Proximal ORF Research Articles

ICTV Virus Taxonomy Profile: Fusariviridae 2024.

Fusariviridae is a family of mono-segmented, positive-sense RNA viruses with genome sizes of 5.9-10.7 kb. Most genomic RNAs are bicistronic, but exceptions have up to four predicted ORFs. In bicistronic genomes, the 5'-proximal ORF codes for a single protein with both RNA-directed RNA polymerase (RdRP) and RNA helicase (Hel) domains; little is known about the protein encoded by the second ORF. Fusarivirids do not appear to form virions. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Fusariviridae, which is available at ictv.global/report/fusariviridae.

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.

Multiple Cis-acting elements modulate programmed -1 ribosomal frameshifting in Pea enation mosaic virus.

Programmed -1 ribosomal frameshifting (-1 PRF) is used by many positive-strand RNA viruses for translation of required products. Despite extensive studies, it remains unresolved how cis-elements just downstream of the recoding site promote a precise level of frameshifting. The Umbravirus Pea enation mosaic virus RNA2 expresses its RNA polymerase by -1 PRF of the 5′-proximal ORF (p33). Three hairpins located in the vicinity of the recoding site are phylogenetically conserved among Umbraviruses. The central Recoding Stimulatory Element (RSE), located downstream of the p33 termination codon, is a large hairpin with two asymmetric internal loops. Mutational analyses revealed that sequences throughout the RSE and the RSE lower stem (LS) structure are important for frameshifting. SHAPE probing of mutants indicated the presence of higher order structure, and sequences in the LS may also adapt an alternative conformation. Long-distance pairing between the RSE and a 3′ terminal hairpin was less critical when the LS structure was stabilized. A basal level of frameshifting occurring in the absence of the RSE increases to 72% of wild-type when a hairpin upstream of the slippery site is also deleted. These results suggest that suppression of frameshifting may be needed in the absence of an active RSE conformation.

The complete genome sequence of a novel mycovirus from Alternaria longipes strain HN28.

The complete nucleotide sequence of Alternaria longipes dsRNA virus 1 (AlRV1), a novel double-stranded RNA (dsRNA) mycovirus, was determined and analyzed. AlRV1-HN28 contains a single dsRNA genome segment 3415 base pairs in length (excluding the 3' poly(A) tail) and was predicted to contain two discontiguous open reading frames (ORFs, ORF A and ORF B). The 5'-proximal ORF A (1182 nt) potentially encodes a protein of 394 amino acids (aa) with a predicted molecular mass of 43kDa; this protein showed no significant similarities to any other sequences in any of the NCBI protein databases. The 3'-proximal ORF B (1737 nt) encodes a protein of 579 aa with a predicted molecular mass of 65kDa; this protein sequence shares similarities with the conserved domains of RNA-dependent RNA polymerases of other mycoviruses. Phylogenetic analysis indicated that AlRV1-HN28 was closely related to four other unclassified viruses, which suggests that the AlRV1-HN28 isolated from Alternaria longipes may belong to a new family of dsRNA mycoviruses. This is the first report of the full-length nucleotide sequence of a mycovirus that infects Alternaria longipes.

A novel virus in the family Hypoviridae from the plant pathogenic fungus Fusarium graminearum

A double-stranded (ds) RNA element, sized at approximately 13kb pairs, was purified from a field isolate, HN10, of Fusarium graminearum. The coding strand of the dsRNA was 13,023 nucleotides (nt) long (excluding the 3′ poly(A) tail) and was predicted to contain two discontiguous open reading frames (ORF A and ORF B). The 5′ proximal ORF A of 531nt encoded a protein of 176 amino acids (aa), and a BLAST search showed it to be similar to the putative papain-like protease domains encoded by Valsa ceratosperma hypovirus 1 (35% identity) and Cryphonectria hypovirus 4 (CHV4) (31% identity). The 3′ proximal ORF B of 11,118nt encoded a large polyprotein with three conserved domains, including papain-like protease, RNA-dependent RNA polymerase and RNA helicase domains. The polyprotein shared significant aa identities with CHV1 (32%) and CHV2 (32%). Both the genome organization and phylogenetic analysis suggested that the characterized RNA represented a novel hypovirus, designated “Fusarium graminearum hypovirus 1 (FgHV1)”, which was closely related to CHV1 and CHV2 in the Hypoviridae family. Elimination of the virus resulted in no dramatic phenotypic alteration of the fungus.

An Internal Ribosome Entry Site Directs Translation of the 3′-Gene from Pelargonium Flower Break Virus Genomic RNA: Implications for Infectivity

Pelargonium flower break virus (PFBV, genus Carmovirus) has a single-stranded positive-sense genomic RNA (gRNA) which contains five ORFs. The two 5′-proximal ORFs encode the replicases, two internal ORFs encode movement proteins, and the 3′-proximal ORF encodes a polypeptide (p37) which plays a dual role as capsid protein and as suppressor of RNA silencing. Like other members of family Tombusviridae, carmoviruses express ORFs that are not 5′-proximal from subgenomic RNAs. However, in one case, corresponding to Hisbiscus chlorotic ringspot virus, it has been reported that the 3′-proximal gene can be translated from the gRNA through an internal ribosome entry site (IRES). Here we show that PFBV also holds an IRES that mediates production of p37 from the gRNA, raising the question of whether this translation strategy may be conserved in the genus. The PFBV IRES was functional both in vitro and in vivo and either in the viral context or when inserted into synthetic bicistronic constructs. Through deletion and mutagenesis studies we have found that the IRES is contained within a 80 nt segment and have identified some structural traits that influence IRES function. Interestingly, mutations that diminish IRES activity strongly reduced the infectivity of the virus while the progress of the infection was favoured by mutations potentiating such activity. These results support the biological significance of the IRES-driven p37 translation and suggest that production of the silencing suppressor from the gRNA might allow the virus to early counteract the defence response of the host, thus facilitating pathogen multiplication and spread.

Plant-feeding insects harbor double-stranded RNA viruses encoding a novel proline-alanine rich protein and a polymerase distantly related to that of fungal viruses

Novel double-stranded RNAs (∼ 8 kbp) were isolated from threecornered alfalfa hopper ( Spissistilus festinus) and beet leafhopper ( Circulifer tenellus), two plant-feeding hemipteran insect pests. The two new viruses, designated Spissistilus festinus virus 1 (SpFV1) and Circulifer tenellus virus 1 (CiTV1), do not appear to be encapsidated in conventional virions and shared a genome organization similar to that of several unclassified fungal viruses. SpFV1 and CiTVl encode a proline-alanine rich protein (PArp) and an RNA-directed RNA polymerase (RdRp). Expression of the 3’-proximal RdRp ORF appears to result from -1 translational frameshifting of the PArp ORF. Phylogenetic analysis of the RdRp indicated that SpFV1 and CiTV1 were most closely related to each other and the unclassified plant virus Cucurbit yellows associated virus, and more distantly related to the unclassified fungal dsRNA viruses Phlebiopsis gigantea virus 2 and Fusarium graminearum virus 3.

Bacterial Hen1 is a 3′ terminal RNA ribose 2′-O-methyltransferase component of a bacterial RNA repair cassette

Hen1 is an RNA ribose 2'-O-methyltransferase that modifies the 3' terminal nucleoside of eukaryal small regulatory RNAs. Here, we report that Hen1 homologs are present in bacterial proteomes from eight different phyla. Bacterial Hen1 is encoded by the proximal ORF of a two-gene operon that also encodes polynucleotide kinase-phosphatase (Pnkp), an RNA repair enzyme. Purified recombinant Clostridium thermocellum Hen1 is a homodimer of a 465-amino acid polypeptide. CthHen1 catalyzes methyl transfer from AdoMet to the 3' terminal nucleoside of an RNA oligonucleotide, but is unreactive with a synonymous DNA oligonucleotide or an RNA with a single 3'-terminal deoxyribose sugar. CthHen1 is optimally active at alkaline pH and dependent on manganese. Activity is inhibited by AdoHcy and abolished by mutations D291A and D316A in the putative AdoMet-binding pocket. The C-terminal fragment, Hen1-(259-465), comprises an autonomous monomeric methyltransferase domain.

Genome sequence and molecular characterization of Homalodisca coagulata virus-1, a novel virus discovered in the glassy-winged sharpshooter (Hemiptera: Cicadellidae)

The complete nucleotide sequence of a novel single-stranded RNA virus infecting the glassy-winged sharpshooter, Homalodisca coagulata, has been determined. In silico analysis of H. coagulata virus-1 ( HoCV-1) revealed a 9321-nt polyadenylated genome encoding two large open reading frames (ORF1 and ORF2) separated by a 182-nt intergenic region (IGR). The deduced amino acid sequence of the 5′-proximal ORF (ORF1, nt 420–5807) exhibited conserved core motifs characteristic of the helicases, cysteine proteases, and RNA-dependent RNA polymerases of other insect-infecting picorna-like viruses. A structural model created using Mfold exposed a series of stem loop (SL) structures immediately preceding the second ORF which are analogous to an internal ribosome entry site (IRES), suggesting that ORF2 begins with a noncognate GCA triplet rather than the canonical AUG. This 3′ ORF2 (5990–8740) showed significant similarity to the structural proteins of members of the family Dicistroviridae, particularly those belonging to the genus Cripavirus. Evidence demonstrating relatedness of these viruses regarding genome organization, amino acid sequence similarity, and putative replication strategy substantiate inclusion of HoCV-1 into this taxonomic position.

Mechanism of ribosome shunting in Rice tungro bacilliform pararetrovirus

In plant pararetroviruses, pregenomic RNA serves both as a template for replication through reverse transcription and a polysictronic mRNA. This RNA has a complex leader sequence preceding the first large ORF. The leader contains multiple short ORFs and strong secondary structure, both inhibiting ribosome scanning. Translation on this RNA is initiated by shunting, in which scanning ribosomes bypass a large portion of the leader with the inhibitory secondary structure and short ORFs. In Cauliflower mosaic virus (CaMV), the ribosome shunting mechanism involves translation of the 5'-proximal short ORF terminating in front of the secondary structure that appears to force ribosomes to take off and resume scanning at a landing site downstream of the structure. Using two plant protoplast systems and shunt-competent wheat-germ extracts, we demonstrate that in Rice tungro bacilliform virus (RTBV) shunting also depends on the first short ORF followed by strong secondary structure. Swapping of the conserved shunt elements between CaMV and RTBV revealed the importance of nucleotide composition of the landing sequence for efficient shunting. The results suggest that the mechanism of ribosome shunting is evolutionary conserved in plant pararetroviruses.

Three unrelated viruses occur in a single isolate of Gremmeniella abietina var. abietina type A

Five enclosed double-stranded RNA (dsRNA) bands in electrophoresis, probably of viral origin, were found from a single isolate (SurS4) of Gremmeniella abietina var. abietina type A. Analysis of the dsRNAs revealed that they represented three different viruses, named as Gremmeniella abietina mitochondrial RNA virus S2 (GaMRV-S2), Gremmeniella abietina RNA virus MS2 (GaRV-MS2) and Gremmeniella abietina RNA virus L2 (GaRV-L2). The genome of GaMRV-S2 was 2587 base pairs (bp) long and had a very low GC content (31%). Sequence variations occurred at both ends. The genome coded for a putative RNA-dependent RNA polymerase (RdRp) under a mitochondrial translation code. The GaRV-MS2 genome was composed of three dsRNA molecules (1781 bp, 1586 bp and 1186 bp). They coded for a putative RdRp, a coat protein (CP) and a protein with an unknown function, respectively. The GaRV-L2 genome was 5129 bp long and contained two ORFs. The 5′-proximal ORF coded for a putative CP, whereas the 3′-proximal ORF encoded for a putative RdRp. The buoyant density of GaRV-MS2 and GaRV-L2 were 1.37 and 1.42 g/ml, respectively. GaMRV-S2, GaRV-MS2 and GaRV-L2 were closely related to the previously described viruses GaMRV-S1, GaRV-MS1 and GaRV-L1, respectively, and are putative members of the genera Mitovirus, Partitivirus and Totivirus, respectively. This is the first report on the occurrence of viruses of all these different genera in a single fungal isolate.

Development of infectious transcripts and genome manipulation of Black queen-cell virus of honey bees.

The South African isolate of Black queen-cell virus (BQCV), a honey bee virus, was previously found to have an 8550 nucleotide genome excluding the poly(A) tail. Its genome contained two ORFs, a 5'-proximal ORF encoding a putative replicase protein and a 3'-proximal ORF encoding a capsid polyprotein. Long reverse transcription (RT)-PCR was used to produce infectious transcripts for BQCV and to manipulate its genome. Primers were designed for the amplification of the complete genome, the in vitro transcription of infectious RNA and PCR-directed mutagenesis. An 18-mer antisense primer was designed for RT to produce full-length single-stranded cDNA (ss cDNA). Unpurified ss cDNA from the RT reaction mixture was used directly as a template to amplify the full genome by long high-fidelity PCR. The SP6 promoter sequence was introduced into the sense primer to transcribe RNA directly from the amplicon. RNA was transcribed in vitro with and without the presence of a cap analogue and injected directly into bee pupae, which were then incubated for 8 days. In vitro transcripts were infectious but the presence of a cap analogue did not increase the amount of virus recovered. A single base mutation abolishing an EcoRI restriction site was introduced by fusion-PCR, to distinguish viral particles recovered from infectious transcripts from wild-type virus (wtBQCV). Mutant virus (mutBQCV) and wtBQCV were indistinguishable by electron microscopy and Western blot analysis. The EcoRI restriction site was present in wtBQCV and not in mutBQCV.

The complete nucleotide sequence and development of a diferential detection assay for a pepper mild mottle virus (PMMoV) isolate that overcomes L3 resistance in pepper

The complete nucleotide sequence of the RNA genome of a Pepper Mild Mottle Virus (PMMoV) isolate that overcomes L3 resistance in pepper ( Capsicum sp.) was determined and compared with the sequence of other Tobamoviruses. The RNA genome consists of 6357 nucleotides and contains four open reading frames. The 5′ proximal ORF encodes a 128 kDa product that terminates in an amber codon which may be readthrough to produce a 180 kDa replication-associated protein (ORF 2). ORF 3 codes for the 28 kDa protein assumed to be involved in cell to cell spread of the virus. The last ORF encodes the coat protein (CP). Amino acid sequence comparison of the CP of this and other PMMoV isolates showed the same substitution (Met to Asn) as found in the Italian isolate of PMMoV and which is assumed to be responsible for L3-resistance breaking. RT-PCR using a common primer pair for PMMoV followed by restriction enzyme analysis with EcoRI allowed the discrimination of resistance breaking from non-L3 resistance breaking virus isolates.

Rubella virus DI RNAs and replicons: requirement for nonstructural proteins acting in cis for amplification by helper virus.

A rubella virus (RUB) replicon was constructed by replacing the 3' proximal structural protein ORF (SP-ORF) in Robo402, a RUB infectious cDNA clone, with a reporter gene, green fluorescent protein (GFP). This replicon, RUBrep/GFP, mimics naturally occurring RUB defective-interfering (DI) RNAs generated during serial undiluted passage that maintain the 5' proximal nonstructural protein ORF (NS-ORF) but contain deletions in the SP-ORF. Following transfection of Vero cells with in vitro RNA transcripts from RUBrep/GFP, replicon replication occurred and the replicon was amplified and spread to other cells in the presence of standard helper virus. GFP expression was a much more sensitive indicator of replicon replication than was Northern analysis to detect replicon-specific RNAs. Most of a series of RUBrep/GFP constructs with deletions in the NS-ORF not only were incapable of self-replication, but were not amplified by standard helper virus. The only exception was a construct with an in-frame deletion between two NotI sites that removed nucleotides 1685-2192 of the genome; this construct did not express GFP by itself, but did express GFP in the presence of standard helper RUB and was spread to other cells. Thus, with the exception of this region, the NS-ORF is required in cis for amplification of RUB replicons by standard helper virus, explaining the selection of DI RNAs that maintain the NS-ORF. Surprisingly, when the NotI deletion was introduced into Robo402, a viable virus resulted that replicated only threefold less efficiently than did Robo402 virus. Thus, the NotI region of the NS-ORF is not necessary for virus replication. This deletion covers a region of the NS-ORF without predicted function, which therefore may function as a spacer or hinge between functional domains. Nevertheless, it was an unexpected finding that a small virus such as RUB could dispense with approximately 10% of its genome.

Additional organizational features of the murine γ-glutamyl hydrolase gene: Two remotely situated exons within the complement C3 gene locus encode an alternate 5′ end and proximal ORF under the control of a bidirectional promoter

Analysis of independently isolated clones from a mouse liver cDNA library identified a splice variant of γ-GH mRNA with novel nucleotide sequence at the 5′ end. Genomic sequencing now shows that this variant (variant II) incorporates two new alternates (exons Bla and Blb) of exon 1 in the murine γ-GH gene remotely situated with respect to the rest of the gene. Further analysis of this variant also showed that it incorporates a small segment at the 3′ end of exon A1, revealing that the previously described exon 1 consists of two individual exons (Ala and Alb) joined at a cryptic splice site. The 5′ UTR and a segment of the ORF of variant II results from splicing of exon Bla to exon Blb which in turn is spliced to exon Alb and through this splicing to the rest of the exons within this gene. Remarkably, this splicing occurs even though exons Bla and Blb are located >45 Kb upstream of exons Ala and Alb. Our results also show that transcription starting at exons Bla and Blb is under the control of a separate and bidirectional promoter (promoter B). Exons Bla and Blb are located on the sense DNA strand within the complement C3 gene locus which is encoded on the antisense strand. This promoter is less efficient than the downstream promoter (promoter A) in regulating transcription at least in the context of reporter gene and primer extension assays. However, in these same contexts, this region of DNA sequence in the reverse orientation is markedly more efficient in driving transcription of an unidentified gene. Deletion of specific regions of sequence within this promoter have different effects depending upon the orientation (forward or reverse) within the reporter gene construct.

Nucleotide sequence of a Dianthovirus RNA1-like RNA found in grassy stunt-diseased rice plants.

A Dianthovirus RNA1-like RNA (DR1L RNA, 4486 nucleotides in length) was found in grassy stunt-diseased rice plants together with Rice grassy stunt virus (RGSV). DR1L RNA has characteristics common to Dianthovirus RNA1 such as (1) presence of a GGAUUUUUAG potential shifty-heptanucleotide at the end of the 5'-proximal ORF, which encodes a 35-kDa protein, followed by a 77-nucleotide sequence capable of forming a stem-loop structure for an efficient--1 frameshift to express the downstream region in a 96-kDa putative replicase protein, (2) presence of nearly identical 17-nucleotide sequences in the 5'-terminal region and in a region upstream of an ORF encoding a 28-kDa, putative capsid protein (CP), and (3) near identity of the 3'-terminal 20 nucleotides to those of Dianthovirus RNAs. Western blot analysis using an antiserum against the C-terminal domain of the putative CP and RT-PCR analysis using primers specific to DR1L RNA of fractions after sucrose density gradient centrifugation of RGSV nucleoproteins indicated that DR1L RNA is associated with the 28-kDa putative CP but not with the 36-kDa RGSV CP. Two additional ORFs for 15-kDa and 33-kDa proteins were present in DR1L RNA although their expression in plants and functions are not known.

Ribosome shunt is essential for infectivity of cauliflower mosaic virus.

Cauliflower mosaic virus (CaMV) is a DNA-containing pararetrovirus replicating by means of reverse transcription of a terminally redundant pregenomic 35S RNA that is also used as a polycistronic mRNA. The leader of 35S RNA is long, highly structured, and contains multiple short ORFs (sORFs), which strongly interfere with the ribosome scanning process. Translation of this RNA is initiated by a ribosome shunt mechanism, in which ribosomes translate the most 5'-proximal short ORF (sORF A), then skip a large region of the leader containing a putative RNA encapsidation signal and reinitiate translation at the first long viral ORF. Here, we demonstrate that the efficiency of the sORF A-mediated ribosome shunt is an important determinant of viral infectivity. Point mutations in sORF A, which reduced the basal level of shunt-dependent expression and the degree of shunt enhancement by a CaMV-encoded translation transactivator (TAV), consequently reduced infectivity of the virus in turnip plants. First- or second-site reversions appeared in the viral progeny. The second-site reversions restored shuntdependent expression to an extent correlating with their relative abundance in the progeny. Mutations that abolished both the basal and TAV-activated components of shunting proved to be lethal. Finally, by using an artificial stem structure that blocks scanning, we obtained direct evidence that ribosome shunt operates during CaMV infection.

Analysis of the complete genome sequence of black queen-cell virus, a picorna-like virus of honey bees.

A virus with picorna-like biophysical properties was isolated from South African honey bees. On the basis of serology, it was identified as an isolate of black queen-cell virus (BQCV). Nucleotide sequence analysis revealed an 8550 nt polyadenylated genome containing two large ORFs. The 5'-proximal ORF (ORF 1) represented 4968 nt while the 3'-proximal ORF (ORF 2) represented 2562 nt. The ORFs were separated by a 208 nt intergenic region and were flanked by a 657 nt 5'-untranslated region and a 155 nt 3'-untranslated region. Deduced amino acid sequences for ORF 1 and ORF 2 were most similar to the non-structural and structural proteins, respectively, of Drosophila C virus (DCV), Rhopalosiphum padi virus (RhPV), Himetobi P virus (HiPV) and Plautia stali intestine virus (PSIV). It is proposed that BQCV belongs to the group of picorna-like, insect-infecting RNA viruses constituted by DCV, RhPV, HiPV and PSIV.

Ribosome shunting in the cauliflower mosaic virus 35S RNA leader is a special case of reinitiation of translation functioning in plant and animal systems

The shunt model predicts that small ORFs (sORFs) within the cauliflower mosaic virus (CaMV) 35S RNA leader and downstream ORF VII are translated by different mechanisms, that is, scanning-reinitiation and shunting, respectively. Wheat germ extract (WGE) and rabbit reticulocyte lysate (RRL) in vitro translation systems were used to discriminate between these two processes and to study the mechanism of ribosomal shunt. In both systems, expression downstream of the leader occurred via ribosomal shunt under the control of a stable stem and a small ORF preceding it. Shunting ribosomes were also able to initiate quite efficiently at non-AUG start codons just downstream of the shunt landing site in WGE but not in RRL. The short sORF MAGDIS from the mammalian AdoMetDC RNA, which conditionally suppresses reinitiation at a downstream ORF, prevented shunting if placed at the position of sORF A, the 5'-proximal ORF of the CaMV leader. We have demonstrated directly that sORF A is translated and that proper termination of translation at the 5'-proximal ORF is absolutely required for both shunting and linear ribosome migration. These findings strongly indicate that shunting is a special case of reinitiation.

Characterization and in vitro translation analysis of pelargonium flower break virus.

Pelargonium flower break virus (PFBV) is one of the common viruses in the glasshouses of Western Europe and has been assigned to the genus Carmovirus. A Spanish isolate obtained from nursery-grown Pelargonium zonale plants (PFBV-m) has been characterized. The molecular weight of genomic RNA and coat protein of PFBV-m were determined to be 1.36 x 10(6) (corresponding to approximately 4 kb) and 36,000, respectively. Only genomic-size RNA was encapsidated in PFBV virions; making necessary to purify double-stranded RNA from infected tissue in order to detect putative PFBV subgenomic RNAs. PFBV RNA directed the synthesis of a major polypeptide of 34 kDa and three other relevant polypeptides of estimated sizes 88-90 kDa, 42 kDa and 35-36 kDa. Antisera specific to PFBV immunoprecipitated the in vitro translated 35-36 kDa polypeptide indicating that this polypeptide is the PFBV coat protein. The PFBV in vitro translation pattern was very similar to that of CarMV although the relative levels of translated coat protein differed dramatically between the two viruses, most probably due to the lack of encapsidation of subgenomic PFBV. In vitro translation studies with a different biological clone obtained from the same PFBV-m isolate revealed a prominent additional polypeptide which is postulated to be a truncation of the 5' proximal ORF.