[Identification of a novel termination release factor eRF3b expressing the eRF3 activity in vitro and in vivo]. / Identifikatsiia novogo faktora terminatsii transliatsii eRF3b, obladaiushchego in vitro i in vivo aktivnost'iu eRF3.

Termination of translation in eukaryotes is governed by the ribosome, a termination codon in the mRNA, and two polypeptide chain release factors (eRF1 and eRF3). We have identified a human protein of 628 amino acids, named eRF3b, which is highly homologous to the known human eRF3 henceforth named eRF3a. At the nucleotide and at the amino acid levels the human eRF3a and eRF3b are about 87% identical. The differences in amino acid sequence are concentrated near the amino terminus. The most important difference in the nucleotide sequence is that eRF3b lacks a GGC repeat close to the initiation codon in eRF3a. We have cloned the cDNA encoding the human eRF3b, purified the eRF3b expressed in Escherichia coli, and found that the protein is active in vitro as a potent stimulator of the release factor activity of human eRFl. Like eRF3a, eRF3b exhibits GTPase activity, which is ribosome- and eRFl-dependent. In vivo assays (based on suppression of readthrough induced by three species of suppressor tRNAs: amber, ochre, and opal) show that the human eRF3b is able to enhance the release factor activity of endogenous and overexpressed eRFl with all three stop codons.

Stop codon recognition in ciliates: Euplotes release factor does not respond to reassigned UGA codon.

In eukaryotes, the polypeptide release factor 1 (eRF1) is involved in translation termination at all three stop codons. However, the mechanism for decoding stop codons remains unknown. A direct interaction of eRF1 with the stop codons has been postulated. Recent studies focus on eRF1 from ciliates in which some stop codons are reassigned to sense codons. Using an in vitro assay based on mammalian ribosomes, we show that eRF1 from the ciliate Euplotes aediculatus responds to UAA and UAG as stop codons and lacks the capacity to decipher the UGA codon, which encodes cysteine in this organism. This result strongly suggests that in ciliates with variant genetic codes eRF1 does not recognize the reassigned codons. Recent hypotheses describing stop codon discrimination by eRF1 are not fully consistent with the set of eRF1 sequences available so far and require direct experimental testing.

Identification of a cellular protein specifically interacting with the precursor of the hepatitis B e antigen.

In hepatitis B virus (HBV) the precore gene encodes a protein from which derives P22, the precursor of the mature secreted hepatitis B virus e antigen (HBeAg). Circumstantial evidences suggest that HBeAg and/or its precursor P22 are important for establishing persistent infection. Although P22 is essentially present in the secretory pathway, a substantial fraction has been found in the cytosol. In order to get new insights into the biological function of P22, we looked for cellular proteins which could strongly associate with this protein. Using immunoprecipitation studies on human cell extracts, we found that a non-secreted cellular protein of about 32 kDa (P32) bound with a high specificity to P22. P32 associated neither with HBeAg nor with the viral core protein P21 which exhibits the same amino acids sequence as P22 but is N-terminally shorter by 10 residues. We also demonstrated that this interaction depended on the presence of the P22 C-terminal domain. Our data argues for a potential biological function of P22.

Complementarity between the mRNA 5' untranslated region and 18S ribosomal RNA can inhibit translation.

In eubacteria, base pairing between the 3' end of 16S rRNA and the ribosome-binding site of mRNA is required for efficient initiation of translation. An interaction between the 18S rRNA and the mRNA was also proposed for translation initiation in eukaryotes. Here, we used an antisense RNA approach in vivo to identify the regions of 18S rRNA that might interact with the mRNA 5' untranslated region (5' UTR). Various fragments covering the entire mouse 18S rRNA gene were cloned 5' of a cat reporter gene in a eukaryotic vector, and translation products were analyzed after transient expression in human cells. For the largest part of 18S rRNA, we show that the insertion of complementary fragments in the mRNA 5' UTR do not impair translation of the downstream open reading frame (ORF). When translation inhibition is observed, reduction of the size of the complementary sequence to less than 200 nt alleviates the inhibitory effect. A single fragment complementary to the 18S rRNA 3' domain retains its inhibitory potential when reduced to 100 nt. Deletion analyses show that two distinct sequences of approximately 25 nt separated by a spacer sequence of 50 nt are required for the inhibitory effect. Sucrose gradient fractionation of polysomes reveals that mRNAs containing the inhibitory sequences accumulate in the fractions with 40S ribosomal subunits, suggesting that translation is blocked due to stalling of initiation complexes. Our results support an mRNA-rRNA base pairing to explain the translation inhibition observed and suggest that this region of 18S rRNA is properly located for interacting with mRNA.

The product of the mammalian orthologue of the Saccharomyces cerevisiae HBS1 gene is phylogenetically related to eukaryotic release factor 3 (eRF3) but does not carry eRF3-like activity.

We describe here the cloning and sequencing of human and mouse cDNAs encoding a putative GTP binding protein. Sequence comparison shows that these cDNAs (named eRFS) are likely to represent the orthologues of the yeast Saccharomyces cerevisiae HBS1 gene and that the C-terminal domains of the encoded proteins share structural features with eukaryotic elongation factor eEF-1A and release factor 3 (eRF3) families. The phylogenetic analysis suggests that eRFS proteins and Hbs1p form a cluster of orthologous sequences branching with the eRF3 family. Nevertheless, in yeast, the human eRFS protein and Hbs1p do not complement eRF3/Sup35p thermosensitive mutation and do not interact with eRF1.

Eukaryotic release factor 1 (eRF1) abolishes readthrough and competes with suppressor tRNAs at all three termination codons in messenger RNA.

It is known from experiments with bacteria and eukaryotic viruses that readthrough of termination codons located within the open reading frame (ORF) of mRNAs depends on the availability of suppressor tRNA(s) and the efficiency of termination in cells. Consequently, the yield of readthrough products can be used as a measure of the activity of polypeptide chain release factor(s) (RF), key components of the translation termination machinery. Readthrough of the UAG codon located at the end of the ORF encoding the coat protein of beet necrotic yellow vein furovirus is required for virus replication. Constructs harbouring this suppressible UAG codon and derivatives containing a UGA or UAA codon in place of the UAG codon have been used in translation experiments in vitro in the absence or presence of human suppressor tRNAs. Readthrough can be virtually abolished by addition of bacterially-expressed eukaryotic RF1 (eRF1). Thus, eRF1 is functional towards all three termination codons located in a natural mRNA and efficiently competes in vitro with endogenous and exogenous suppressor tRNA(s) at the ribosomal A site. These results are consistent with a crucial role of eRF1 in translation termination and forms the essence of an in vitro assay for RF activity based on the abolishment of readthrough by eRF1.

Overexpression of human release factor 1 alone has an antisuppressor effect in human cells.

Two eukaryotic proteins involved in translation termination have recently been characterized in in vitro experiments. Eukaryotic release factor 1 (eRF1) catalyzes the release of the polypeptide chain without any stop codon specificity. The GTP-binding protein eRF3 confers GTP dependence to the termination process and stimulates eRF1 activity. We used tRNA-mediated nonsense suppression at different stop codons in a cat reporter gene to analyze the polypeptide chain release factor activities of the human eRF1 and eRF3 proteins overexpressed in human cells. In a chloramphenicol acetyltransferase assay, we measured the competition between the suppressor tRNA and the human release factors when a stop codon was present in the ribosomal A site. Whatever the stop codon (UAA, UAG, or UGA) present in the cat open reading frame, the overexpression of human eRF1 alone markedly decreased translational readthrough by suppressor tRNA. Thus, like the procaryotic release factors RF1 and RF2 in Escherichia coli, eRF1 seems to have an intrinsic antisuppressor activity in human cells. Levels of antisuppression of overexpression of both eRF3 and eRF1 were almost the same as those of overexpression of eRF1 alone, suggesting that eRF1-eRF3 complex-mediated termination may be controlled by the expression level of eRF1. Surprisingly, when overexpressed alone, eRF3 had an inhibitory effect on cat gene expression. The results of cat mRNA stability studies suggest that eRF3 inhibits gene expression at the transcriptional level. This indicates that in vivo, eRF3 may perform other functions, including the stimulation of eRF1 activity.

Is there a human [psi]?

The yeast Sup35p protein which is responsible for the [psi] phenotype, is a GTP-binding protein involved in translation termination. It was suggested recently that the [psi] determinant has prion-like properties that were localized in the 114 N-terminal amino acids of the protein. In this study, we show that the 5' end of the human SUP35 gene open reading frame is longer than previously reported by 138 codons. This N-terminal sequence presents similarities with the N-terminus of S. cerevisiae Sup35p protein, involved in [psi] maintenance. By transfection of human cells and Western blotting, we demonstrate that translation is initiated at the first AUG encountered at the 5' end of the human SUP35 gene. The longest form of the protein, which contains the N-terminal extension, is the major form of Sup35p protein in non transfected cells. Moreover, an analog of the long form of Sup35p protein is found in various mouse tissues. We suggest that the protein encoded by SUP35 gene could have, at least in human, the properties described for the yeast [psi] element.

Importance of the C terminus of the hepatitis B virus precore protein in secretion of HBe antigen.

The hepatitis B virus (HBV) e antigen (HBeAg) is a 15 kDa soluble antigen derived from a precursor protein (precore protein) by two processing events, cleavage of the N-terminal signal peptide and cleavage of the C-terminal 34 amino acids. So far, the role of the C-terminal sequences in secretion has not been analysed in full. In this study deletion of the last 60 amino acids was found to abrogate HBeAg secretion whereas deletions of the last 10, 25 or 39 amino acids decreased its secretion rate. These data demonstrate that C-terminal precore protein sequences are crucial for HBe secretion and determine its secretion rate.

Characterization and biosynthesis of the woodchuck hepatitis virus e antigen.

The biosynthesis of the secretory core gene product of the woodchuck hepatitis virus (WHV) was studied in human cells. We have shown that the WHV e antigen was a N-glycosylated (most likely a diglycosylated) protein, with an apparent M(r) of 24K. To demonstrate that the WHV precore protein was correctly processed in human cells, we engineered chimeric proteins in which signal peptides or arginine-rich domains of WHV and hepatitis B virus (HBV) precore proteins were exchanged. Our results showed that both the signal peptide and the arginine-rich region of WHV precore protein were cleaved off during the secretion pathway, as previously reported for precore protein of human HBV and duck HBV. These observations demonstrate that the maturation process of the e antigen is conserved in hepadnaviruses. In addition, on the basis of inhibition experiments, we suggest that the cleavage of the carboxy terminus of the WHV precore protein occurred in a post-endoplasmic reticulum compartment, most likely beyond the medial Golgi, and that this cleavage was catalysed by an aspartyl protease.

Translation of the hepatitis B virus P gene by ribosomal scanning as an alternative to internal initiation.

The hepatitis B virus (HBV) P gene which encodes the reverse transcriptase and other proteins required for replication is expressed on the bicistronic mRNA pregenome which also encodes the capsid protein in its first cistron. Recent results have suggested that the hepadnaviral P gene is translated by internal entry of ribosomes upstream from the P gene, in the overlapping C gene. Using a reporter gene fused to the HBV C or P gene, we demonstrate that the C sequence does not allow internal initiation of translation. Alternatively, our results support a model in which the HBV P gene is translated by ribosomes which scan from the capped extremity of the bicistronic mRNA pregenome. The mechanism by which the ribosomes scan past four AUGs before they initiate translation at the P AUG was analyzed. Our results show that these AUGs are skipped via two mechanisms: leaky scanning on AUGs in a weak or suboptimal initiation context and translation of an out-of-C-frame minicistron followed by reinitiation at P AUG. The minicistron translation allows ribosomes to bypass an AUG in a favorable context that would otherwise be used as a start codon for translation of a truncated capsid protein. Our results suggest that this elaborated scanning mechanism permits the coordinate expression of the HBV C and P genes on the viral bicistronic mRNA pregenome.

Significance of anti-HBx antibodies in hepatitis B virus infection.

Serological responses to hepatitis B virus-X determinants have been noted in human sera, but conflicting findings concerning the correlation of anti-HBx antibodies with different stages of hepatitis B virus infection or pathological sequelae have been reported. Using an adenovirus-based eukaryotic vector, the 17 kD X protein was efficiently expressed in 293 cells. Cellular extracts containing the eukaryotic X protein have been used to screen for anti-HBx antibodies by immunoblot analysis in a large panel of sera from patients affected by hepatitis B virus chronic hepatitis, hepatocellular carcinoma and acute viral hepatitis. Sera from 32 of 171 (19%) chronic hepatitis B virus patients were positive for anti-HBx antibodies. Only one of thirty-two (3%) HBsAg-negative, anti-HBs/anti-HBc-positive chronic hepatitis serum was anti-HBx positive. Very few sera from primary hepatocellular carcinoma patients showed positivity for anti-HBx (8 of 149 or 5%). Anti-HBx were also detected in 8 of 48 (17%) acute viral hepatitis patients. In the four cases that were followed up weekly, anti-HBx antibodies appeared 3 to 4 wk after the onset of the clinical signs. To compare the X protein expressed in eukaryotic and prokaryotic cells as a substrate for anti-HBx antibody detection, 171 sera were screened with HBx fusion proteins expressed in Escherichia coli. The prokaryotic cell extract test seems to be more sensitive. During the chronic phase of hepatitis B virus infection, the presence of anti-HBx antibodies detected with the eukaryotic cell extract correlates with the presence of well-established markers of ongoing viral replication: serum hepatitis B virus-DNA (p less than 0.001) and intrahepatic HBcAg expression (p less than 0.001).

The DNA polymerase from the archaebacterium Sulfolobus acidocaldarius: a thermophilic and thermoresistant enzyme which can perform automated polymerase chain reaction.

A DNA polymerase purified from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius was used to perform automated DNA amplification at 70 degrees C as well as site directed mutagenesis by Polymerase Chain Reaction (P.C.R.). The yield of amplification performed at optimum MgCl2 concentration for the Taq or the S. acidocaldarius DNA polymerase, for the same DNA target, was equivalent. The ability of S. acidocaldarius DNA polymerase to perform P.C.R. under less stringent requirement of MgCl2 concentration gives this enzyme a non-negligible advantage over the Taq DNA polymerase.

Hepatitis B virus (HBV) X gene expression in human cells and anti-HBx antibodies detection in chronic HBV infection.

All mammalian hepatitis B virus genomes contain an open reading frame X (X-ORF) of unknown function which could encode a protein of 17 kDa. Using a plasmid containing the entire X-ORF preceded by the adenovirus type 2 major late promoter and its tripartite leader sequence efficient expression of the HBV X-gene was achieved. The X protein of 17 kDa was characterized by immunoblotting and immunoprecipitated with an antiserum prepared against a X fusion protein produced in E. coli. By cell fractionation and indirect immunofluorescence the X-protein was found at least in part associated with nuclei. Human cell extracts containing the X protein have been used to screen human sera for anti-HBx antibodies. Such antibodies were detected in sera from patients with active chronic hepatitis with ongoing viral replication. The efficient expression of the HBV X protein obtained will facilitate its functional analysis.

Internal entry of ribosomes and ribosomal scanning involved in hepatitis B virus P gene expression.

The recent demonstration that the synthesis of duck hepatitis B virus (HBV) reverse transcriptase does not require translational frameshifting and the finding that poliovirus mRNA translation occurs in a cap-independent manner by internal binding of ribosomes in the 5' noncoding region led us to design experiments to test the hypothesis of internal entry of ribosomes on C gene mRNA for HBV P gene expression. We show that in human cells, translation can be initiated at the first AUG of the HBV P gene by entry of ribosomes in a region located upstream of the P gene. Moreover, the leaky scanning of ribosomes observed on the first AUG of the HBV P gene could be responsible for the synthesis of the two forms of reverse transcriptase described for HBV particles.

Biosynthesis of hepatitis B virus e antigen: directed mutagenesis of the putative aspartyl protease site.

The C gene products of all mammalian hepadnaviruses contain a region with sequence similarities to the catalytic center of the aspartyl proteases. This region could have the capacity to cleave precore proteins, leading to the synthesis of e antigen. By site-directed mutagenesis on a plasmid containing the hepatitis B virus C gene, we have replaced either the Asp residue of the putative aspartyl protease catalytic center or an Asp residue located 3 amino acids upstream. Transient expression of the mutated hepatitis B virus C gene in human and mouse cells showed that none of these mutations prevented the secretion of an accurately processed HBe antigen. Thus, we demonstrated that the aspartyl protease responsible for e antigen precursor processing is not C gene encoded but is more likely to be a cellular enzyme. From these results, we suggest a model for the mechanism of e antigen synthesis.

Expression mechanism of the hepatitis B virus (HBV) C gene and biosynthesis of HBe antigen.

The C gene of the hepatitis B virus, which contains two in-phase initiation codons delimiting the pre-C sequence and the C region, directs the synthesis of the major protein of the capsid (HBcAg) and of a precore protein which upon processing results in the secretion of the HBeAg. We used an adenovirus-based vector to study in the human 293 cell line the C gene products, the intermediates of the precore protein processing and the kind of protease involved in this processing. The synthesis of the 21-kDa HBcAg polypeptide was dependent on the deletion of the pre-C sequence suggesting that a pre-C mRNA is not used for the synthesis of the major capsid protein. With the construct containing the complete C gene, two proteins of 25 and 22 kDa were detected intracellularly, corresponding to the unprocessed and partially processed precore protein, respectively. In addition, a 15-kDa protein (HBeAg) was secreted in the culture medium. Using pepstatin, an inhibitor specific for aspartyl proteinases, reduction of HBeAg secretion and accumulation of the 22-kDa processing intermediate were observed, suggesting the involvement of an aspartyl proteinase in the conversion of the 22-kDa protein into HBeAg.

Expression of an enzymatically active murine retroviral reverse transcriptase in human cells.

The region of the pol gene of the Moloney murine leukemia virus (M-MuLV) encoding the reverse transcriptase and RNase H activities was inserted in an eukaryotic expression vector and transiently expressed in human cultured cells. This results in the expression of high levels of reverse transcriptase activity. This enzyme, partially purified, also carries a RNase H activity, has the biochemical requirements of the viral enzyme and is recognized and inhibited by antibodies directed against a M-MuLV reverse transcriptase expressed in Escherichia coli.