Proximal Open Reading Frames Research Articles

A new tetra-segmented splipalmivirus with divided RdRP domains from Cryphonectria naterciae, a fungus found on chestnut and cork oak trees in Europe

Positive-sense (+), single-stranded (ss) RNA viruses with divided RNA-dependent RNA polymerase (RdRP) domains have been reported from diverse filamentous ascomycetes since 2020. These viruses are termed splipalmiviruses or polynarnaviruses and have been characterized largely at the sequence level, but ill-defined biologically. Cryphonectria naterciae, from which only one virus has been reported, is an ascomycetous fungus potentially plant-pathogenic to chestnut and oak trees. We molecularly characterized multiple viruses in a single Portuguese isolate (C0614) of C. naterciae, taking a metatranscriptomic and conventional double-stranded RNA approach. Among them are a novel splipalmivirus (Cryphonectria naterciae splipalmivirus 1, CnSpV1) and a novel fusagravirus (Cryphonectria naterciae fusagravirus 1, CnFGV1). This study focused on the former virus. CnSpV1 has a tetra-segmented, (+)ssRNA genome (RNA1 to RNA4). As observed for other splipalmiviruses reported in 2020 and 2021, the RdRP domain is separately encoded by RNA1 (motifs F, A and B) and RNA2 (motifs C and D). A hypothetical protein encoded by the 5′-proximal open reading frame of RNA3 shows similarity to a counterpart conserved in some splipalmiviruses. The other RNA3-encoded protein and RNA4-encoded protein show no similarity with known proteins in a blastp search. The tetra-segment nature was confirmed by the conserved terminal sequences of the four CnSpV1 segments (RNA1 to RNA4) and their 100% coexistence in over 100 single conidial isolates tested. The experimental introduction of CnSpV1 along with CnFGV1 into a virus free strain C0754 of C. naterciae vegetatively incompatible with C0614 resulted in no phenotypic alteration, suggesting asymptomatic infection. The protoplast fusion assay indicates a considerably narrow host range of CnSpV1, restricted to the species C. naterciae and C. carpinicola. This study contributes to better understanding of the molecular and biological properties of this unique group of viruses.

Revisiting the cysteine-rich proteins encoded in the 3'-proximal open reading frame of the positive-sense single-stranded RNA of some monopartite filamentous plant viruses: functional dissection of p15 from grapevine virus B.

A reexamination of proteins with conserved cysteines and basic amino acids encoded by the 3'-proximal gene of the positive-sense single-stranded RNA of some monopartite filamentous plant viruses has been carried out. The cysteines are involved in a putative Zn-finger domain, which, together with the basic amino acids, form part of the nuclear or nucleolar localization signals. An in-depth study of one of these proteins, p15 from grapevine B virus (GVB), has shown: (i) a three-dimensional structure with four α-helices predicted by two independent in silico approaches, (ii) the nucleolus as the main accumulation site by applying confocal laser microscopy to a fusion between p15 and the green fluorescent protein, (iii) the involvement of the basic amino acids and the putative Zn-finger domain, mapping at the N-terminal region of p15, in the nucleolar localization signal, as revealed by the effect of six alanine substitution mutations, (iv) the p15 suppressor function of sense-mediated RNA silencing as revealed by agroinfiltration in a transgenic line of Nicotiana benthamiana, and (v) the enhancer activity of p15 on viral pathogenicity in N. benthamiana when expressed from a potato virus X vector. In addition, we elaborate on an evolutionary scenario for these filamentous viruses, invoking takeover by a common ancestor(s) of viral or host genes coding for those cysteine-rich proteins, followed by divergence, which would also explain why they are encoded in the 3'-proximal gene of the genomic single-stranded viral RNA.

Ribosomes guide pachytene piRNA formation on long intergenic piRNA precursors.

PIWI-interacting RNAs (piRNAs) are a class of small non-coding RNAs essential for fertility. In adult mouse testes, most piRNAs are derived from long single-stranded RNAs lacking annotated open reading frames (ORFs). The mechanisms underlying how piRNA sequences are defined during the cleavages of piRNA precursors remain elusive. Here, we show that 80S ribosomes translate the 5´-proximal short ORFs (uORFs) of piRNA precursors. MOV10L1/Armitage RNA helicase then facilitates the translocation of ribosomes into the uORF-downstream regions (UDRs). The ribosome-bound UDRs are targeted by piRNA processing machinery, with the processed ribosome-protected-regions becoming piRNAs. The dual modes of interaction between ribosomes and piRNA precursors underlies the distinct piRNA biogenesis requirements at uORFs and UDRs. Ribosomes also mediate piRNA processing in roosters and green lizards, implying this mechanism is evolutionarily conserved in amniotes. Our results uncover a function for ribosomes on non-coding regions of RNAs and reveal the mechanisms underlying how piRNAs are defined.

A new polycipivirus identified in Colobopsis shohki.

A new polycipivirus was identified in the arboreal ant Colobopsis shohki. The viral RNA was 11,855 nt in length with five 5'-proximal open reading frames (ORFs) encoding structural proteins and a long 3' ORF encoding the replication polyprotein. The protein sequences of these ORFs had significant similarity to those of the polycipiviruses Lasius niger virus 1 and Solenopsis invicta virus 2. The results of phylogenetic analysis and its genome organization suggested that this virus belongs to the genus Sopolycivirus in the family Polycipiviridae. The name "Colobopsis shohki virus 1" (CshV1) is proposed for the new virus.

ICTV Virus Taxonomy Profile: Polycipiviridae.

Polycipiviridae is a family of picorna-like viruses with non-segmented, linear, positive-sense RNA genomes of approximately 10–12 kb. Unusually for viruses within the order Picornavirales, their genomes are polycistronic, with four (or more) consecutive 5′-proximal open reading frames (ORFs) encoding structural (and possibly other) proteins and a long 3′ ORF encoding the replication polyprotein. Members of species within the family have all been detected in ants or via arthropod transcriptomic datasets. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the Polycipiviridae, which is available at www.ictv.global/report/polycipiviridae.

Molecular characteristics of tomato mosaic virus infecting tomato in Uganda

Viral diseases are part of the limiting factors to tomato (Solanum lycopersicum L.) cultivation worldwide, reducing both the quality and quantity of yield. Tomato mosaic virus (ToMV) is one of the damaging viruses of tomato. This paper describes molecular characteristics of the full length genome of ToMV isolated from tomato in Uganda (ToMV-Ug). The genomic, ribonucleic acid (RNA), of this isolate is 6383 nucleotides (nts) in length, encoding four open reading frames (ORFs). Based on the homology with other ToMV strains, the 5’ proximal 130 kilo dalton (kDa) ORF and its read-through product (180 kDa) are expected to encode two proteins required for viral genome replication; while the 30 kDa middle ORF and the 17.5 kDa 3’ proximal ORF are expected to encode the movement protein (MP) and coat protein (CP), respectively. The 5’- and 3’- untranslated regions (UTRs) are 71 and 201 nts, respectively. Comparison with previously published ToMV sequences showed that ToMV-Ug is 99% identical to ToMV strains from Africa (Egypt and Zimbabwe), as well as diverse locations such as China, Australia, Germany and Japan; suggesting high levels of sequence conservation within this virus. This is the first report detailing molecular analysis of a ToMV isolate from Uganda and the Eastern and Central Africa regions. Keywords : Ribonucleic acid, Solanum lycopersicum, Tobamovirus

Alfalfa virus S, a new species in the family Alphaflexiviridae.

A new species of the family Alphaflexiviridae provisionally named alfalfa virus S (AVS) was discovered in alfalfa samples originating from Sudan. A complete nucleotide sequence of the viral genome consisting of 8,349 nucleotides excluding the 3’ poly(A) tail was determined by high throughput sequencing (HTS) on an Illumina platform. NCBI BLAST searches revealed that the virus shares the greatest degree of sequence identity with members of the family Alphaflexiviridae, genus Allexivirus. The AVS genome contains six computationally-predicted open reading frames (ORF) encoding viral replication protein, triple gene block protein 1 (TGB1), TGB2, TGB3-like protein, unknown 38.4 kDa protein resembling serine-rich 40 kDa protein characteristic for allexiviruses, and coat protein (CP). AVS lacks a clear 3’ proximal ORF that encodes a nucleic acid-binding protein typical for allexiviruses. The identity of the virus was confirmed by RT-PCR with primers derived from the HTS-generated sequence, dot blot hybridization with DIG-labeled virus-specific RNA probes, and Western blot analysis with antibodies produced against a peptide derived from the CP sequence. Transmission electron microscopic observations of the infected tissues showed the presence of filamentous particles similar to allexiviruses in their length and appearance. To the best of our knowledge, this is the first report on the identification of a putative allexivirus in alfalfa (Medicago sativa). The genome sequence of AVS has been deposited in NCBI GenBank on 03/02/2016 as accession № KY696659.

Analysis of the complete genome sequence of black queen cell virus JL1 from infected honeybees in China.

There are six strains of the complete genomic sequences of black queen cell virus (BQCV) published in the GenBank, including South Africa (AF183905), South Korea (JX149531), Hungary 10 (EF517515), Poland 4 (EF517519), Poland 5 (EF517520) and Poland 6 (EF517521). Based on the six BQCV strains published in the GenBank, ten pairs of primers were designed in the present study using reverse transcription polymerase chain reaction to obtain the first complete genome sequence of a BQCV strain in China, called the BQCV China-JL1 strain (KP119603). A phylogenetic tree was then built to analyse their genetic relationships. The BQCV China-JL1 strain showed 86-93% similarity with the six strains published in the GenBank. The BQCV China-JL1 strain consisted of 8358 nucleotides (nt). The 5'-proximal open reading frame (ORF1) initiated at nt position 546 and terminated at nt position 4676, ORF3 initiated at nt position 4891 and terminated at nt position 5433, and the 3'-proximal ORF (ORF2) was located between nt positions 5750 and 8203.

Investigations on the Tobacco Necrosis Virus D p60 Replicase Protein

Tobacco Necrosis Virus D (TNV-D), in the genus Betanecrovirus (family Tombusviridae), possesses a single-stranded, positive-sense RNA genome containing six open reading frames (ORFs). Two 5'-proximal ORFs (1 and 2) encode overlapping polypeptides of 22 and 82 kDa (p22 and p82, respectively) which are both required for replication. The p22 auxiliary protein contains no replication motifs but the C-terminal region, downstream of a leaky stop codon, encodes a 60 kDa polypeptide (p60) which contains conserved RNA-dependent RNA polymerase (RdRP) motifs. Here we have expressed and purified recombinant p60 and show that in vitro it binds and efficiently synthesises both TNV-D RNA and Satellite tobacco necrosis virus C RNA. Alanine scanning mutagenesis of conserved amino acids in characteristic motifs in p60 revealed that some mutations significantly reduced RNA synthesis but mutating the second asparagine residue in the conserved GDD box was lethal. The effects of mutating identical amino acids in p60 on virus replication in vivo were examined in Nicotiana benthamiana plants following infection with RNA transcribed from wild type (wt) and mutant constructs. In inoculated leaves the behaviour of the mutants paralleled the in vitro data but systemic infection was precluded in all but one mutant which had reverted to wt. This study is the first to demonstrate the nucleic acid-binding and synthetic capabilities of a betanecrovirus polymerase.

Confirmation of the Genome Position and Mass of the Viral Protein, Vp3, ofSolenopsis invictaVirus 1 (Picornavirales: Dicistroviridae) Infecting the Red Imported Fire Ant (Hymenoptera: Formicidae)

Solenopsis invicta virus 1 (SINV-1) is the first virus to be discovered and characterized in any ant species (Valles et al. 2004). The virus exhibits a host range limited to fire ants in the Solenopsis genus (Valles et al. 2007). The single-stranded RNA genome is composed of 8,026 nucleotides containing 2 large open reading frames (ORFs) in the sense orientation with an untranslated region (UTR) at each end and between the two ORFs. The 5’-proximal ORF encodes for the non-structural proteins, helicase, protease, and RNA-dependent RNA polymerase (RdRp) and the 3’-proximal ORF for the structural (or capsid) proteins. ORF 2 was originally reported to commence at nucleotide position 4,390 at a canonical AUG start codon. However, it was later revealed empirically to actually start at codon GCU (genome position 4423-4425) which encodes for an alanine (Valles and Hashimoto 2008). SDS-PAGE analysis of purified SINV-1 particles yields 3 major (VP1, VP2, VP3) and one minor (VP4) capsid proteins. N-terminal analysis has revealed the margins of the capsid proteins and the corresponding scissile bonds within the structural polyprotein (Valles and Hashimoto 2008). Amino acid residues at these junctions were consistent with other dicistroviruses and unclassified picorna-like insect-infecting viruses (Liljas et al. 2002). The objective of this short communication was to confirm production and genome location of VP3 within the SINV-1 structural polyprotein using Western analysis. Polyclonal antibodies were synthesized from a recombinant peptide sequence obtained from the predicted amino acid sequence of VP3 (i.e., SRGGYRYKFFADDN). The peptide sequence is located at amino acid positions 9971010 of the structural polyprotein (Fig. 1A). Polyclonal antibodies were raised in a rabbit host by GenScript USA, Inc. (Piscataway, NJ) according to the company’s standard protocols. Ants used for purification of virus were collected by excavating nests from locations in Gainesville, FL. Colonies were examined by RT-PCR methods to identify ants exclusively infected with

Isolation and characterization of Solenopsis invicta virus 3, a new positive-strand RNA virus infecting the red imported fire ant, Solenopsis invicta

We report the discovery of a new virus from the red imported fire ant, Solenopsis invicta. Solenopsis invicta virus 3 (SINV-3) represents the third virus discovered from this ant species using the metagenomics approach. The single (positive)-strand RNA, monopartite, bicistronic genome of SINV-3 was sequenced in entirety (GenBank accession number FJ528584), comprised of 10,386 nucleotides, and polyadenylated at the 3' terminus. This genome size was confirmed by Northern analysis. The genome revealed 2 large open reading frames (ORFs) in the sense orientation with an untranslated region (UTR) at each end and between the two ORFs. The 5' proximal ORF (ORF 1) encoded a predicted protein of 299.1 kDa (2580 amino acids). The 3' proximal ORF (ORF 2) encoded a predicted protein of 73.2 kDa (651 amino acids). RNA-dependent RNA polymerase (RdRp), helicase, and protease domains were recognized in ORF 1. SDS-PAGE separation of purified SINV-3 particles yielded 2 bands (ostensibly capsid proteins) with a combined molecular mass of 77.3 kDa which was similar to the mass predicted by ORF 2 (73.2 kDa). Phylogenetic analysis of the conserved amino acid sequences containing domains I to VIII of the RdRp from dicistroviruses, iflaviruses, plant small RNA viruses, picornaviruses, and 4 unassigned positive-strand RNA viruses revealed a trichotomous phenogram with SINV-3 and Kelp fly virus comprising a unique cluster. Electron microscopic examination of negatively stained samples of SINV-3 revealed isometric particles with apparent projections and a diameter of 27.3+/-1.3 nm. SINV-3 was successfully transmitted to uninfected workers by feeding. The minus (replicative) strand of SINV-3 was detected in worker ants indicating replication of the virus. The possibility of using SINV-3 as a microbial control agent for fire ants is discussed.

Depurination of Brome Mosaic Virus RNA3 in Vivo Results in Translation-dependent Accelerated Degradation of the Viral RNA

Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein isolated from the pokeweed plant (Phytolacca americana) that exhibits antiviral activity against several plant and animal viruses. We have shown previously that PAP depurinates Brome mosaic virus (BMV) RNAs in vitro and that prior incubation of these RNAs with PAP reduced their synthesis in barley protoplasts. To investigate the post-transcriptional effect of PAP on viral RNA in vivo, we transcribed BMV RNA3 and expressed PAP in the yeast, Saccharomyces cerevisiae, which is a surrogate host for BMV. With an inducible transcription system, we show that the half-life of RNA3 in PAP-expressing cells was significantly less than in cells expressing PAPx, its enzymatically inactive form. PAP bound to RNA3 and depurinated the RNA within open reading frames 3 and 4 and within untranslated regions of the RNA. The depurinated RNA was associated with polysomes, caused ribosomes to stall at the point of depurination, and was targeted for accelerated degradation by components of the No-go decay pathway. As a consequence of translation elongation arrest and increased RNA degradation, expression of PAP in yeast also decreased the level of protein 3a, encoded by the 5'-proximal open reading frame 3 of BMV RNA3. These data provide the first evidence of viral RNA depurination in vivo by any ribosome-inactivating protein and support our hypothesis that depurination contributes to the antiviral activity of PAP, by enhancing viral RNA degradation and reducing translation of viral protein product.

Functional analysis of structural motifs in dicistroviruses

The family Dicistroviridae is composed of positive-stranded RNA viruses which have monopartite genomes. These viruses carry genome-linked virus proteins (VPg) and poly (A) tails. The 5′ untranslated region (UTR) is approximately 500 nucleotides and contains an internal ribosome entry site (IRES). These features resemble those of vertebrate picornaviruses, but dicistroviruses have other distinct characteristics. Picornaviruses have a single large open reading frame (ORF) encoding the capsid proteins at the 5′-end and the replicases at the 3′-end. In contrast, dicistroviruses have two nonoverlapping ORFs. The 5′-proximal ORF encodes the replicases and the 3′-proximal ORF encodes the capsid proteins. Usually, positive-stranded viruses which have capsid protein genes in the 3′ part of the genome produce subgenomic RNA for synthesis of the capsid proteins, because abundant quantities of the capsid proteins are required for the viral replication cycle. In dicistroviruses, translation of the capsid proteins is controlled by an additional IRES. This IRES is located in the intergenic region (IGR) between the replicase and capsid coding regions, and mediates the initiation of translation for the capsid proteins. The IGR-IRES has a multiple stem-loop structure containing three pseudoknots. We describe the characteristics of dicistroviruses, including the RNA elements and viral proteins.

A NEW MEMBER OF THE FAMILY FLEXIVIRIDAE FROM PHLOMIS FRUCTICOSA

SUMMARY A virus recovered in 2006 from Phlomis fructicosa L. (family Lamiacee) by mechanical transmission to Nicotiana occidentalis had flexuous particles ca. 850 nm long with an outward aspect recalling that of some members of the family Flexiviridae. Sequencing of a 3035 nt fragment of the viral genome, spanning from part of the RdRp gene to the 3’ terminal poly(A) tract, showed a structural organization comparable with that of species in the genus Trichovirus. The 5’ proximal open reading frame (ORF1) encoding the RdRp protein, was followed by two ORFs showing homologies with movement proteins of the p30 superfamily (ORF2) and the coat protein of flexiviruses (ORF3). These polypeptides had a substantial level of similarity with the corresponding proteins of members of the genus Trichovirus. A putative ORF, showing homology with nucleic acid binding proteins (NB) of some flexiviruses, was identified at the extreme 3’region of the viral genome. Its origin and expression are discussed.

Characterization of structural proteins of Solenopsis invicta virus 1

Purification of Solenopsis invicta virus 1 (SINV-1) from its host, S. invicta, and subsequent examination by electron microscopy revealed a homogeneous fraction of spherical particles with a diameter of 30–35 nm. Quantitative PCR with SINV-1-specific oligonucleotide primers verified that this fraction contained high copy numbers of the SINV-1 genome. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the SINV-1 purified fraction revealed three major and one minor protein bands. The protein bands were labeled VP1 (40.8 ± 1.4 kDa), VP2 (35.7 ± 2.8 kDa), VP3 (25.2 ± 1.8 kDa), and VP4 (22.2 ± 2.5 kDa) based on mass. N-terminal sequence was acquired successfully for VP1, VP2, and VP3, but not VP4, and delineated each capsid protein within the 3′-proximal open reading frame of SINV-1. Positional organization of the viral proteins within the SINV-1 structural polyprotein was consistent with dicistroviruses (when based on sequence similarity). Blastp analysis of SINV-1 VP1, VP2, and VP3 revealed significant identity with corresponding structural capsid proteins of positive-strand RNA viruses, particularly acute bee paralysis virus (ABPV), Kashmir bee virus (KBV) and Israeli acute paralysis virus (IAPV). Amino acid residues about the scissile bonds for VP1 and VP3 were consistent with dicistroviruses and insect-infecting picorna-like viruses. N-terminal sequencing of VP2 also established that translation initiation of the SINV-1 structural polyprotein was mediated by an internal ribosomal entry site and is AUG-independent.

Complete nucleotide sequence of Nootka lupine vein-clearing virus

The complete genome sequence of Nootka lupine vein-clearing virus (NLVCV) was determined to be 4,172 nucleotides in length containing four open reading frames (ORFs) with a similar genetic organization of virus species in the genus Carmovirus, family Tombusviridae. The order and gene product size, starting from the 5'-proximal ORF consisted of: (1) polymerase/replicase gene, ORF1 (p27) and ORF1RT (readthrough) (p87), (2) movement proteins ORF2 (p7) and ORF3 (p9), and, (3) the 3'-proximal coat protein ORF4, (p37). The genomic 5'- and 3'-proximal termini contained a short (59 nt) and a relatively longer 405 nt untranslated region, respectively. The longer replicase gene product contained the GDD motif common to RNA-dependent RNA polymerases. Phylogenetically, NLVCV formed a subgroup with the following four carmoviruses when separately comparing the amino acids of the coat protein or replicase protein: Angelonia flower break virus (AnFBV), Carnation mottle virus (CarMV), Pelargonium flower break virus (PFBV), and Saguaro cactus virus (SgCV). Whole genome nucleotide analysis (percent identities) among the carmoviruses with NLVCV suggested a similar pattern. The species demarcation criteria in the genus Carmovirus for the amino acid sequence identity of the polymerase (<52%) and coat (<41%) protein genes restricted NLVCV as a distinct species, and instead, placed it as a tentative strain of CarMV, PFBV, or SgCV when both the polymerase and CP were used as the determining factors. In contrast, the species criteria that included different host ranges with no overlap and lack of serology relatedness between NLVCV and the carmoviruses, suggested that NLVCV was a distinct species. The relatively low cutoff percentages allowed for the polymerase and CP genes to dictate the inclusion/exclusion of a distinct carmovirus species should be reevaluated. Therefore, at this time we have concluded that NLVCV should be classified as a tentative new species in the genus Carmovirus, family Tombusviridae.

Severe Acute Respiratory Syndrome Coronavirus Protein 6 Accelerates Murine Coronavirus Infections

One or more of the unique 3'-proximal open reading frames (ORFs) of the severe acute respiratory syndrome (SARS) coronavirus may encode determinants of virus virulence. A prime candidate is ORF6, which encodes a 63-amino-acid membrane-associated peptide that can dramatically increase the lethality of an otherwise attenuated JHM strain of murine coronavirus (L. Pewe, H. Zhou, J. Netland, C. Tangudu, H. Olivares, L. Shi, D. Look, T. Gallagher, and S. Perlman, J. Virol. 79:11335-11342, 2005). To discern virulence mechanisms, we compared the in vitro growth properties of rJ.6, a recombinant JHM expressing the SARS peptide, with isogenic rJ.6-KO, which has an inactive ORF containing a mutated initiation codon and a termination codon at internal position 27. The rJ.6 infections proceeded rapidly, secreting progeny about 1.5 h earlier than rJ.6-KO infections did. The rJ.6 infections were also set apart by early viral protein accumulation and by robust expansion via syncytia, a characteristic feature of JHM virus dissemination. We found no evidence for protein 6 operating at the virus entry or assembly stage, as virions from either infection were indistinguishable. Rather, protein 6 appeared to operate by fostering viral RNA and protein synthesis, as RNA quantifications by reverse transcription-quantitative PCR revealed viral RNA levels in the rJ.6 cultures that were five to eight times higher than those lacking protein 6. Furthermore, protein 6 coimmunoprecipitated with viral RNAs and colocalized on cytoplasmic vesicles with replicating viral RNAs. The SARS coronavirus encodes a novel membrane protein 6 that can accelerate replication of a related mouse virus, a property that may explain its ability to increase in vivo virus virulence.

Competitive Translation Efficiency at the Picornavirus Type 1 Internal Ribosome Entry Site Facilitated by ViralcisandtransFactors

Enteroviruses (EVs) overcome their host cells by usurping the translation machinery to benefit viral gene expression. This is accomplished through alternative translation initiation in a cap-independent manner at the viral internal ribosomal entry site (IRES). We have investigated the role of cis- and trans-acting viral factors in EV IRES translation in living cells. We observed that considerable portions of the viral genome, including the 5'-proximal open reading frame and the 3' untranslated region, contribute to stimulation of IRES-mediated translation. With the IRES in proper context, translation via internal initiation in uninfected cells is as efficient as at capped messages with short, unstructured 5' untranslated regions. IRES function is enhanced in cells infected with the EV coxsackievirus B3, but the related poliovirus has no significant stimulatory activity. This differential is due to the inherent properties of their 2A protease and is not coupled to 2A-mediated proteolytic degradation of the eukaryotic initiation factor 4G. Our results suggest that the efficiency of alternative translation initiation at EV IRESs depends on a properly configured template rather than on targeted alterations of the host cell translation machinery.

Complete nucleotide sequence and genome organization of Pelargonium flower break virus.

The complete nucleotide sequence of Pelargonium flower break virus (PFBV) has been determined. The genomic RNA is 3923 nucleotides (nt) long and contains five open reading frames (ORFs). The 5'-proximal ORF encodes a 27 kDa protein (p27) and terminates with an amber codon which may be read-through into an in-frame p56 ORF to generate a 86 kDa protein (p86) containing the viral RNA dependent-RNA polymerase motifs. Two small ORFs, located in the central part of the viral genome, encode polypeptides of 7 (p7) and 12 kDa (p12), respectively, which are very likely involved in virus movement. Interestingly, p12 presents a leucine zipper motif that has not been previously reported in related proteins. The 3'-proximal ORF encodes a 37 kDa capsid protein (CP). The p12 ORF is in-frame with the p86 ORF and a double read-through protein of 99 kDa (p99) may be produced. Amino acid sequence comparisons revealed that the proteins encoded by ORFs 2, 3 and 4 are more similar to the corresponding gene products of Carnation mottle virus than to those of other carmoviruses, whereas the p27 and the CP show higher identity with the equivalent proteins of Saguaro cactus virus. Phylogenetic analysis conducted with the different viral products confirmed the assignment of PFBV to the genus Carmovirus.

Two unrelated double-stranded RNA molecule patterns in Gremmeniella abietina type A code for putative viruses of the families Totiviridae and Partitiviridae.

Two double stranded (ds) RNA molecule patterns, probably of viral origin, were sequenced from Gremmeniella abietina var. abietina type A. The genome of Gremmeniella abietina RNA virus L1 (GaRV-L1) from isolate HR2 was 5133 bp and contained two open reading frames (ORFs). The 5'-proximal ORF coded for a putative coat protein (CP) and the 3'-proximal ORF encoded putative RNA-dependent RNA polymerase (RdRp). GaRV-L1 had sequence similarities with a previously described totivirus (Helminthosporium victoriae 190S virus) and two unclassified members of family Totiviridae ( Sphaeropsis sapinea RNA virus 1 and Sphaeropsis sapinea RNA virus 2). The genome of Gremmeniella abietina RNA virus MS1 (GaRV-MS1) from isolate C5 was composed of three dsRNA molecules coding for a putative RdRp (dsRNA1), a putative CP (dsRNA2) and protein of unknown function (dsRNA3). The lengths of these dsRNA molecules were 1782, 1586 and 1181 bp, respectively. Sequence comparisons indicated that the GaRV-MS1 dsRNA pattern comprises a putative virus that is highly similar to Discula destructiva virus 1, Discula destructiva virus 2 and Fusarium solani virus 1 of the family Partitiviridae.