Sequence and organization of barley yellow dwarf virus genomic RNA.

The nucleotide sequence of the genomic RNA of barley yellow dwarf virus, PAV serotype was determined, except for the 5'-terminal base, and its genome organization deduced. The 5,677 nucleotide genome contains five large open reading frames (ORFs). The genes for the coat protein (1) and the putative viral RNA-dependent RNA polymerase were identified. The latter shows a striking degree of similarity to that of carnation mottle virus (CarMV). By comparison with corona- and retrovirus RNAs, it is proposed that a translational frameshift is involved in expression of the polymerase. An ORF encoding an Mr 49,797 protein (50K ORF) may be translated by in-frame readthrough of the coat protein stop codon. The coat protein, an overlapping 17K ORF, and a 3'6.7K ORF are likely to be expressed via subgenomic mRNAs.     

Complete genome sequence and analyses of the subgenomic RNAs of sweet potato chlorotic stunt virus reveal several new features for the genus Crinivirus

The complete nucleotide sequences of genomic RNA1 (9,407 nucleotides [nt]) and RNA2 (8,223 nt) of Sweet potato chlorotic stunt virus (SPCSV; genus Crinivirus, family Closteroviridae) were determined, revealing that SPCSV possesses the second largest identified positive-strand single-stranded RNA genome among plant viruses after Citrus tristeza virus. RNA1 contains two overlapping open reading frames (ORFs) that encode the replication module, consisting of the putative papain-like cysteine proteinase, methyltransferase, helicase, and polymerase domains. RNA2 contains the Closteroviridae hallmark gene array represented by a heat shock protein homologue (Hsp70h), a protein of 50 to 60 kDa depending on the virus, the major coat protein, and a divergent copy of the coat protein. This grouping resembles the genome organization of Lettuce infectious yellows virus (LIYV), the only other crinivirus for which the whole genomic sequence is available. However, in striking contrast to LIYV, the two genomic RNAs of SPCSV contained nearly identical 208-nt-long 3' terminal sequences, and the ORF for a putative small hydrophobic protein present in LIYV RNA2 was found at a novel position in SPCSV RNA1. Furthermore, unlike any other plant or animal virus, SPCSV carried an ORF for a putative RNase III-like protein (ORF2 on RNA1). Several subgenomic RNAs (sgRNAs) were detected in SPCSV-infected plants, indicating that the sgRNAs formed from RNA1 accumulated earlier in infection than those of RNA2. The 5' ends of seven sgRNAs were cloned and sequenced by an approach that provided compelling evidence that the sgRNAs are capped in infected plants, a novel finding for members of the Closteroviridae.     

The 3' end of Norwalk virus mRNA contains determinants that regulate the expression and stability of the viral capsid protein VP1: a novel function for the VP2 protein

Norwalk virus (NV) is the prototype strain of a group of noncultivable human caliciviruses responsible for epidemic outbreaks of acute gastroenteritis. The capsid protein VP1 is synthesized from a subgenomic RNA that contains two open reading frames (ORFs), ORF2 and ORF3, and the 3' untranslated region (UTR). ORF2 and ORF3 code for the capsid protein (VP1) and a small structural basic protein (VP2), respectively. We discovered that the yields of virus-like particles (VLPs) composed of VP1 are significantly reduced when this protein is expressed from ORF2 alone. To determine how the 3' terminus of the NV subgenomic RNA regulates VP1 expression, we compared VP1 expression levels by using recombinant baculovirus constructs containing different 3' elements. High VP1 levels were detected by using a recombinant baculovirus that contained ORF2, ORF3, and the 3'UTR (ORF2+3+3'UTR). In contrast, expression of VP1 from constructs that lacked the 3'UTR (ORF2+3), ORF3 (ORF2+3'UTR), or both (ORF2 alone) was highly reduced. Elimination of VP2 synthesis from the subgenomic RNA by mutation resulted in VP1 levels similar to those obtained with the ORF2 construct alone, suggesting a cis role for VP2 in upregulation of VP1 expression levels. Comparisons of the kinetics of RNA and capsid protein expression levels by using constructs with or without ORF3 or the 3'UTR revealed that the 3'UTR increased the levels of VP1 RNA, whereas the presence of VP2 resulted in increased levels of VP1. Furthermore, VP2 increased VP1 stability and protected VP1 from disassembly and protease degradation. The increase in VP1 expression levels caused by the presence of VP2 in cis was also observed in mammalian cells.     

Molecular characterization of a dsRNA mycovirus, Fusarium graminearum virus-DK21, which is phylogenetically related to hypoviruses but has a genome organization and gene expression strategy resembling those of plant potex-like viruses

Fusarium graminearum causes a serious scab disease of small grains in Korea. The nucleotide sequence of the genomic RNA of a double-stranded RNA (dsRNA) virus, Fusarium graminearum virus-DK21 (FgV-DK21), from F. graminearum strain DK21, which is associated with hypovirulence in F. graminearum, was determined and compared to the genome sequences of other mycoviruses, including Cryponectria hypoviruses. The FgV-DK21 dsRNA consists of 6,624 nucleotides, excluding the 3'-terminal poly(A) tail. The viral genome has 53- and 46-nucleotide 5' and 3' untranslated regions (UTRs), respectively, and five putative open reading frames. A phylogenetic analysis of the deduced amino acid sequence of ORF1, which encodes a putative RNA-dependent RNA polymerase, and those of other mycoviruses revealed that this organism forms a distinct virus clade with other hypoviruses, and is more distantly related to other mycoviruses (3.8 to 24.0% identity). However, pairwise sequence comparisons of the nucleotide and deduced amino acid sequences of ORFs 2 through 5 revealed no close relationships to other protein sequences currently available in GenBank. Analyses of RNA accumulation by Northern blot and primer extension indicated that these putative gene products are expressed from at least two different subgenomic RNAs (sgRNAs), in contrast to the cases in other hypoviruses. This study suggests the existence of a new, as yet unassigned, genus of mycoviruses that exhibits a potex-like genome organization and sgRNA accumulation.     

The ORF2 protein of hepatitis E virus binds the 5' region of viral RNA

Hepatitis E virus (HEV) is a major human pathogen in much of the developing world. It is a plus-strand RNA virus with a 7.2-kb polyadenylated genome consisting of three open reading frames, ORF1, ORF2, and ORF3. Of these, ORF2 encodes the major capsid protein of the virus and ORF3 encodes a small protein of unknown function. Using the yeast three-hybrid system and traditional biochemical techniques, we have studied the RNA binding activities of ORF2 and ORF3, two proteins encoded in the 3' structural part of the genome. Since the genomic RNA from HEV has been postulated to contain secondary structures at the 5' and 3' ends, we used these two terminal regions, besides other regions within the genome, in this study. Experiments were designed to test for interactions between the genomic RNA fusion constructs with ORF2 and ORF3 hybrid proteins in a yeast cellular environment. We show here that the ORF2 protein contains RNA binding activity. The ORF2 protein specifically bound the 5' end of the HEV genome. Deletion analysis of this protein showed that its RNA binding activity was lost when deletions were made beyond the N-terminal 111 amino acids. Finer mapping of the interacting RNA revealed that a 76-nucleotide (nt) region at the 5' end of the HEV genome was responsible for binding the ORF2 protein. This 76-nt region included the 51-nt HEV sequence, conserved across alphaviruses. Our results support the requirement of this conserved sequence for interaction with ORF2 and also indicate an increase in the strength of the RNA-protein interaction when an additional 44 bases downstream of this 76-nt region were included. Secondary-structure predictions and the location of the ORF2 binding region within the HEV genome indicate that this interaction may play a role in viral encapsidation.     

Analysis of astrovirus serotype 1 RNA, identification of the viral RNA-dependent RNA polymerase motif, and expression of a viral structural protein.

We report the results from sequence analysis and expression studies of the gastroenteritis agent astrovirus serotype 1. We have cloned and sequenced 5,944 nucleotides (nt) of the estimated 7.2-kb RNA genome and have identified three open reading frames (ORFs). ORF-3, at the 3' end, is 2,361 nt in length and is fully encoded in both the genomic and subgenomic viral RNAs. Expression of ORF-3 in vitro yields an 87-kDa protein that is immunoprecipitated with a monoclonal antibody specific for viral capsids. This protein comigrates with an authentic 87-kDa astrovirus protein immunoprecipitated from infected cells, indicating that this region encodes a viral structural protein. The adjacent upstream ORF (ORF-2) is 1,557 nt in length and contains a viral RNA-dependent RNA polymerase motif. The viral RNA-dependent RNA polymerase motifs from four astrovirus serotypes are compared. Partial sequence (2,018 nt) of the most 5' ORF (ORF-1) reveals a 3C-like serine protease motif. The ORF-1 sequence is incomplete. These results indicate that the astrovirus genome is organized with nonstructural proteins encoded at the 5' end and structural proteins at the 3' end. ORF-2 has no start methionine and is in the -1 frame compared with ORF-1. We present sequence evidence for a ribosomal frameshift mechanism for expression of the viral polymerase.     

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.     

The 5' untranslated region of Rhopalosiphum padi virus contains an internal ribosome entry site which functions efficiently in mammalian, plant, and insect translation systems

Rhopalosiphum padi virus (RhPV) is one of several picorna-like viruses that infect insects; sequence analysis has revealed distinct differences between these agents and mammalian picornaviruses. RhPV has a single-stranded positive-sense RNA genome of about 10 kb; unlike the genomes of Picornaviridae, however, this genome contains two long open reading frames (ORFs). ORF1 encodes the virus nonstructural proteins, while the downstream ORF, ORF2, specifies the structural proteins. Both ORFs are preceded by long untranslated regions (UTRs). The intergenic UTR is known to contain an internal ribosome entry site (IRES) which directs non-AUG-initiated translation of ORF2. We have examined the 5' UTR of RhPV for IRES activity by translating synthetic dicistronic mRNAs containing this sequence in a variety of systems. We now report that the 5' UTR contains an element which directs internal initiation of protein synthesis from an AUG codon in mammalian, plant, and Drosophila in vitro translation systems. In contrast, the encephalomyocarditis virus IRES functions only in the mammalian system. The RhPV 5' IRES element has features in common with picornavirus IRES elements, in that no coding sequence is required for IRES function, but also with cellular IRES elements, as deletion analysis indicates that this IRES element does not have sharply defined boundaries.     

Sequential Partially Overlapping Gene Arrangement in the Tricistronic S1 Genome Segments of Avian Reovirus and Nelson Bay Reovirus: Implications for Translation Initiation

Previous studies of the avian reovirus strain S1133 (ARV-S1133) S1 genome segment revealed that the open reading frame (ORF) encoding the final sigmaC viral cell attachment protein initiates over 600 nucleotides distal from the 5' end of the S1 mRNA and is preceded by two predicted small nonoverlapping ORFs. To more clearly define the translational properties of this unusual polycistronic RNA, we pursued a comparative analysis of the S1 genome segment of the related Nelson Bay reovirus (NBV). Sequence analysis indicated that the 3'-proximal ORF present on the NBV S1 genome segment also encodes a final sigmaC homolog, as evidenced by the presence of an extended N-terminal heptad repeat characteristic of the coiled-coil region common to the cell attachment proteins of reoviruses. Most importantly, the NBV S1 genome segment contains two conserved ORFs upstream of the final sigmaC coding region that are extended relative to the predicted ORFs of ARV-S1133 and are arranged in a sequential, partially overlapping fashion. Sequence analysis of the S1 genome segments of two additional strains of ARV indicated a similar overlapping tricistronic gene arrangement as predicted for the NBV S1 genome segment. Expression analysis of the ARV S1 genome segment indicated that all three ORFs are functional in vitro and in virus-infected cells. In addition to the previously described p10 and final sigmaC gene products, the S1 genome segment encodes from the central ORF a 17-kDa basic protein (p17) of no known function. Optimizing the translation start site of the ARV p10 ORF lead to an approximately 15-fold increase in p10 expression with little or no effect on translation of the downstream final sigmaC ORF. These results suggest that translation initiation complexes can bypass over 600 nucleotides and two functional overlapping upstream ORFs in order to access the distal final sigmaC start site.     

戊型肝炎病毒亚基因组RNA的研究

本文利用同位素代谢标记在HEV感染85～10.5,6.5～7.5h分别检测到1及2个亚基因组RNA,而感染21h后及在成熟的病毒颗粒内未能检测到亚基因组RNA。通过杂交实验,发现HEV的亚基因组RNA具有典型的共3′端的半套式结构,且基因组RNA与亚基因组RNA的5′端不存在共同的引导序列。通过紫外转录图谱发现HEV的亚基因组RNA是通过独立转录的方式产生的。利用引物延伸反应发现两种亚基因组RNA的转录起始位点分别位于RNA聚合酶区及非结构区、结构区的基因间序列。     

The complete nucleotide sequence of PEBV RNA2 reveals the presence of a novel open reading frame and provides insights into the structure of tobraviral subgenomic promoters.

The 3374 nucleotide sequence of RNA2 from the British PEBV strain SP5 has been determined. The RNA includes three open reading frames flanked by 5' and 3' noncoding regions of 509 and 480 nucleotides. The open reading frames specify coat protein, a 29.6K product homologous to the 29.1K product of TRV(TCM) RNA2 and a 23K product not homologous to any previously described protein. The homology demonstrated between the coat proteins of PRV, TRV and PEBV indicates a common evolutionary origin for these proteins. Upstream of each ORF are located sequences homologous to those with which subgenomic RNAs of other tobraviruses start. Subgenomic RNAs for the expression of the three ORFs may start at these points. On all five tobraviral RNA2 molecules sequenced to date, these sequences were found upstream of the coat protein ORF in association with a strongly-conserved potential secondary structural element. Similar potential structures were identified upstream of other tobraviral ORFs. These structures may contribute to the activity of the tobraviral subgenomic promoter.     

A bicistronic subgenomic mRNA encodes both the ORF2 and ORF3 proteins of hepatitis E virus

Hepatitis E virus replicons containing the neomycin resistance gene expressed from open reading frames (ORFs) 2 and 3 were transfected into Huh-7 cells, and stable cell lines containing functional replicons were selected by constant exposure to G418 sulfate. Northern blot analyses detected full-length replicon RNA and a single subgenomic RNA. This subgenomic RNA, which was capped, initiated at nucleotide 5122 downstream of the first two methionine codons in ORF3 and was bicistronic; two closely spaced methionine codons in different reading frames were used for the initiation of ORF3 and ORF2 translation.     

Discovery of the first insect nidovirus, a missing evolutionary link in the emergence of the largest RNA virus genomes

Nidoviruses with large genomes (26.3-31.7 kb; 'large nidoviruses'), including Coronaviridae and Roniviridae, are the most complex positive-sense single-stranded RNA (ssRNA+) viruses. Based on genome size, they are far separated from all other ssRNA+ viruses (below 19.6 kb), including the distantly related Arteriviridae (12.7-15.7 kb; 'small nidoviruses'). Exceptionally for ssRNA+ viruses, large nidoviruses encode a 3'-5'exoribonuclease (ExoN) that was implicated in controlling RNA replication fidelity. Its acquisition may have given rise to the ancestor of large nidoviruses, a hypothesis for which we here provide evolutionary support using comparative genomics involving the newly discovered first insect-borne nidovirus. This Nam Dinh virus (NDiV), named after a Vietnamese province, was isolated from mosquitoes and is yet to be linked to any pathology. The genome of this enveloped 60-80 nm virus is 20,192 nt and has a nidovirus-like polycistronic organization including two large, partially overlapping open reading frames (ORF) 1a and 1b followed by several smaller 3'-proximal ORFs. Peptide sequencing assigned three virion proteins to ORFs 2a, 2b, and 3, which are expressed from two 3'-coterminal subgenomic RNAs. The NDiV ORF1a/ORF1b frameshifting signal and various replicative proteins were tentatively mapped to canonical positions in the nidovirus genome. They include six nidovirus-wide conserved replicase domains, as well as the ExoN and 2'-O-methyltransferase that are specific to large nidoviruses. NDiV ORF1b also encodes a putative N7-methyltransferase, identified in a subset of large nidoviruses, but not the uridylate-specific endonuclease that - in deviation from the current paradigm - is present exclusively in the currently known vertebrate nidoviruses. Rooted phylogenetic inference by Bayesian and Maximum Likelihood methods indicates that NDiV clusters with roniviruses and that its branch diverged from large nidoviruses early after they split from small nidoviruses. Together these characteristics identify NDiV as the prototype of a new nidovirus family and a missing link in the transition from small to large nidoviruses.     

Sequence of the region coding for virion proteins C and E2 and the carboxy terminus of the nonstructural proteins of rubella virus: comparison with alphaviruses

The sequence of the 3' 4508 nucleotides (nt) of the genomic RNA of the Therien strain of rubella virus (RV) was determined for cDNA clones. The sequence contains a 3189-nt open reading frame (ORF) which codes for the structural proteins C, E2 and E1. C is predicted to have a length of 300 amino acids (aa). The N-terminal half of the C protein is highly basic and hydrophilic in nature, and is putatively the region of the protein which interacts with the virion RNA. At the C terminus of the C protein is a stretch of 20 hydrophobic aa which also serves as the signal sequence for E2, indicating that the cleavage of C from the polyprotein precursor may be catalyzed by signalase in the lumen of the endoplasmic reticulum. E2 is 282 aa in length and contains four potential N-linked glycosylation sites and a putative transmembrane domain near its C terminus. The sequence of E1 has been previously described [Frey et al., Virology 154 (1986) 228-232]. No homology could be detected between the amino acid sequence of the RV structural proteins and the amino acid sequence of the alphavirus structural proteins. From the position of a region of 30 nt in the RV genomic sequence which exhibited significant homology with the sequence in the alphavirus genome at which subgenomic RNA synthesis is initiated, the RV subgenomic RNA is predicted to be 3346 nt in length and the nontranslated region from the 5' end of the subgenomic RNA to the structural protein ORF is predicted to be 98 nt. In a different translation frame beginning at the 5' end of the RV nt sequence reported here is a 1407 nt ORF which is the C terminal region of the nonstructural protein ORF. This ORF overlaps the structural protein ORF by 149 nt. A low level of homology could be detected between the predicted amino acid sequence of the C-terminus of the RV nonstructural protein ORF and the replicase proteins of several positive RNA viruses of animals and plants, including nsp4 of the alphaviruses, the protein encoded by the C-terminal region of the alphavirus nonstructural ORF. However, the overall homology between RV and the alphaviruses in this region of the genome was only 18%, indicating that these two genera of the Togavirus family are only distantly related. Intriguingly, there is a 2844-nt ORF present in the negative polarity orientation of the RV sequence which could encode a 928-aa polyprotein.     

Examination of potato virus X proteins synthesized in infected tobacco plants.

Nucleotide sequence analysis of potato virus X (PVX) genomic RNA predicts five open reading frames (ORFs). Previous analysis of total RNAs from PVX-infected leaf tissue suggested that six subgenomic RNAs are synthesized during infection. However, the proteins encoded by the genomic RNA, the subgenomic RNAs, or the predicted ORFs have not been identified in vivo. To characterize the coding properties of the viral RNA, particularly to determine whether the five predicted ORFs function in vivo, total protein extracts prepared from PVX-infected leaf tissue were analyzed by using antibodies raised against virus-specific synthetic peptides and against the virus capsid protein. Dot blot analyses showed that these antibodies reacted to PVX-infected extracts, indicating in vivo expression of the five predicted ORFs. In addition, Western blot (immunoblot) analysis of the extracts showed that ORF 1, 2, 3, and 4 peptide antisera and coat protein antiserum detect predominantly a single protein.