Translation Of ORF1 Research Articles

Amalga-like virus infecting Antonospora locustae, a microsporidian pathogen of grasshoppers, plus related viruses associated with other arthropods

A previously reported Expressed Sequence Tag (EST) library from spores of microsporidian Antonospora locustae includes a number of clones with sequence similarities to plant amalgaviruses. Reexamining the sequence accessions from that library, we found additional such clones, contributing to a 3247-nt contig that approximates the length of an amalga-like virus genome. Using A. locustae spores stored from that previous study, and new ones obtained from the same source, we newly visualized the putative dsRNA genome of this virus and obtained amplicons yielding a 3387-nt complete genome sequence. Phylogenetic analyses suggested it as prototype strain of a new genus in family Amalgaviridae. The genome contains two partially overlapping long ORFs, with downstream ORF2 in the +1 frame relative to ORF1 and a proposed motif for +1 ribosomal frameshifting in the region of overlap. Subsequent database searches using the predicted fusion protein sequence of this new amalga-like virus identified related sequences in the transcriptome of a basal hexapod, the springtail species Tetrodontophora bielanensis. We speculate that this second new amalga-like virus (contig length, 3475 nt) likely also derived from a microsporidian, or related organism, which was associated with the springtail specimens at the time of sampling for transcriptome analysis. Other findings of interest include evidence that the ORF1 translation products of these two new amalga-like viruses contain a central region of predicted α-helical coiled coil, as recently reported for plant amalgaviruses, and transcriptome-based evidence for another new amalga-like virus in the transcriptome of another basal hexapod, the two-pronged bristletail species Campodea augens.

A +1 ribosomal frameshifting motif prevalent among plant amalgaviruses

Sequence accessions attributable to novel plant amalgaviruses have been found in the Transcriptome Shotgun Assembly database. Sixteen accessions, derived from 12 different plant species, appear to encompass the complete protein-coding regions of the proposed amalgaviruses, which would substantially expand the size of genus Amalgavirus from 4 current species. Other findings include evidence for UUU_CGN as a +1 ribosomal frameshifting motif prevalent among plant amalgaviruses; for a variant version of this motif found thus far in only two amalgaviruses from solanaceous plants; for a region of α-helical coiled coil propensity conserved in a central region of the ORF1 translation product of plant amalgaviruses; and for conserved sequences in a C-terminal region of the ORF2 translation product (RNA-dependent RNA polymerase) of plant amalgaviruses, seemingly beyond the region of conserved polymerase motifs. These results additionally illustrate the value of mining the TSA database and others for novel viral sequences for comparative analyses.

Cellular mRNAs access second ORFs using a novel amino acid sequence-dependent coupled translation termination–reinitiation mechanism

Polycistronic transcripts are considered rare in the human genome. Initiation of translation of internal ORFs of eukaryotic genes has been shown to use either leaky scanning or highly structured IRES regions to access initiation codons. Studies on mammalian viruses identified a mechanism of coupled translation termination-reinitiation that allows translation of an additional ORF. Here, the ribosome terminating translation of ORF-1 translocates upstream to reinitiate translation of ORF-2. We have devised an algorithm to identify mRNAs in the human transcriptome in which the major ORF-1 overlaps a second ORF capable of encoding a product of at least 50 aa in length. This identified 4368 transcripts representing 2214 genes. We investigated 24 transcripts, 22 of which were shown to express a protein from ORF-2 highlighting that 3' UTRs contain protein-coding potential more frequently than previously suspected. Five transcripts accessed ORF-2 using a process of coupled translation termination-reinitiation. Analysis of one transcript, encoding the CASQ2 protein, showed that the mechanism by which the coupling process of the cellular mRNAs was achieved was novel. This process was not directed by the mRNA sequence but required an aspartate-rich repeat region at the carboxyl terminus of the terminating ORF-1 protein. Introduction of wobble mutations for the aspartate codon had no effect, whereas replacing aspartate for glutamate repeats eliminated translational coupling. This is the first description of a coordinated expression of two proteins from cellular mRNAs using a coupled translation termination-reinitiation process and is the first example of such a process being determined at the amino acid level.

Lost in translation

“Young” APE-type non-LTR retrotransposons (non-LTRs) typically encode two open reading frames (ORFs 1 and 2). The shorter ORF1 translation product (ORF1p) comprises an RNA binding activity, thought to bind to non-LTR transcript RNA, protect against nuclease degradation and specify nuclear import of the ribonuclear protein complex (RNP). ORF2 encodes a multifunctional protein (ORF2p) comprising apurinic/apyrimidinic endonuclease (APE) and reverse-transcriptase (RT) activities, responsible for genome replication and re-integration into chromosomal DNA. However, some clades of APE-type non-LTRs only encode a single ORF—corresponding to the multifunctional ORF2p outlined above (and for simplicity referred-to as ORF2 below). The absence of an ORF1 correlates with the acquisition of a 2A oligopeptide translational recoding element (some 18–30 amino acids) into the N-terminal region of ORF2p. In the case of non-LTRs encoding two ORFs, the presence of ORF1 would necessarily downregulate the translation of ORF2. We argue that in the absence of an ORF1, 2A could provide the corresponding translational downregulation of ORF2. While multiple molecules of ORF1p are required to decorate the non-LTR transcript RNA in the cytoplasm, conceivably only a single molecule of ORF2p is required for target-primed reverse transcription/integration in the nucleus. Why would the translation of ORF2 need to be controlled by such mechanisms? An “excess” of ORF2p could result in disadvantageous levels of genome instability by, for example, enhancing short, interspersed, element (SINE) retrotransposition and the generation of processed pseudogenes. If so, the acquisition of mechanisms—such as 2A—to control ORF2p biogenesis would be advantageous.

Reselection of a Genomic Upstream Open Reading Frame in Mouse Hepatitis Coronavirus 5′-Untranslated-Region Mutants

An AUG-initiated upstream open reading frame (uORF) encoding a potential polypeptide of 3 to 13 amino acids (aa) is found within the 5' untranslated region (UTR) of >75% of coronavirus genomes based on 38 reference strains. Potential CUG-initiated uORFs are also found in many strains. The AUG-initiated uORF is presumably translated following genomic 5'-end cap-dependent ribosomal scanning, but its function is unknown. Here, in a reverse-genetics study with mouse hepatitis coronavirus, the following were observed. (i) When the uORF AUG-initiating codon was replaced with a UAG stop codon along with a U112A mutation to maintain a uORF-harboring stem-loop 4 structure, an unimpaired virus with wild-type (WT) growth kinetics was recovered. However, reversion was found at all mutated sites within five virus passages. (ii) When the uORF was fused with genomic (main) ORF1 by converting three in-frame stop codons to nonstop codons, a uORF-ORF1 fusion protein was made, and virus replicated at WT levels. However, a frameshifting G insertion at virus passage 7 established a slightly 5'-extended original uORF. (iii) When uAUG-eliminating deletions of 20, 30, or 51 nucleotides (nt) were made within stem-loop 4, viable but debilitated virus was recovered. However, a C80U mutation in the first mutant and an A77G mutation in the second appeared by passage 10, which generated alternate uORFs that correlated with restored WT growth kinetics. In vitro, the uORF-disrupting nondeletion mutants showed enhanced translation of the downstream ORF1 compared with the WT. These results together suggest that the uORF represses ORF1 translation yet plays a beneficial but nonessential role in coronavirus replication in cell culture.

Low dependency of retrotransposition on the ORF1 protein of the zebrafish LINE, ZfL2-1

The zebrafish long interspersed element (LINE), ZfL2-1, which belongs to the L2 clade, contains two open reading frames, ORF1 and ORF2. ORF1 encodes a protein containing a coiled-coil motif and an esterase domain, whereas ORF2 encodes a protein containing an endonuclease and a reverse transcriptase domain. To elucidate the functional significance of ORF1 in retrotransposition, we constructed many variants of ZfL2-1 and examined their retrotransposition ability. We concluded: 1) the ORF1 protein is not essential for ZfL2-1 retrotransposition in cultured cells; 2) the translation of ORF1 is required for the translation of ORF2; and 3) ORF2 translation probably occurs via suppression of the ORF1 stop codon, the efficiency of which is influenced by the context of the sequence juxtaposed to the 3′ side of the stop codon. These results offer a new perspective on the evolution of the L2 clade LINEs.

Pseudo-polyprotein translated from the full-length ORF1 of capillovirus is important for pathogenicity, but a truncated ORF1 protein without variable and CP regions is sufficient for replication

The first open-reading frame (ORF) of the genus Capillovirus encodes an apparently chimeric polyprotein containing conserved regions for replicase (Rep) and coat protein (CP), while other viruses in the family Flexiviridae have separate ORFs encoding these proteins. To investigate the role of the full-length ORF1 polyprotein of capillovirus, we generated truncation mutants of ORF1 of apple stem grooving virus by inserting a termination codon into the variable region located between the putative Rep- and CP-coding regions. These mutants were capable of systemic infection, although their pathogenicity was attenuated. In vitro translation of ORF1 produced both the full-length polyprotein and the smaller Rep protein. The results of in vivo reporter assays suggested that the mechanism of this early termination is a ribosomal −1 frame-shift occurring downstream from the conserved Rep domains. The mechanism of capillovirus gene expression and the very close evolutionary relationship between the genera Capillovirus and Trichovirus are discussed.

Mammalian Gene PEG10 Expresses Two Reading Frames by High Efficiency –1 Frameshifting in Embryonic-associated Tissues

Paternally expressed gene 10 (PEG10) is a mammalian gene that is essential for embryonic development in mice. The gene contains two overlapping open reading frames (ORF1 and ORF2) and is derived from a retroelement that acquired a cellular function. It is not known if both reading frames are required for PEG10 function. Synthesis of ORF2 would be possible only if programmed -1 frameshifting occurred during ORF1 translation. In this study the frameshifting activity of PEG10 was analyzed in vivo, and a potential role for ORF2 was investigated. Phylogenetic analysis demonstrated that PEG10 is highly conserved in therian mammals, with all species retaining the elements necessary for frameshifting as well as functional motifs in each ORF. The frameshift site of PEG10 was highly active in cultured cells and produced the ORF1-2 protein. In mice, endogenous ORF1 and an ORF1-2 frameshift protein were detected in the developing placenta and amniotic membrane from 9.5 days post-coitus through to term with a very high frameshift efficiency (>60%). Mutagenesis of the active site motif of a putative protease within ORF2 showed that this enzyme is active and participates in post-translational processing of PEG10 ORF1-2. Both PEG10 proteins were also detected in first trimester human placenta. By contrast, neither protein expression nor frameshifting was detected in adult mouse tissues. These studies imply that the ORF1-2 protein, synthesized utilizing the most efficient -1 frameshift mechanism yet documented in vivo, will have an essential function that is intrinsic to the importance of PEG10 in mammals.

Analysis of the complete genome of peach chlorotic mottle virus: identification of non-AUG start codons, in vitro coat protein expression, and elucidation of serological cross-reactions

The entire genome of peach chlorotic mottle virus (PCMV), originally identified as Prunus persica cv. Agua virus (4N6), was sequenced and analysed. PCMV cross-reacts with antisera to diverse viruses, such as plum pox virus (PPV), genus Potyvirus, family Potyviridae; and apple stem pitting virus (ASPV), genus Foveavirus, family Flexiviridae. The PCMV genome consists of 9005 nucleotides (nts), excluding a poly(A) tail at the 3' end of the genome. Five open reading frames (ORFs) were identified with four untranslated regions (UTR) including a 5', a 3', and two intergenic UTRs. The genome organisation of PCMV is similar to that of ASPV and the two genomes share a nucleotide (nt) sequence identity of 58%. PCMV ORF1 encodes the replication-associated protein complex (Mr 241,503), ORF2-ORF4 code for the triple gene block proteins (TGBp; Mr 24,802, 12,370, and 7320, respectively), and ORF5 encodes the coat protein (CP) (Mr 42,505). Two non-AUG start codons participate in the initiation of translation: 35AUC and 7676AUA initiate translation of ORF1 and ORF5. In vitro expression with subsequent Western blot analysis confirmed ORF5 as the CP-encoding gene and confirmed that the codon AUA is able to initiate translation of the CP. Expression of a truncated CP fragment (Mr 39, 689) was demonstrated, and both proteins are expressed in vivo, since both were observed in Western blot analysis of PCMV-infected peach and Nicotiana occidentalis. The expressed proteins cross-reacted with an antiserum against ASPV. The amino acid sequences of the CPs of PCMV and ASPV CP share only 37% identity, but there are 11 shared peptides 4-8 aa residues long. These may constitute linear epitopes responsible for ASPV antiserum cross reactions. No significant common linear epitopes were associated with PPV. Extensive phylogenetic analysis indicates that PCMV is closely related to ASPV and is a new and distinct member of the genus Foveavirus.

The ORFO product of Potato leafroll virus is indispensable for virus accumulation.

Using a cDNA expression cassette in combination with agroinoculation of potato leaf discs we have investigated the role the protein encoded by ORF0 of Potato leafroll virus (PLRV) and have shown its importance for virus accumulation. Two mutations introduced into ORF0 by site-directed mutagenesis prevented expression of the corresponding protein and completely abolished virus accumulation in plant cells. They did not, however, affect translation of ORF1 and ORF2. We therefore conclude that ORF0 of PLRV produces a protein essential for virus accumulation, a hitherto undescribed finding.

Full or Near Full Length Nucleotide Sequences of TT Virus Variants (Types SANBAN and YONBAN) and the TT Virus-Like Mini Virus

TT virus (TTV) is a common virus and consists of many genotypes and variants. In addition, there exists a virus which both differs greatly from and retains a considerable resemblance to TTV, such as the TTV-like mini virus (TLMV) as we reported previously. Here we report the near full length genomic sequences of 4 isolates of a new variant of TTV (designated YONBAN) along with the full length sequences of 2 isolates of the TTV-SANBAN lineage and 7 isolates of the TLMV species derived from human sera. The TTV-YONBAN sequences showed only about 50% identity at the nucleotide level to those of the prototype TTV (TA278) and to SANBAN, and even less to TLMV. Moreover, the ORF1 of YONBAN lacked the ATG initiation codon which is shared by all the TTV and TLMV isolates so far identified in humans; instead, YONBAN had a Kozak’s rule-compatible ACG codon as the candidate initiation site for the ORF1 translation. Nevertheless, the overall genetic structure and the conserved amino acid motifs within the ORF1 and the ORF2 were well shared among the prototype TTV strains, the SANBAN and YONBAN variants, and TLMV. The most conserved nucleotide sequence was found in the noncoding region just upstream from the ORF2, allowing construction of a phylogenetic tree which implied that the TTV genotypes and variants, the TLMV, and chicken anemia virus could be coclassified under a superfamily for which we proposed the name of ‘Paracircoviridae’ in our previous report.

Sequence analysis of a cDNA containing the gag and prot regions of the soybean retrovirus-like element, SIRE-1.

SIRE-1 is a family of several hundred dispersed copies of a very large DNA element from Glycine max that has features characteristic of retroviruses and retrotransposons. A 2.4 kb SIRE-1-specific fragment was recovered from a soybean cDNA library and sequenced. The sequence contains two ORFs. Theoretical translation of ORF1 produces a gag-prot-like polyprotein containing highly conserved motifs found in retroelement nucleocapsids (CX2CX4HX4C) and aspartic proteases (LDSG). The second ORF is foreshortened. The cDNA also contains nearly 200 bp of a putative 5' LTR just upstream of a tRNA primer-binding site.

Translation of the human LINE-1 element, L1Hs.

Full-length RNA transcribed from the human LINE-1 (L1) element L1 Homo sapiens (L1Hs) has a 900-nt, G+C-rich, 5'-untranslated region (UTR). The 5' UTR is followed by two long open reading frames, ORF1 and ORF2, which are separated from each other by an inter-ORF region of 33 nt that includes two or three in-frame stop codons. We examine here the mechanism(s) by which the translation of L1Hs ORF1 and ORF2 is initiated. A stable hairpin structure (delta G = -74.8 kcal/mol), inserted at nt 661 of the 5' UTR, caused a 3- to 8-fold decrease in the in vitro and in vivo translation of either a lacZ reporter gene for ORF1 or the ORF1 polypeptide product, p40, but translation of a lacZ reporter gene in ORF2 was increased. The results are compatible with a model for ORF1 translation initiation in which the majority of ribosomes scan from a point 5' of nt 661 but suggest that ORF2 is not translated by attached ribosomes that reinitiate after the termination of ORF1 translation. Our data are compatible with a model whereby the translation of L1Hs ORF2 is initiated internally.

Translation of LINE-1 DNA elements in vitro and in human cells.

The LINE-1 (L1) family of interspersed DNA sequences found throughout the human genome (L1 Homo sapiens, L1Hs) includes active transposable elements. Current models for the mechanism of transposition involve reverse transcription of an RNA intermediate and utilization of element-encoded proteins. We report that an antiserum against the polypeptide encoded by the L1Hs 5' open reading frame (ORF1) detects, in human cells, an endogenous ORF1 protein as well as the ORF1 product of an appropriate transfecting recombinant vector. The endogenous polypeptide is most abundant in teratocarcinoma and choriocarcinoma cells, among those cell lines tested; it appears to be a single species of approximately 38 kDa. In contrast, RNAs synthesized in vitro from cDNAs representing full-length, polyadenylylated cytoplasmic L1Hs RNA yield, upon in vitro translation, ORF1 products of slightly different sizes. This is consistent with the fact that the various cDNAs are different and represent transcription of different genomic L1Hs elements. In vitro studies additionally suggest that translation of ORF1 is initiated at the first AUG codon. Finally, in no case was an ORF1-ORF2 fusion protein detected.

Overlapping Reading Frames at the LYS5 Locus in the Yeast Yarrowia lipolytica

Mutants affected at the LYS5 locus of Yarrowia lipolytica lack detectable dehydrogenase (SDH) activity. The LYS5 gene has previously been cloned, and we present here the sequence of the 2.5-kilobase-pair (kb) DNA fragment complementing the lys5 mutation. Two large antiparallel open reading frames (ORF1 and ORF2) were observed, flanked by potential transcription signals. Both ORFs appear to be transcribed, but several lines of evidence suggest that only ORF2 is translated and encodes SDH. (i) The global amino acid compositions of Saccharomyces cerevisiae SDH and of the putative ORF2 product are similar and that of ORF1 is dissimilar. (ii) An in-frame translational fusion of ORF2 with the Escherichia coli lacZ gene was introduced into yeast cells and resulted in a beta-galactosidase activity regulated similarly to SDH; no beta-galactosidase activity was obtained with an in-frame fusion of ORF1 with lacZ. (iii) The introduction of a stop codon at the beginning of ORF2 prevented SDH expression in yeast cells, whereas no phenotypic effect was observed when ORF1 translation was blocked.

Overlapping reading frames at the LYS5 locus in the yeast Yarrowia lipolytica.

Mutants affected at the LYS5 locus of Yarrowia lipolytica lack detectable dehydrogenase (SDH) activity. The LYS5 gene has previously been cloned, and we present here the sequence of the 2.5-kilobase-pair (kb) DNA fragment complementing the lys5 mutation. Two large antiparallel open reading frames (ORF1 and ORF2) were observed, flanked by potential transcription signals. Both ORFs appear to be transcribed, but several lines of evidence suggest that only ORF2 is translated and encodes SDH. (i) The global amino acid compositions of Saccharomyces cerevisiae SDH and of the putative ORF2 product are similar and that of ORF1 is dissimilar. (ii) An in-frame translational fusion of ORF2 with the Escherichia coli lacZ gene was introduced into yeast cells and resulted in a beta-galactosidase activity regulated similarly to SDH; no beta-galactosidase activity was obtained with an in-frame fusion of ORF1 with lacZ. (iii) The introduction of a stop codon at the beginning of ORF2 prevented SDH expression in yeast cells, whereas no phenotypic effect was observed when ORF1 translation was blocked.