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PubMed2006

Sequence analysis and organization of the Neodiprion abietis nucleopolyhedrovirus genome.

Of 30 baculovirus genomes that have been sequenced to date, the only nonlepidopteran baculoviruses include the dipteran Culex nigripalpus nucleopolyhedrovirus and two hymenopteran nucleopolyhedroviruses that infect the sawflies Neodiprion lecontei (NeleNPV) and Neodiprion sertifer (NeseNPV). This study provides a complete sequence and genome analysis of the nucleopolyhedrovirus that infects the balsam fir sawfly Neodiprion abietis (Hymenoptera, Symphyta, Diprionidae). The N. abietis nucleopolyhedrovirus (NeabNPV) is 84,264 bp in size, with a G+C content of 33.5%, and contains 93 predicted open reading frames (ORFs). Eleven predicted ORFs are unique to this baculovirus, 10 ORFs have a putative sequence homologue in the NeleNPV genome but not the NeseNPV genome, and 1 ORF (neab53) has a putative sequence homologue in the NeseNPV genome but not the NeleNPV genome. Specific repeat sequences are coincident with major genome rearrangements that distinguish NeabNPV and NeleNPV. Genes associated with these repeat regions encode a common amino acid motif, suggesting that they are a family of repeated contiguous gene clusters. Lepidopteran baculoviruses, similarly, have a family of repeated genes called the bro gene family. However, there is no significant sequence similarity between the NeabNPV and bro genes. Homologues of early-expressed genes such as ie-1 and lef-3 were absent in NeabNPV, as they are in the previously sequenced hymenopteran baculoviruses. Analyses of ORF upstream sequences identified potential temporally distinct genes on the basis of putative promoter elements.

Amino Acid Motifs↗

PubMed1995

Translational inhibition by a human cytomegalovirus upstream open reading frame despite inefficient utilization of its AUG codon.

The second of three short upstream open reading frames (uORF2) in the transcript leader of the human cytomegalovirus gp48 (gpUL4) virion glycoprotein gene inhibits downstream translation approximately 10-fold. Remarkably, this inhibition depends on the amino acid coding information of uORF2. In the current studies we demonstrate that expression of the cistron downstream from uORF2 depends on ribosomes bypassing the uORF2 AUG codon (AUG2) by a leaky scanning mechanism. Replacing the nucleotides surrounding the wild-type AUG2 codon with those optimal for translation initiation reduces downstream translation approximately 10-fold. Analyses of mutants in which uORF2 either overlaps or is in frame with the downstream reading frame reveal that the initiation frequency at the wild-type AUG2 codon is surprisingly low; rather, the majority of ribosomal subunits bypass the wild-type AUG2 codon because of its suboptimal context. We propose a model to explain this unprecedented example of a paradoxically strong inhibitory effect of an upstream ORF despite inefficient utilization of its initiation codon.

Base Sequence↗

PubMed1995

Analysis of human papillomavirus type 16 E5 oncogene expression in vitro and from bicistronic messenger RNAs.

During a human papillomavirus infection (HPV), a set of spliced messenger RNAs (mRNA) are produced from the coding strand of the 8-kilobase virus DNA genome. These mRNAs have been isolated from cervical intraepithelial neoplasia biopsies, tumor biopsies and HPV type 16 (HPV-16)-containing cell lines, and subsequent analyses have shown that the different mRNAs are obtained by often complex splicing events. In the present study we have investigated the coding capacity of the HPV-16 E5 open reading frame (ORF) in an in vitro translation system and by the expression of a HPV-16 E5-CAT fusion gene from bicistronic messenger RNAs derived from polycistronic mRNAs which have previously been identified by others. Our results show that the HPV-16 E5 protein synthesis can only be initiated from the authentic ATG codon in the presence of E2 ORF translation. The E5 gene is found to be expressed only from a bicistronic mRNA with an E2 ORF upstream of the E5 gene indicating that the E5 protein is synthesized coordinated with the E2 protein. The supposition that the E5 protein is synthesized early in infection is discussed.

3T3 Cells↗

PubMed2006

Significant expansion of Vicia pannonica genome size mediated by amplification of a single type of giant retroelement.

Amplification and eventual elimination of dispersed repeats, especially those of the retroelement origin, account for most of the profound size variability observed among plant genomes. In most higher plants investigated so far, differential accumulation of various families of elements contributes to these differences. Here we report the identification of giant Ty3/gypsy-like retrotransposons from the legume plant Vicia pannonica, which alone make up approximately 38% of the genome of this species. These retrotransposons have structural features of the Ogre elements previously identified in the genomes of pea and Medicago. These features include extreme size (25 kb), the presence of an extra ORF upstream of the gag-pol region, and a putative intron dividing the prot and rt coding sequences. The Ogre elements are evenly dispersed on V. pannonica chromosomes except for terminal regions containing satellite repeats, their individual copies show extraordinary sequence similarity, and at least part of them are transcriptionally active, which suggests their recent amplification. Similar elements were also detected in several other Vicia species but in most cases in significantly lower numbers. However, there was no obvious correlation of the abundance of Ogre sequences with the genome size of these species.

Base Sequence↗

PubMed2000

The ram-dependence of Streptomyces lividans differentiation is bypassed by copper.

The onset of morphological differentiation in Streptomyces lividans is intrinsically delayed in comparison to Streptomyces coelicolor, but can be advanced by adding extra copper to the medium. Copper-specific chelators block aerial hyphae formation in both strains illustrating the crucial role of copper in morphogenesis. The S. coelicolor ram cluster was isolated as a clone that complements the copper-dependent differentiation of S. lividans. The S. lividans ram cluster was cloned and shown to be 99.6% identical to the S. coelicolor clone. The difference in development between S. lividans and S. coelicolor could neither be related to functional differences between the two ram clusters nor to differences in the transcription level. In both strains the low level of ramAB transcription correlated with aerial mycelium formation and was coupled to the upstream ORF ramS. An increased ramAB expression level in S. lividans by the introduction of an extra copy of ram stimulated the development. In S. lividans disruption of ramABR resulted in the inability to produce aerial hyphae. Conversely, the identical mutant of S. coelicolor retained its developmental capacities, indicating the presence of a ram-independent developmental route that is not present or not activated in S. lividans. Aerial hyphae and spore formation in the S. lividans ramABR mutant was restored when grown near wild-type strains, suggesting that the ram gene products are involved in transport of a factor essential for normal development. In addition, an elevated copper concentration in the medium also relieved the developmental block of these mutants. These findings suggest that higher copper concentrations render this ram-associated factor obsolete.

ATP-Binding Cassette Transporters↗

PubMed2005

A role for poly(dA)poly(dT) tracts in directing activity of the Plasmodium falciparum calmodulin gene promoter.

Expression of the Plasmodium falciparum calmodulin gene (pfcam) is developmentally regulated throughout the blood-stage cycle. The promoter lies within approximately 1 kb of intergenic sequence that separates the pfcam open reading frame (ORF) from an upstream inverted ORF encoding a product homologous to the co-chaperone STI1. Using the oligo-capping method, which selectively reverse-transcribes cDNA from only full-length, capped transcript, we have mapped multiple transcription-initiation sites for both genes. Transcription of the pfSTI1 gene initiates over a 150 bp region centred approximately 350 bp upstream of the ORF. The pfcam transcription start sites cluster into four approximately 30 bp regions lying within 180 bp upstream of the pfcam ORF, generating transcripts with 5' untranslated regions (UTR) of 3-173 nucleotides in length. Remarkably, splicing was found to be related to UTR length, with apparent preferential splicing of longer transcripts. Activity of the pfcam promoter diminished in a linear fashion to undetectable levels upon step-wise removal of sequence between 625 and 230 bp upstream of the start ATG. Electromobility-shift assays demonstrated nuclear factor binding to eight oligonucleotide probes spanning 657 bp of the pfcam ORF proximal upstream sequence. The degree of binding correlated with the density of poly(dA)poly(dT) tracts within the probes, and in all cases could be inhibited by excess synthetic poly(dA)poly(dT), but not by poly(dAdT)poly(dAdT). The multiple transcription-initiation sites of both pfSTI1 and pfcam genes lie just downstream of 25 bp-long poly(dA)poly(dT) tracts, and the intergenic region contains over 20 poly(dA)poly(dT) tracts of 4 bp or more. Our results suggest that the basal pfcam promoter is situated between approximately -300 and -230 bp upstream of the pfcam ORF and that the P. falciparum transcription-initiation complex has a low degree of sequence-specificity for the sites of initiation but preferentially acts downstream of long poly(dA)poly(dT) tracts.

5' Untranslated Regions↗

PubMed2006

The dicistronic RNA from the mouse LINE-1 retrotransposon contains an internal ribosome entry site upstream of each ORF: implications for retrotransposition.

Most eukaryotic mRNAs are monocistronic and translated by cap-dependent initiation. LINE-1 RNA is exceptional because it is naturally dicistronic, encoding two proteins essential for retrotransposition, ORF1p and ORF2p. Here, we show that sequences upstream of ORF1 and ORF2 in mouse L1 function as internal ribosome entry sites (IRESes). Deletion analysis of the ORF1 IRES indicates that RNA structure is critical for its function. Conversely, the ORF2 IRES localizes to 53 nt near the 3' end of ORF1, and appears to depend upon sequence rather than structure. The 40 nt intergenic region (IGR) is not essential for ORF2 IRES function or retrotransposition. Because of strong cis-preference for both proteins during L1 retrotransposition, correct stoichiometry of the two proteins can only be achieved post-transcriptionally. Although the precise stoichiometry is unknown, the retrotransposition intermediate likely contains hundreds of ORF1ps for every ORF2p, together with one L1 RNA. IRES-mediated translation initiation is a well-established mechanism of message-specific regulation, hence, unique mechanisms for the recognition and control of these two IRESes in the L1 RNA could explain differences in translational efficiency of ORF1 and ORF2. In addition, translational regulation may provide an additional layer of control on L1 retrotransposition efficiency, thereby protecting the integrity of the genome.

Animals↗

PubMed1994

Cloning and sequencing of a Bordetella pertussis serum resistance locus.

We have characterized a new virulence factor in Bordetella pertussis: serum resistance. Compared with Escherichia coli HB101, wild-type B. pertussis was relatively resistant to classical-pathway, complement-dependent killing by normal human serum. However, a mutant of B. pertussis (BPM2041) which is less virulent in mice and which has Tn5 lac inserted in a previously uncharacterized bvg-regulated gene was found to be at least 10-fold more susceptible to serum killing than the wild type. We have named this locus brk, for Bordetella resistance to killing. We have cloned and sequenced the brk locus, and it encodes two divergently transcribed open reading frames (ORFs), termed BrkA and BrkB. Both ORFs are necessary for serum resistance. Within the 300 bases which separate the two ORFs and upstream of each ORF are putative sites for BvgA binding. BrkA shows 29% identity to pertactin and has two RGD motifs in addition to a conserved proteolytic processing site and an outer membrane targeting signal. Like pertactin, BrkA is involved in adherence and invasion. Despite the similarities, a pertactin mutant was found to be not as sensitive to serum killing as the BrkA or BrkB mutants. BrkB is similar to ORFs in E. coli and Mycobacterium leprae and displays domains of homology to various transporters. On the basis of its hydropathy profile, BrkB is predicted to be a cytoplasmic membrane protein. By Southern blot, brk sequences were found in Bordetella bronchiseptica and Bordetella parapertussis but not in Bordetella avium.

Amino Acid Sequence↗

PubMed1987

Identification and mapping of human papillomavirus type 1 RNA transcripts recovered from plantar warts and infected epithelial cell cultures.

Multiple spliced transcripts of human papillomavirus type 1 were detected by electron microscopic analysis of R-loops formed with total RNA extracted from plantar warts and with poly(A)+ RNA isolated from cultured keratinocytes infected with human papillomavirus type 1. The 5' ends of the RNAs were mapped to sites in the E7 open reading frame (ORF), just upstream of the E6 ORF and in the upstream regulatory region. Species with 5' ends in E7 accounted for over 95% of all transcripts seen. Two polyadenylation sites were used, one at the end of the early (E) region of the viral DNA, the other at the end of the late (L) region. The most abundant species had a short 5' exon of approximately 100 nucleotides spanning the junction of the E7 and E1 ORFs spliced to a 3' exon of 800 nucleotides in the region with overlapping E2 and E4 ORFs; it was polyadenylated at the end of the E region. This species probably encodes the abundant E4 protein found in plantar warts (F. Breitburd, O. Croissant, and G. Orth, Cancer Cells, vol. 5, in press; J. Doorbar, D. Campbell, R. J. A. Grand, and P. H. Gallimore, EMBO J. 5:355-362, 1986). Other transcripts had exons spanning the E6-E7 ORFs, the E4-E5-L2-L1 ORFs, or the L1 ORF. The infrequent L1 transcript, probably the mRNA coding for the major capsid protein, had the same 5' exon in E7 as the abundant mRNA spliced from E1 and E4 ORFs, suggesting genetic regulation via the choice of the alternative polyadenylation sites or mRNA processing.

Base Sequence↗

PubMed2001

[lambda N gene expression regulated by the leader sequence].

It was reported recently that changing the TIR (translational initiation region) of lambda N gene resulted in the increasing expression of lambda N gene and it was regulated at translational level. According to the alignment, the leader sequence of lambda N gene had three parts: a code region for ORF lambda N, the upstream sequence of ORF lambda N and 17 bp of spacer between ORF lambda N and downstream of lambda N gene. ORF lambda N is an open reading frame located at upstream of lambda N gene coding a peptide of 19 amino acids. To study the mechanism of regulation of lambda N gene expression, three serials of plasmids with mutant leader region of lambda N gene were constructed. (1) pMC1403-XT, in which the start codon or the partial code of ORF lambda N was connected with lacZ to obtain the ORF lambda N-lacZ fusion gene and in which the ORF lambda N-lacZ expression was under the control of a strong trp/lac promoter; (2) The ORF lambda N mutants in which the termination codon TAA was introduced into ORF lambda N at different positions by site-directed mutagenesis to preterminate the ORF lambda N on plasmid pMC1403N; (3) Mutants in which a deletion was located at upstream ORF lambda N in the ORF lambda N mutants above. The results obtained from determination of the beta-galactosidase activity in the transformants harboring the different plasmids showed that the ORF lambda N-lacZ expression was suppressed by the ORF lambda N code region and the lambda N expression was increased in both the second and third serials of mutants. At the same time the results from RNA-DNA dot hybridization showed that the lambda N gene expression was regulated at translational level. Therefore it was predicted that the reason of relatively low expression for lambda N gene in pMC1403N was due to the low efficiency of ORF lambda N translation. There are two ways to increase the expression of lambda N gene. One is to preterminate the translation of ORF lambda N at a suitable position to decrease the ORF lambda N effects on downstream lambda N gene translation; the other is to change the upstream sequence of ORF lambda N to improve its translation efficiency.

5' Untranslated Regions↗

PubMed1993

Biochemical and genetic analysis of the unidirectional theta replication of the S. agalactiae plasmid pIP501.

pIP501 is a broad-host-range plasmid originating from Streptococcus agalactiae. In this report we show that (i) it replicates by a theta mechanism initiating at the 3' end of the gene encoding the replication protein RepR and progressing in the direction of transcription of this gene; (ii) its replication origin lies within or a few nucleotides downstream from ORF R and not upstream from it as suggested in the literature (Brantl et al. (1990) Nucleic Acids Res. 18, 4783-4789); (iii) the RepR protein positively regulates pIP501 copy number; and (vi) the main function ensured by the sequences located upstream from ORF R is to express this ORF. Since the replication properties of pIP501 are indistinguishable from those of the highly related Enterococcus faecalis plasmid pAM beta 1, we conclude that these elements form the first family of theta replicating plasmids in gram-positive bacteria. Based on sequence similarities, we extend this family to the S. pyogenes plasmid pSM19035 and to 12 other plasmids isolated from streptococci or enterococci.

Bacterial Proteins↗

PubMed2001

Inhibition of corticotropin releasing hormone type-1 receptor translation by an upstream AUG triplet in the 5' untranslated region.

The influence of an upstream open reading frame (ORF) in the 5'-untranslated region (UTR) of the mRNA on corticotropin-releasing hormone receptor type 1 (CRHR1) translation was studied in constructs containing the 5'-UTR of CRHR1, with or without an ATG-to-ATA mutation in the upstream ORF, and the main ORF of luciferase or CRHR1. Upstream mutation in luciferase constructs increased luciferase activity when transfected into COS-7 or AtT20 cells compared with the native 5'-UTR. Transfection of CRHR1 constructs containing the upstream mutation into AtT20 or LVIP2.0zc reporter cells, resulted in higher (125)I-Tyr-oCRH binding and corticotropin-releasing hormone-stimulated cAMP production, without changes in CRHR1 mRNA levels (measured by RNase protection assay). In vitro translation of luciferase or CRHR constructs with or without mutation of the upstream ATG, and Western blot analysis with anti-luciferase and anti-CRHR1 antibodies confirmed that mutation of the upstream ATG increases translation of the main ORF. The mechanism by which the upstream ORF inhibits translation may involve translation of the upstream peptide, because in vitro translation, or transfection into LVIP2.0zc cells of a fusion construct of the upstream ORF and green fluorescent protein (GFP) yielded a band consistent with the molecular size of GFP protein. The study shows that the upstream AUG in 5'-UTR of CRHR1 mRNA inhibits receptor expression by inhibiting mRNA translation and suggests the short open reading frame in the 5'-UTR plays a role in regulating translation of the CRH receptor.

5' Untranslated Regions↗

PubMed1991

Putative 65 kDa protein of beet yellows closterovirus is a homologue of HSP70 heat shock proteins.

A portion of the RNA genome of beet yellows closterovirus (BYV) has been sequenced encompassing a complete long open reading frame (ORF) potentially encoding a 65 kDa protein. The sequence of this putative protein was strikingly similar to those of HSP70-related heat shock proteins. The counterparts of all the eight segments strongly conserved in HSP70s could be confidently identified in the BYV 65 kDa protein. It is suggested that some of these segments might be the ATP-binding site(s) and that, similarly to the heat shock proteins, the 65 kDa is probably ATP-binding. Generally, however, the divergence between the 65 kDa sequence and the sequences of the HSP70s was much more pronounced than that between any two members of the latter family, allowing a clearer delineation of clusters of conserved residues that might be crucial for protein function. It is suggested that these observations will be helpful in functional dissection of the proteins of the HSP70 family. Analysis of the sequence of a portion of the ORF found upstream from the 65 kDa ORF showed that the C-terminal domain of the encoded protein could be an RNA-dependent RNA polymerase closely related to those of tricornaviruses, a family of RNA plant viruses with three component genomes.

Adenosine Triphosphate↗

PubMed2004

Regulation of vasopressin V1b receptors and stress adaptation.

Vasopressin (VP) regulates pituitary corticotroph function by acting upon plasma membrane G-protein receptors of the V1b subtype (V1bR), coupled to calcium-phospholipid signaling. The number of these receptors in the anterior pituitary varies during stress in direct correlation with corticotroph responsiveness, suggesting that the V1bR plays an important role during adaptation of the hypothalamic-pituitary-adrenal (HPA) axis to stress. The molecular regulation of pituitary V1bR involves transcriptional and translational mechanisms. V1bR gene transcription, which is necessary to maintain V1bR mRNA levels, depends on a number of responsive elements in the promoter region, of which the stretch of GA repeats near the transcription start point (GAGA box) is essential. Although transcriptional activation is necessary to maintain V1bR mRNA levels, the lack of correlation between VP binding and V1bR mRNA suggests that V1bR content is mainly regulated at the translational level. Two potential mechanisms by which the 5' untranslated region (5'UTR) of the V1bR mediates negative and positive regulation of V1bR translation were identified. This includes the repressor effect of small open reading frames (ORF) present upstream of the main V1bR ORF, and an internal ribosome entry site (IRES), which activates V1bR translation. The existence of multiple loci of regulation for the V1bR at transcriptional and translational levels provides a mechanism to facilitate plasticity of regulation of the number of pituitary vasopressin receptors according to physiological demand.

Adaptation, Physiological↗

PubMed1997

Translation of an uncapped mRNA involves scanning.

tat, an essential gene of human immunodeficiency virus, when placed under the control of the RNA polymerase III promoter from the adenovirus VA RNA1 gene, is transcribed into an uncapped and nonpolyadenylated mRNA. This VA-Tat RNA is translated to produce functional Tat protein in transfected mammalian cells (Gunnery, S., and Mathews, M. B. (1995) Mol. Cell. Biol. 15, 3597-3607). The presence of an upstream open reading frame (ORF) in VA-Tat RNA is inhibitory to the translation of the Tat ORF, suggesting that the RNA is scanned during translation even though it is uncapped. Because the effect of the upstream ORF is relatively small (about 2-fold), we sought more definitive evidence of scanning by introducing secondary structures of varying stabilities into the 5'-untranslated region of VA-Tat RNA. The results of transfection experiments showed that highly stable secondary structure was inhibitory to Tat synthesis, whereas structures of lower stability were not inhibitory, confirming that uncapped mRNA is subject to scanning. Furthermore, translation of the downstream ORF was reduced but not eliminated by mutations that caused the upstream ORF to overlap the Tat ORF. Extending the overlap of the two ORFs further decreased the translation of the downstream ORF. This observation implies that ribosomes reinitiate after termination, possibly after migrating in a 3' to 5' direction through the overlap region of the mRNA. Similar results were obtained with a capped polymerase II transcript, indicating that the translation of polymerase II and polymerase III transcripts occurs through similar mechanisms.

Genes, tat↗

PubMed2002

The human herpesvirus-8 (Kaposi's sarcoma-associated herpesvirus) ORF 40/41 region encodes two distinct transcripts.

The human herpesvirus-8 (HHV-8) locus encoding ORFs 40/41 is a candidate homologue for the Epstein-Barr virus BBLF 2/3 gene, which encodes the putative primase-associated factor. Northern blot data revealed that two transcripts originated from the HHV-8 ORF 40/41 region. The sizes of these transcripts (2.2 and 0.7 kb) suggested that one transcript was the result of a spliced form of ORFs 40 and 41 and the second transcript originated from a region within ORF 41. cDNA sequence and 5' RACE analysis revealed the removal of an intron between ORFs 40 and 41 and a transcriptional start site 82 nt upstream of ORF 40. The start of transcription for the smaller transcript was mapped to within ORF 41. Regions upstream of the transcriptional start sites were subcloned into a luciferase reporter vector, and transient luciferase assays indicated that distinct promoters drive the expression of each transcript.

Animals↗

PubMed1996

Genomic cloning and characterization of the nonoccupied allele corresponding to the integration site of human papillomavirus type 16 DNA in the cervical cancer cell line SiHa.

Human papillomavirus (HPV) type 16 DNA sequences have been found integrated into the host cell genome in a large number of cervical tumors and cell lines derived therefrom. In this study, we have cloned and analyzed the nonoccupied allele corresponding to the integration site of HPV-16 in the cervical cancer cell line SiHa. Our mapping analyses revealed an approximately 7.8-kb deletion of cellular DNA upon viral integration. Computer analysis of 2.3 kb of DNA sequences from the deleted genomic region as well as 1.0 kb of sequences upstream of the viral integration site showed no significant homology to any known human sequences. DNase I mapping experiments on native chromatin demonstrated the existence of two hypersensitive sites in both the HPV-16-containing and nonoccupied alleles located approximately 1.1 and 1.7 kb upstream of the viral integration site. This suggests that viral integration occurred close to putative regulatory sequences and that recombination with host cellular DNA was not followed by a reorganization of the chromatin structure upstream of the integration site. Nuclear run-on and RT-PCR experiments showed HPV-specific transcription spanning the E2, E4, E5, and L1/L2 open reading frames (ORFs) located upstream of the HPV-16 regulatory region (URR). Taken together, our data suggest that the cellular DNA region upstream of the HPV-16 integration site in the SiHa cell line contains regulatory elements affecting transcription of HPV-16 ORFs located upstream of the HPV-16 URR.

Alleles↗

PubMed1993

Transcriptional analysis of the virion-sense genes of the geminivirus beet curly top virus.

The genome of the geminivirus beet curly top virus (BCTV) consists of a single circular DNA containing overlapping open reading frames (ORFs) located on both the virion-sense and complementary-sense DNA strands. To investigate the expression of these ORFs, RNA extracted from infected Nicotiana benthamiana and Beta vulgaris has been examined for the presence of viral transcripts. An abundant 1.1-kb virion-sense polyadenylated RNA and four complementary-sense polyadenylated RNAs of 1.7, 1.5, 1.3, and 0.7 kb have been identified by northern blot hybridization, confirming the bidirectional transcription strategy implied by the arrangement of ORFs. We previously demonstrated that two overlapping virion-sense ORFs are involved in coat protein synthesis (ORF V1) and viral single-stranded DNA accumulation (ORF V2). Mutants of a third virion-sense ORF (ORF V3), located upstream and overlapping ORFs V1 and V2, retain the ability to replicate efficiently in N. benthamiana leaf discs but produce an asymptomatic infection in N. benthamiana and B. vulgaris at low frequency, associated with reduced levels of viral DNA compared to wild-type infection. Our data support the recent suggestion that ORF V3 participates in virus movement. The 1.1 kb virion-sense RNA comprises a population of overlapping transcripts with 5' termini suitably positioned for the expression of ORFs V1, V2, and V3. The overlapping arrangement of the transcripts and juxtaposition of putative regulatory elements could provide a means for the temporal control of virion-sense gene expression.

Amino Acid Sequence↗