Eukaryotic initiation factor (eIF)-4F. Implications for a role in internal initiation of translation

In order to study the eukaryotic translation initiation mechanisms of "internal initiation," "re-initiation," and/or "coupled internal initiation," a series of model mRNAs have been constructed which contain two non-overlapping open reading frames (ORFs) that encode different lengths of rabbit alpha globin. These mRNAs, along with the bicistronic constructs TK/CAT and TK/P2CAT developed by Pelletier and Sonenberg (Pelletier, J., and Sonenberg, N. (1988) Nature 334, 320-325, 1988), were used to program an in vitro rabbit reticulocyte lysate translation system. Cap-dependent and cap-independent translation were distinguished by monitoring translation in the presence or absence of exogenously added cap analog (m7GTP). Messenger RNAs which translate both ORF1 and ORF2 by a cap-dependent mechanism, as well as mRNAs that translate ORF2 by a cap-independent mechanism while still translating ORF1 in a cap-dependent fashion have been obtained. These same alpha globin mRNAs differ by no more than 45 nucleotides in intercistronic length. Initiation factor addition studies were performed in this same in vitro translation system. Both eukaryotic initiation factor (eIF)-4F and, to a lesser extent, eIF-4B can stimulate translation of an internally located ORF independent of upstream ORF translation and in a manner not dependent on mRNA cap recognition. This indicates that the cap-recognition initiation factor, eIF-4F, and eIF-4B facilitate cap-independent and internal initiation of an open reading frame.     

Mechanism of translation of monocistronic and multicistronic human immunodeficiency virus type 1 mRNAs.

We have used a panel of cDNA clones expressing wild-type and mutant human immunodeficiency virus type 1 (HIV-1) mRNAs to study translation of these mRNAs in eucaryotic cells. The tat open reading frame (ORF) has a strong signal for translation initiation, while rev and vpu ORFs have weaker signals. The expression of downstream ORFs is inhibited in mRNAs that contain the tat ORF as the first ORF. In contrast, downstream ORFs are expressed efficiently from mRNAs that have rev or vpu as the first ORF. All env mRNAs contain the upstream vpu ORF. Expression of HIV-1 Env protein requires a weak vpu AUG, which allows leaky scanning to occur, thereby allowing ribosomes access to the downstream env ORF. We concluded that HIV-1 mRNAs are translated by the scanning mechanism and that expression of more than one protein from each mRNA was caused by leaky scanning at the first AUG of the mRNA.     

Evidence for a Dual Strategy in the Expression of Cauliflower Mosaic Virus Open Reading Frames I and IV

Studies have indicated that cauliflower mosaic virus (CaMV) gene expression is mediated by the translation of polycistronic 35S pregenomic RNA, but the involvement of some minor subgenomic RNA species is also suspected. We examined the involvement of the 35S promoter in the expression of CaMV open reading frames (ORFs) I and IV using both 35S RNA-driven and promoter-less ORF I- and ORF IV-β-glucuronidase (GUS) fusion constructs. In addition to the 35S promoter-dependent expression of both ORF I- and IV-GUS fusions, we detected the 35S promoter-independent expression of both fusion genes via subgenomic mRNAs, which were detected by Northern blotting in the protoplasts transfected with the 35S promoter-driven constructs as well as in those transfected with the promoter-less constructs. These results suggest the involvement of subgenomic RNAs in the expression of CaMV ORFs I and IV, and the operation of a dual strategy in the expression of two viral genes.     

Equine arteritis virus is not a togavirus but belongs to the coronaviruslike superfamily.

The nucleotide sequence of the genome of equine arteritis virus (EAV) was determined from a set of overlapping cDNA clones and was found to contain eight open reading frames (ORFs). ORFs 2 through 7 are expressed from six 3'-coterminal subgenomic mRNAs, which are transcribed from the 3'-terminal quarter of the viral genome. A number of these ORFs are predicted to encode structural EAV proteins. The organization and expression of the 3' part of the EAV genome are remarkably similar to those of coronaviruses and toroviruses. The 5'-terminal three-quarters of the genome contain the putative EAV polymerase gene, which also shares a number of features with the corresponding gene of corona- and toroviruses. The gene contains two large ORFs, ORF1a and ORF1b, with an overlap region of 19 nucleotides. The presence of a "shifty" heptanucleotide sequence in this region and a downstream RNA pseudoknot structure indicate that ORF1b is probably expressed by ribosomal frameshifting. The frameshift-directing potential of the ORF1a/ORF1b overlap region was demonstrated by using a reporter gene. Moreover, the predicted ORF1b product was found to contain four domains which have been identified in the same relative positions in coronavirus and torovirus ORF1b products. The sequences of the EAV and coronavirus ORF1a proteins were found to be much more diverged. The EAV ORF1a product contains a putative trypsinlike serine protease motif. Our data indicate that EAV, presently considered a togavirus, is evolutionarily related to viruses from the coronaviruslike superfamily.     

Efficiency of reinitiation of translation on human immunodeficiency virus type 1 mRNAs is determined by the length of the upstream open reading frame and by intercistronic distance.

In this study, we examined the mechanism of translation of the human immunodeficiency virus type 1 tat mRNA in eucaryotic cells. This mRNA contains the tat open reading frame (ORF), followed by rev and nef ORFs, but only the first ORF, encoding tat, is efficiently translated. Introduction of premature stop codons in the tat ORF resulted in efficient translation of the downstream rev ORF. We show that the degree of inhibition of translation of rev is proportional to the length of the upstream tat ORF. An upstream ORF spanning 84 nucleotides was predicted to inhibit 50% of the ribosomes from initiating translation at downstream AUGs. Interestingly, the distance between the upstream ORF and the start codon of the second ORF also played a role in efficiency of downstream translation initiation. It remains to be investigated if these conclusions relate to translation of mRNAs other than human immunodeficiency virus type 1 mRNAs. The strong inhibition of rev translation exerted by the presence of the tat ORF may reflect the different roles of Tat and Rev in the viral life cycle. Tat acts early to induce high production of all viral mRNAs. Rev induces a switch from the early to the late phase of the viral life cycle, resulting in production of viral structural proteins and virions. Premature Rev production may result in entrance into the late phase in the presence of suboptimal levels of viral mRNAs coding for structural proteins, resulting in inefficient virus production.     

Leaky scanning and scanning-independent ribosome migration on the tricistronic S1 mRNA of avian reovirus

The S1 genome segments of avian and Nelson Bay reovirus encode tricistronic mRNAs containing three sequential partially overlapping open reading frames (ORFs). The translation start site of the 3'-proximal ORF encoding the sigmaC protein lies downstream of two ORFs encoding the unrelated p10 and p17 proteins and more than 600 nucleotides distal from the 5'-end of the mRNA. It is unclear how translation of this remarkable tricistronic mRNA is regulated. We now show that the p10 and p17 ORFs are coordinately expressed by leaky scanning. Translation initiation events at these 5'-proximal ORFs, however, have little to no effect on translation of the 3'-proximal sigmaC ORF. Northern blotting, insertion of upstream stop codons or optimized translation start sites, 5'-truncation analysis, and poliovirus 2A protease-mediated cleavage of eIF4G indicated sigmaC translation derives from a full-length tricistronic mRNA using a mechanism that is eIF4G-dependent but leaky scanning- and translation reinitiation-independent. Further analysis of artificial bicistronic mRNAs failed to provide any evidence that sigmaC translation derives from an internal ribosome entry site. Additional features of the S1 mRNA and the mechanism of sigmaC translation also differ from current models of ribosomal shunting. Translation of the tricistronic reovirus S1 mRNA, therefore, is dependent both on leaky scanning and on a novel scanning-independent mechanism that allows translation initiation complexes to efficiently bypass two functional upstream ORFs.     

Post-transcriptional gene silencing in the root system of the actinorhizal tree Allocasuarina verticillata

In recent years, RNA interference has been exploited as a tool for investigating gene function in plants. We tested the potential of double-stranded RNA interference technology for silencing a transgene in the actinorhizal tree Allocasuarina verticillata. The approach was undertaken using stably transformed shoots expressing the beta-glucuronidase (GUS) gene under the control of the constitutive promoter 35S; the shoots were further transformed with the Agrobacterium rhizogenes A4RS containing hairpin RNA (hpRNA) directed toward the GUS gene, and driven by the 35S promoter. The silencing and control vectors contained the reporter gene of the green fluorescent protein (GFP), thus allowing a screening of GUS-silenced composite plantlets for autofluorescence. With this rapid procedure, histochemical data established that the reporter gene was strongly silenced in both fluorescent roots and actinorhizal nodules. Fluorometric data further established that the level of GUS silencing was usually greater than 90% in the hairy roots containing the hairpin GUS sequences. We found that the silencing process of the reporter gene did not spread to the aerial part of the composite A. verticillata plants. Real-time quantitative polymerase chain reaction showed that GUS mRNAs were substantially reduced in roots and, thereby, confirmed the knock-down of the GUS transgene in the GFP(+) hairy roots. The approach described here will provide a versatile tool for the rapid assessment of symbiotically related host genes in actinorhizal plants of the Casuarinaceae family.     

Regulation of tomato leaf curl viral gene expression in host tissues

The regulation of expression of the two virion-sense (V1 and V2) and four complementary-sense (C1, C2, C3, and C4) open reading frames (ORFs) of Tomato leaf curl virus (TLCV) was studied in both stably and transiently transformed Nicotiana tabacum tissues with fusions with the beta-glucuronidase (GUS) reporter gene. GUS-expressing transgenic lines were obtained with each of the four complementary-sense gene-GUS fusion constructs and with truncated versions of the virion-sense gene-GUS fusion constructs (V1GUSdeltaC and V2GUSdeltaC) lacking complementary-sense sequences encoding the C1, C2, and C3 ORFs. However, little or no GUS expression was observed in kanamycin-resistant plants transformed with full-length, virion-sense gene constructs (V1GUS and V2GUS) constituting the complete viral genome. In contrast, V1GUS and V2GUS were found to direct high-level GUS expression in transient assays with tobacco protoplasts, suggesting that integration of viral constructs containing functional, complementary-sense genes may lead to repression or deletion of the introduced constructs in transgenic tissues. V2GUS expression in the transient protoplast assay was found to be severely curtailed by specific mutation of the C2 ORF, supporting a role for the C2 protein in transactivation of TLCV virion-sense gene expression. TLCV ORF-GUS constructs displayed distinctive tissue expression patterns in transgenic tobacco plants that could be divided into constitutive (C1, C4, and V2GUSdeltaC), predominantly vascular (C2, C3), or reduced expression in cells associated with the vascular bundles (V1GUSdeltaC). The significance of these results is discussed in terms of current models of gene function and regulation in geminiviruses.     

Identification of DEBS 1, DEBS 2 and DEBS 3, the multienzyme polypeptides of the erythromycin-producing polyketide synthase from Saccharopolyspora erythraea.

The ery A region of the erythromycin biosynthetic gene cluster of Saccharopolyspora erythraea has previously been shown to contain three large open reading frames (ORFs) that encode the components of 6-deoxyerythronolide B synthase (DEBS). Polyclonal antibodies were raised against recombinant proteins obtained by overexpression of 3' regions of the ORF2 and ORF3 genes. In Western blotting experiments, each antiserum reacted strongly with a different high molecular weight protein in extracts of erythromycin-producing S. erythraea cells. These putative DEBS 2 and DEBS 3 proteins were purified and subjected to N-terminal sequence analysis. The protein sequences were entirely consistent with the and DEBS 3 proteins were purified and subjected to N-terminal sequence analysis. The protein sequences were entirely consistent with the translation start sites predicted from the DNA sequences of ORFs 2 and 3. A third high molecular weight protein co-purified with DEBS 2 and DEBS 3 and had an N-terminal sequence that matched a protein sequence translated from the DNA sequence some 155 base pairs upstream from the previously proposed start codon of ORF1.     

Organization and nucleotide sequences of two lactococcal bacteriocin operons

Two distinct regions of the Lactococcus lactis subsp. cremoris 9B4 plasmid p9B4-6, each of which specified bacteriocin production as well as immunity, have been sequenced and analyzed by deletion and frameshift mutation analyses. On a 1.8-kb ScaI-ClaI fragment specifying low antagonistic activity, three open reading frames (ORFs) were present, which were organized in an operon. The first two ORFs, containing 69 and 77 codons, respectively, were involved in bacteriocin activity, whereas the third ORF, containing 154 codons, was essential for immunity. Primer extension analysis indicated the presence of a promoter upstream of the ORFs. Two ORFs were present on a 1.3-kb ScaI-HindII fragment specifying high antagonistic activity. The first ORF, containing 75 codons, specified bacteriocin activity. The second ORF, containing 98 codons, specified immunity. The nucleotide sequences of both fragments upstream of the first ORFs as well as the first 20 bp of the first ORF of both bacteriocin operons appeared to be identical.