Efficient homologous RNA recombination and requirement for an open reading frame during replication of equine arteritis virus defective interfering RNAs

Equine arteritis virus (EAV), the prototype arterivirus, is an enveloped plus-strand RNA virus with a genome of approximately 13 kb. Based on similarities in genome organization and protein expression, the arteriviruses have recently been grouped together with the coronaviruses and toroviruses in the newly established order Nidovirales. Previously, we reported the construction of pEDI, a full-length cDNA copy of EAV DI-b, a natural defective interfering (DI) RNA of 5.6 kb (R. Molenkamp et al., J. Virol. 74:3156-3165, 2000). EDI RNA consists of three noncontiguous parts of the EAV genome fused in frame with respect to the replicase gene. As a result, EDI RNA contains a truncated replicase open reading frame (EDI-ORF) and encodes a truncated replicase polyprotein. Since some coronavirus DI RNAs require the presence of an ORF for their efficient propagation, we have analyzed the importance of the EDI-ORF in EDI RNA replication. The EDI-ORF was disrupted at different positions by the introduction of frameshift mutations. These were found either to block DI RNA replication completely or to be removed within one virus passage, probably due to homologous recombination with the helper virus genome. Using recombination assays based on EDI RNA and full-length EAV genomes containing specific mutations, the rates of homologous RNA recombination in the 3'- and 5'-proximal regions of the EAV genome were studied. Remarkably, the recombination frequency in the 5'-proximal region was found to be approximately 100-fold lower than that in the 3'-proximal part of the genome.     

Recombination and Coronavirus Defective Interfering RNAs

Naturally occurring defective interfering RNAs have been found in 4 of 14 coronavirus species. They range in size from 2.2 kb to approximately 25 kb, or 80% of the 30-kb parent virus genome. The large DI RNAs do not in all cases appear to require helper virus for intracellular replication and it has been postulated that they may on their own function as agents of disease. Coronavirus DI RNAs appear to arise by internal deletions (through nonhomologous recombination events) on the virus genome or on DI RNAs of larger size by a polymerase strand-switching (copy-choice) mechanism. In addition to their use in the study of virus RNA replication and virus assembly, coronavirus DI RNAs are being used in a major way to study the mechanism of a high-frequency, site-specific RNA recombination event that leads to leader acquisition during virus replication (i.e., the leader fusion event that occurs during synthesis of subgenomic mRNAs, and the leader-switching event that can occur during DI RNA replication), a distinguishing feature of coronaviruses (and arteriviruses). Coronavirus DI RNAs are also being engineered as vehicles for the generation of targeted recombinants of the parent virus genome.     

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.     

Location of the 5' Termini of Tobacco Necrosis Virus Strain D Subgenomic mRNAs

Northern blot analysis of double-stranded (ds) RNA from bean-leaf tissue infected with tobacco necrosis virus strain D (TNV-D) detected the 4 kb genomic RNA and two subgenomic RNAs of about 1.5 kb and 1.2 kb; RNA extracted from virus particles only contained the genomic species. Blotting and probing with a range of probes indicated the approximate locations of the 5'ends of subgenomic RNA so that primers to fine-map the ends could be designed. When both singlestranded and ds RNA, extracted from TNV-D infected and healthy bean leaves were used as templates for primer extension using primers complementary to sequences at, or upstream of, the initiation codons of, respectively, the coat protein and the p7a genes, major infectionspecific products were detected. Both subgenomic RNAs start at G residues. The larger subgenomic RNA is 1547 nucleotides in length with a leader sequences of 36 nucleotides upstream of the p7a gene, and the smaller subgenomic RNA has a 90 nucleotide leader upstream of the coat protein AUG and is 1202 nucteotides long. An analysis of the 5'terminal locations of both subgenomic RNAs and the previously mapped analogous subgenomic RNAs associated with infection with the related TNV-A isolate, revealed a marked degree of homology downstream of the initiation sites for each RNA. This homology was maintained at the 5'termini of both virion RNAs and could be extended to another isolate of TNV for which partial sequence data, but not subgenomic mapping RNA data are available.     

Replication and packaging of Turnip yellow mosaic virus RNA containing Flock house virus RNA1 sequence

Turnip yellow mosaic virus (TYMV) is a spherical plant virus that has a single 6.3 kb positive strand RNA as a genome. In this study, RNA1 sequence of Flock house virus (FHV) was inserted into the TYMV genome to test whether TYMV can accommodate and express another viral entity. In the resulting construct, designated TY-FHV, the FHV RNA1 sequence was expressed as a TYMV subgenomic RNA. Northern analysis of the Nicotiana benthamiana leaves agroinfiltrated with the TY-FHV showed that both genomic and subgenomic FHV RNAs were abundantly produced. This indicates that the FHV RNA1 sequence was correctly expressed and translated to produce a functional FHV replicase. Although these FHV RNAs were not encapsidated, the FHV RNA having a TYMV CP sequence at the 3’-end was efficiently encapsidated. When an eGFP gene was inserted into the B2 ORF of the FHV sequence, a fusion protein of B2-eGFP was produced as expected. [BMB Reports 2014; 47(6): 330-335]     

Cucumber mosaic virus, a model for RNA virus evolution

Taxonomic relationships: Cucumber mosaic virus (CMV) is the type member of the Cucumovirus genus, in the family Bromoviridae . Additional members of the genus are Peanut stunt virus (PSV) and Tomato aspermy virus (TAV). The RNAs 3 of all members of the genus can be exchanged and still yield a viable virus, while the RNAs 1 and 2 can only be exchanged within a species.
Physical properties: The virus particles are about 29 nm in diameter, and are composed of 180 subunits (T = 3 icosahedral symmetry). The particles sediment with an s value of approximately 98. The virions contain 18% RNA, and are highly labile, relying on RNA–protein interactions for their integrity. The three genomic RNAs, designated RNA 1 (3.3 kb in length), RNA 2 (3.0 kb) and RNA 3 (2.2 kb) are packaged in individual particles; a subgenomic RNA, RNA 4 (1.0 kb), is packaged with the genomic RNA 3, making all the particles roughly equivalent in composition. In some strains an additional subgenomic RNA, RNA 4A is also encapsidated at low levels. The genomic RNAs are single stranded, plus sense RNAs with 5' cap structures, and 3' conserved regions that can be folded into tRNA-like structures.
Satellite RNAs: CMV can harbour molecular parasites known as satellite RNAs (satRNAs) that can dramatically alter the symptom phenotype induced by the virus. The CMV satRNAs do not encode any proteins but rely on the RNA for their biological activity.
Hosts: CMV infects over 1000 species of hosts, including members of 85 plant families, making it the broadest host range virus known. The virus is transmitted from host to host by aphid vectors, in a nonpersistent manner.
Useful web sites: http://mmtsb.scripps.edu/viper/1f15.html (structure); http://www.ncbi.nlm.nih.gov/ICTVdb/ICTVdB/10040001.htm (general information)     

RNA/cDNA Hybridization and Infectivity Tests Suggest that Barley Yellow Mosaic Virus Isolate M has a Bipartite Genome

Northern blot analysis with random-primed cDNAs revealed that RNAs 1 and 2 of barley yellow mosaic virus (Ba YMV) isolate M have no extensive base sequence homologies. Both RNAs are needed for infection. RNA 2 is therefore neither a subgenomic nor a, satellite RNA, but rather an essential part of the viral genome. Ba YMV isolate M has thus a bipartite genome.     

Coronavirus multiplication strategy. II. Mapping the avian infectious bronchitis virus intracellular RNA species to the genome.

Avian infectious bronchitis virus, a coronavirus, directed the synthesis of six major single-stranded polyadenylated RNA species in infected chicken embryo kidney cells. These RNAs include the intracellular form of the genome (RNA F) and five smaller RNA species (RNAs A, B, C, D, and E). Species A, B, C, and D are subgenomic RNAs and together with the genome form a nested sequence set, with the sequences of each RNA contained within every larger RNA species (D. F. Stern and S. I. T. Kennedy, J. Virol 34:665-674, 1980). In the present paper we show by RNase T1 oligonucleotide fingerprinting that RNA E is also a member of the nested set. Partial alkaline fragmentation of the genome followed by sucrose fractionation, oligodeoxythymidylate-cellulose chromatography, and RNase T1 fingerprinting gave a partial 3'-to-5' oligonucleotide spot order. A comparison of the oligonucleotides of each of the five subgenomic RNAs with this spot order established that all of the RNAs are comprised of nucleotide sequences inward from the 3' end of the genome. This result is discussed in relation to the multiplication strategy both of coronaviruses and of other RNA-containing viruses.     

Alterations of the conformation of the RNAs of alfalfa mosaic virus upon binding of a few coat protein molecules

Structural changes in the single-stranded genome RNAs (RNAs 1, 2 and 3) and the subgenomic coat protein messenger (RNA 4) of alfalfa mosaic virus upon addition of a few coat protein molecules of the virus were investigated by measuring the fluorescent intensity of bound ethidium bromide and by circular dichroism. No effect could be observed in the case of the genome RNAs. However, in RNA 4, which is of much less complexity than the genome RNAs, a reduction of the ethidium bromide binding by 30% was found, whereas the positive molar ellipticity at 265 nm was reduced by 9% upon binding of the coat protein. Both changes point to a reduction of the ordered structure of the RNA. Since the protein is known to bind first at the 3′-terminus of RNA 4 and probably also of the genome RNAs, the conformational changes observed could be those thought to be necessary for replicase recognition in this positive-stranded RNA virus which needs the coat protein for starting an infection cycle.     

Complete genome sequence of garlic latent virus,a member of the carlavirus family

The complete genome sequence of the garlic latent virus (GLV) has been determined. The whole GLV genome consists of 8,353 nucleotides, excluding the 3'-end poly(A)+ tail, and contains six open-reading frames (ORFs). Putative proteins that were encoded by the reading frames contain the motifs that were conserved in carlavirus-specific RNA replicases, NTP-dependent DNA helicases, two viral membrane-bound proteins, a viral coat protein, and a zinc-finger. Overall, the GLV genome shows structural features that are common in carlaviruses. An in vitro translation analysis revealed that the zinc-finger protein is not produced as a transframe protein with the coat protein by ribosomal frameshifting. A Northern blot analysis showed that GLV-specific probes hybridized to garlic leaf RNA fragments of about 2.6 and 1.5 kb long, in addition to the 8.5 kb whole genome. The two subgenomic RNAs might be encapsidated into smaller viral particles. In garlic plants, 700 nm long flexuous rod-shaped virus particles were observed in the immunoelectron microscopy using polyclonal antibodies against the GLV coat proteins.