and monitor their effects on sg RNA synthesis without seriously affecting genome replication and translation. The leader TRS present in the duplicated sequence was used and yielded novel sg mRNAs with significantly extended leaders. Our combined findings suggest that the leader TRS hairpin (LTH) and its immediate flanking sequences are essential for efficient sg RNA synthesis and form an independent functional entity that could be moved 300 nucleotides downstream of its original position in the genome. We hypothesize that a conformational switch in the LTH region regulates the role of the 5'-proximal region of the arterivirus genome in subgenomic RNA synthesis.

Development and characterization of a transient-replication assay for the genotype 2a hepatitis C virus subgenomic replicon.

Abstract: Dicistronic, subgenomic hepatitis C virus (HCV) replicons were constructed containing sequences from JFH1, a genotype 2a strain, that also incorporated the firefly luciferase gene under the control of the HCV internal ribosome entry site element. Luciferase activity in Huh-7 cell extracts containing in vitro-transcribed subgenomic JFH1 RNA was monitored over a 72 h period to examine early stages of HCV replication in the absence of any selective pressure. Enzyme activities produced by the replicon were almost 200-fold greater than those generated from corresponding genotype 1b replicons and correlated with an accumulation of NS5A protein and replicon RNA. Transient replication was sensitive to IFN treatment in a dose-dependent manner and, in addition to Huh-7 cells, the U2OS human osteosarcoma cell line supported efficient replication of the JFH1 replicon. Thus, this system based on JFH1 sequences offers improvements over prior genotype 1b replicons for quantitative measurement of viral RNA replication.

Interactions between Rubella Virus Capsid and Host Protein p32 Are Important for Virus Replication

Abstract: The distribution and morphology of mitochondria are dramatically affected during infection with rubella virus (RV). Expression of the capsid, in the absence of other viral proteins, was found to induce both perinuclear clustering of mitochondria and the formation of electron-dense intermitochondrial plaques, both hallmarks of RV-infected cells. We previously identified p32, a host cell mitochondrial matrix protein, as a capsid-binding protein. Here, we show that two clusters of arginine residues within capsid are required for stable binding to p32. Mutagenic ablation of the p32-binding site in capsid resulted in decreased mitochondrial clustering, indicating that interactions with this cellular protein are required for capsid-dependent reorganization of mitochondria. Recombinant viruses encoding arginine-to-alanine mutations in the p32-binding region of capsid exhibited altered plaque morphology and replicated to lower titers. Further analysis indicated that disruption of stable interactions between capsid and p32 was associated with decreased production of subgenomic RNA and, consequently, infected cells produced significantly lower amounts of viral structural proteins under these conditions. Together, these results suggest that capsid-p32 interactions are important for nonstructural functions of capsid that include regulation of virus RNA replication and reorganization of mitochondria during infection.

Genomic RNA sequence of Feline coronavirus strain FIPV WSU-79/1146.

Abstract: A consensus sequence of the Feline coronavirus (FCoV) (strain FIPV WSU-79/1146) genome was determined from overlapping cDNA fragments produced by RT-PCR amplification of viral RNA. The genome was found to be 29 125 nt in length, excluding the poly(A) tail. Analysis of the sequence identified conserved open reading frames and revealed an overall genome organization similar to that of other coronaviruses. The genomic RNA was analysed for putative cis-acting elements and the pattern of subgenomic mRNA synthesis was analysed by Northern blotting. Comparative sequence analysis of the predicted FCoV proteins identified 16 replicase proteins (nsp1-nsp16) and four structural proteins (spike, membrane, envelope and nucleocapsid). Two mRNAs encoding putative accessory proteins were also detected. Phylogenetic analyses confirmed that FIPV WSU-79/1146 belongs to the coronavirus subgroup G1-1. These results confirm and extend previous findings from partial sequence analysis of FCoV genomes.

Antiviral activity of morpholino oligomers designed to block various aspects of Equine arteritis virus amplification in cell culture.

Abstract: The antiviral efficacy of ten antisense phosphorodiamidate morpholino oligomers (PMOs) directed against Equine arteritis virus (EAV), a nidovirus belonging to the family Arteriviridae, was evaluated in mammalian (Vero-E6) cells. Peptide-conjugated PMOs (P-PMOs) supplied in cell culture medium at micromolar concentrations were efficiently taken up by Vero-E6 cells and were minimally cytotoxic. The P-PMOs were designed to base pair to RNA sequences involved in different aspects of EAV amplification: genome replication, subgenomic mRNA synthesis, and translation of genome and subgenomic mRNAs. A novel recombinant EAV, expressing green fluorescent protein as part of its replicase polyproteins, was used to facilitate drug screening. A moderate reduction of EAV amplification was observed with relatively high concentrations of P-PMOs designed to anneal to the 3'-terminal regions of the viral genome or antigenome. To determine if the synthesis of subgenomic mRNAs could be specifically reduced, transcription-regulating sequences essential for their production, but not for the production of genomic RNA, were targeted, but these P-PMOs were found to be ineffective at transcription interference. In contrast, all four P-PMOs designed to base pair with targets in the genomic 5' untranslated region markedly reduced virus amplification in a sequence-specific and dose-responsive manner. At concentrations in the low micromolar range, some of the P-PMOs tested completely inhibited virus amplification. In vitro translation assays showed that these P-PMOs were potent inhibitors of translation. The data suggest that these compounds could be useful as reagents for exploring the molecular mechanics of nidovirus translation and have anti-EAV potential at relatively low concentrations.

Hairpin ribozymes in combination with siRNAs against highly conserved hepatitis C virus sequence inhibit RNA replication and protein translation from hepatitis C virus subgenomic replicons

Abstract: Chronic hepatitis C virus (HCV) infection is a clinically important liver disease with limited therapeutic options in a significant proportion of patients. Therefore, novel efficient therapeutic agents are needed. Because the 5′- and 3′-untranslated regions (UTRs) of HCV are highly conserved and functionally important for HCV replication, they are attractive targets for RNA-cleaving ribozymes or small interfering RNAs (siRNAs). In this study hairpin ribozymes (Rz) targeting HCV 5′- and 3′-UTR sequences were expressed from a retroviral vector transcript under control of two different RNA polIII promoters (tRNAVal, U6). Ribozymes were evaluated in monocistronic, subgenomic I389/hyg-ubi/NS3-3′/5.1 HCV replicon cells as single agents or in combination with siRNAs against HCV 5′- or 3′-UTR recently demonstrated to inhibit HCV replicons. Additionally, ribozyme constructs were generated with the 3′-terminus of the ribozyme flanked by constitutive transport element (CTE) sequences, an RNA motif that has previously been shown to enhance cleavage activity of hammerhead ribozymes. In our study, tRNAVal as well as U6 promoter-driven Rzs markedly reduced HCV replicon RNA expression and HCV internal ribosome entry site (IRES)-mediated HCV NS5B protein translation from monocistronic subgenomic replicons. However, attachment of CTE sequences to the 3′-terminus did not significantly enhance activity of Rzs tested in this study. Interestingly, we detected additive HCV inhibitory effects for combinations of tRNAVal-driven Rzs and U6-derived siRNAs both directed against highly conserved 5′- and 3′-UTR sequence, suggesting that a dual strategy of ribozymes and siRNAs might become a powerful molecular tool to specifically silence HCV RNA replication.

Coronaviruses as Vectors: Stability of Foreign Gene Expression

Abstract: Coronaviruses are enveloped, positive-stranded RNA viruses considered to be promising vectors for vaccine development, as (i) genes can be deleted, resulting in attenuated viruses; (ii) their tropism can be modified by manipulation of their spike protein; and (iii) heterologous genes can be expressed by simply inserting them with appropriate coronaviral transcription signals into the genome For any live vector, genetic stability is an essential requirement However, little is known about the genetic stability of recombinant coronaviruses expressing foreign genes In this study, the Renilla and the firefly luciferase genes were systematically analyzed for their stability after insertion at various genomic positions in the group 1 coronavirus feline infectious peritonitis virus and in the group 2 coronavirus mouse hepatitis virus It appeared that the two genes exhibit intrinsic differences, the Renilla gene consistently being maintained more stably than the firefly gene This difference was not caused by genome size restrictions, by different effects of the encoded proteins, or by different consequences of the synthesis of the additional subgenomic mRNAs The loss of expression of the firefly luciferase was found to result from various, often large deletions of the gene, probably due to RNA recombination The extent of this process appeared to depend strongly on the coronaviral genomic background, the luciferase gene being much more stable in the feline than in the mouse coronavirus genome It also depended significantly on the particular genomic location at which the gene was inserted The data indicate that foreign sequences are more stably maintained when replacing nonessential coronaviral genes

Complete nucleotide sequence and genome organization of Pelargonium line pattern virus and its relationship with the family Tombusviridae

Abstract: The complete nucleotide sequence of Pelargonium line pattern virus (PLPV) has been determined. The PLPV genomic RNA comprises 3884 nt and contains six open reading frames (ORFs) potentially encoding proteins of 27 (p27), 13 (p13), 87 (p87), 7 (p7), 6 (p6), and 37 kDa (p37), respectively. The arrangement of these ORFs on the PLPV genome closely resembles that of members of the genus Carmovirus in the family Tombusviridae and, moreover, most of the putative PLPV gene products showed high identity with proteins of this viral group. However, several striking differences were noticed. Carmoviruses generate two subgenomic RNAs whereas PLPV produces a single one. In addition, only p7 showed similarity with movement proteins of carmoviruses whereas p6 (as p13) has no viral (or other) homologs. This protein might be expressed from a non-canonical start codon or, alternatively, through a −1 frameshift (FS) mechanism. Both, the production of one subgenomic RNA and the likely involvement of a −1 FS for expression of an internal ORF parallel the translation strategies reported for the unique species of the genus Panicovirus, belonging also to the family Tombusviridae. Overall, the results support the placement of PLPV in this family although its peculiar characteristics preclude its direct assignment to any of the current genera.