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T Hohn

Publications and source records attributed to T Hohn.

At least 37 records · Page 2Linked to original sources
PubMed2000

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.

Amino Acid Sequence↗
PubMed2000

Interaction of the cauliflower mosaic virus coat protein with the pregenomic RNA leader.

Using the yeast three-hybrid system, the interaction of the Cauliflower mosaic virus (CaMV) pregenomic 35S RNA (pgRNA) leader with the viral coat protein, its precursor, and a series of derivatives was studied. The purine-rich domain in the center of the pgRNA leader was found to specifically interact with the coat protein. The zinc finger motif of the coat protein and the preceding basic domain were essential for this interaction. Removal of the N-terminal portion of the basic domain led to loss of specificity but did not affect the strength of the interaction. Mutations of the zinc finger motif abolished not only the interaction with the RNA but also viral infectivity. In the presence of the very acidic C-terminal domain, which is part of the preprotein but is not present in the mature CP, the interaction with the RNA was undetectable.

5' Untranslated Regions↗
PubMed2000

The rice tungro bacilliform virus gene II product interacts with the coat protein domain of the viral gene III polyprotein.

Rice tungro bacilliform virus (RTBV) is a plant pararetrovirus whose DNA genome contains four genes encoding three proteins and a large polyprotein. The function of most of the viral proteins is still unknown. To investigate the role of the gene II product (P2), we searched for interactions between this protein and other RTBV proteins. P2 was shown to interact with the coat protein (CP) domain of the viral gene III polyprotein (P3) both in the yeast two-hybrid system and in vitro. Domains involved in the P2-CP association have been identified and mapped on both proteins. To determine the importance of this interaction for viral multiplication, the infectivity of RTBV gene II mutants was investigated by agroinoculation of rice plants. The results showed that virus viability correlates with the ability of P2 to interact with the CP domain of P3. This study suggests that P2 could participate in RTBV capsid assembly.

Amino Acid Sequence↗
PubMed2000

Termination and peptide release at the upstream open reading frame are required for downstream translation on synthetic shunt-competent mRNA leaders.

We have shown recently that a stable hairpin preceded by a short upstream open reading frame (uORF) promotes nonlinear ribosome migration or ribosome shunt on a synthetic mRNA leader (M. Hemmings-Mieszczak and T. Hohn, RNA 5:1149-1157, 1999). We have now used the model mRNA leader to study further the mechanism of shunting in vivo and in vitro. We show that a full cycle of translation of the uORF, including initiation, elongation, and termination, is a precondition for the ribosome shunt across the stem structure to initiate translation downstream. Specifically, AUG recognition and the proper release of the nascent peptide are necessary and sufficient for shunting. Furthermore, the stop codon context must not impede downstream reinitiation. Translation of the main ORF was inhibited by replacement of the uORF by coding sequences repressing reinitiation but stimulated by the presence of the virus-specific translational transactivator of reinitiation (cauliflower mosaic virus pVI). Our results indicate reinitiation as the mechanism of translation initiation on the synthetic shunt-competent mRNA leader and suggest that uORF-dependent shunting is more prevalent than previously anticipated. Within the above constraints, uORF-dependent shunting is quite tolerant of uORF and stem sequences and operates in systems as diverse as plants and fungi.

5' Untranslated Regions↗
PubMed1999

A stable hairpin preceded by a short open reading frame promotes nonlinear ribosome migration on a synthetic mRNA leader.

The regulation of cauliflower mosaic virus (CaMV) pregenomic 35S RNA translation occurs via nonlinear ribosome migration (ribosome shunt) and is mediated by an elongated hairpin structure in the leader. The replacement of the viral leader by a series of short, low-energy stems in either orientation supports efficient ribosomal shunting, showing that the stem per se, and not its sequence, is recognized by the translation machinery. The requirement for cis-acting sequences from the unstructured terminal regions of the viral leader was analyzed: the 5'-terminal polypyrimidine stretch and the short upstream open reading frame (uORF) A stimulate translation, whereas the 3'-flanking region seems not to be essential. Based on these results, an artificial leader was designed with a stable stem flanked by unstructured sequences derived from parts of the 5'- and 3'-proximal regions of the CaMV 35S RNA leader. This artificial leader is shunt-competent in translation assays in vivo and in vitro, indicating that a low-energy stem, broadly used as a device to successfully interfere with ribosome scanning, can efficiently support translation, if preceded by a short uORF. The synthetic 140-nt leader can functionally replace the CaMV 35S RNA 600-nt leader, thus implicating the universal role that nonlinear ribosome scanning could play in translation initiation in eukaryotes.

5' Untranslated Regions↗
PubMed1999

Upstream and downstream sequence elements determine the specificity of the rice tungro bacilliform virus promoter and influence RNA production after transcription initiation.

The contribution of sequences upstream and downstream of the transcription start site to the strength and specificity of the promoter of rice tungro bacilliform virus was analysed in transgenic rice plants. The promoter is strongly stimulated by downstream sequences which include an intron and is active in all vascular and epidermal cells. Expression in the vascular tissue requires a promoter element located between -100 and -164 to which protein(s) from rice nuclear extracts bind. Elimination of this region leads to specificity for the epidermis. Due to the presence of a polyadenylation signal in the intron, short-stop RNA is produced from the promoter in addition to full-length primary transcript and its spliced derivatives. The ratio between short-stop RNA and full-length or spliced RNA is determined by upstream promoter sequences, suggesting the assembly of RNA polymerase complexes with different processivity on this promoter.

Badnavirus↗
PubMed1999

Nuclear targeting of the cauliflower mosaic virus coat protein.

The entry of the viral genomic DNA of cauliflower mosaic virus into the nucleus is a critical step of viral infection. We have shown by transient expression in plant protoplasts that the viral coat protein (CP), which is processed from the product of open reading frame IV, contains an N-terminal nuclear localization signal (NLS). The NLS is exposed on the surface of the virion and is thus available for interaction with a putative NLS receptor. Phosphorylation of the matured CP did not influence the nuclear localization of the protein but improved protein stability. Mutation of the NLS completely abolished viral infectivity, thus indicating its importance in the virus life cycle. The NLS seems to be regulated by the N terminus of the precapsid, which inhibits its nuclear targeting. This regulation could be important in allowing virus assembly in the cytoplasm.

Amino Acid Sequence↗
PubMed1998

The open reading frame III product of cauliflower mosaic virus forms a tetramer through a N-terminal coiled-coil.

The open reading frame III product of cauliflower mosaic virus is a protein of 15 kDa (p15) that is essential for the virus life cycle. It was shown that the 34 N-terminal amino acids are sufficient to support protein-protein interaction with the full-length p15 in the yeast two-hybrid system. A corresponding peptide was synthesized and a recombinant p15 was expressed in Escherichia coli and purified. Circular dichroism spectroscopy showed that the peptide and the full-length protein can assume an alpha-helical conformation. Analytical centrifugation allowed to determine that p15 assembles as a rod-shaped tetramer. Oxidative cross-linking of N-terminal cysteines of the peptide generated specific covalent oligomers, indicating that the N terminus of p15 is a coiled-coil that assembles as a parallel tetramer. Mutation of Lys22 into Asp destabilized the tetramer and put forward the presence of a salt bridge between Lys22 and Asp24 in a model building of the stalk. These results suggest a model in which the stalk segment of p15 is located at its N terminus, followed by a hinge that provides the space for presenting the C terminus for interactions with nucleic acids and/or proteins.

Amino Acid Sequence↗
PubMed1998

Nonlinear ribosome migration on cauliflower mosaic virus 35S RNA in transgenic tobacco plants.

Cauliflower mosaic virus (CaMV) uses a specialised translation mechanism to bypass the long leader sequence of the 35S RNA. The effect of the CaMV 35S RNA leader sequence on the expression of a downstream beta-glucuronidase (GUS) reporter gene was studied in transgenic tobacco plants. Enzymatic GUS assays of these transgenic plants show that a shunt mechanism of translation indeed occurs in planta with an average efficiency of 5% compared with the leaderless construct. Histological GUS analyses indicate that the shunt mechanism occurs throughout the whole plant and at all developmental stages.

Blotting, Southern↗
PubMed1998

Ribosome shunting in cauliflower mosaic virus. Identification of an essential and sufficient structural element.

A wheat germ cell-free system was used to study details of ribosome shunting promoted by the cauliflower mosaic virus 35 S RNA leader. By testing a dicistronic construct with the leader placed between two coding regions, we confirmed that the 35 S RNA leader does not include an internal ribosome entry site of the type observed with picornavirus RNAs. A reporter gene fused to the leader was shown to be expressed by ribosomes that had followed the bypass route (shunted) and, with lower efficiency, by ribosomes that had scanned through the whole region. Stem section 1, the most stable of the three stem sections of the leader, was shown to be an important structural element for shunting. Mutations that abolished formation of this stem section drastically reduced reporter gene expression, whereas complementary mutations that restored stem section 1 also restored shunting. A micro-leader capable of shunting consisting of stem section 1 and flanking sequences could be defined. A small open reading frame preceding stem section 1 enhances shunting.

Base Composition↗
PubMed1998

The cauliflower mosaic virus capsid protein: assembly and nucleic acid binding in vitro.

The capsid protein of the cauliflower mosaic virus (CaMV) was expressed in a bacterial system to study CaMV assembly. Bacterial lysates contained soluble particulate material and insoluble inclusion bodies that were both used for analysis. In vitro renaturation of pIV derivatives lead to the appearance of folded sheets or large tubular structures in electron microscopy. The region between amino acid positions 77 and 332 is sufficient for self-aggregation of pIV in vitro. C-terminal deletion to amino acid position 265 still allowed dimerization but prevented further aggregation. Nucleic acid binding assays of immobilized pIV derivatives demonstrated that a region located upstream of the retroviral "zinc finger-like" motif is involved in unspecific binding dsDNA, ssDNA and RNA.

Amino Acid Sequence↗
PubMed1998

Forced evolution reveals the importance of short open reading frame A and secondary structure in the cauliflower mosaic virus 35S RNA leader.

Cauliflower mosaic virus pregenomic 35S RNA begins with a long leader sequence containing an extensive secondary structure and up to nine short open reading frames (sORFs), 2 to 35 codons in length. To test whether any of these sORFs are required for virus viability, their start codons were mutated either individually or in various combinations. The resulting viral mutants were tested for infectivity on mechanically inoculated turnip plants. Viable mutants were passaged several times, and the stability of the introduced mutations was analyzed by PCR amplification and sequencing. Mutations at the 5'-proximal sORF A and in the center of the leader resulted in delayed symptom development and in the appearance of revertants. In the central leader region, the predicted secondary structure, rather than the sORF organization, was restored, while true reversions or second-site substitutions in response to mutations of sORF A restored this sORF. Involvement of sORF A and secondary structure of the leader in the virus replication cycle, and especially in translation of the 35S RNA via ribosome shunting, is discussed.

Base Sequence↗
PubMed1998

Regulation of CaMV 35 S RNA translation is mediated by a stable hairpin in the leader.

The cauliflower mosaic virus (CaMV) 35 S RNA functions as both messenger and pregenomic RNA under the control of its 600-nt leader, which contains regulatory elements involved in splicing, polyadenylation, translation, reverse transcription, and packaging. We have recently documented that the 35 S RNA leader adopts an elongated hairpin conformation and additional higher-order structures, a long-range pseudoknot and a dimer. Alternative structures might coexist, probably fulfilling specialized functions. In this paper, we analyze the biological significance of the elongated hairpin structure. We have introduced a spectrum of large deletions and small substitutions in the 35 S RNA leader and characterized their impact on the structure by temperature gradient gel electrophoresis. This analysis showed that the elongated hairpin consists of three sections of different stability (stem section I, II, and III). The overall secondary structure is relatively stable in the range of 10-32 degrees C. It melts between 32 and 38 degrees C in a manner indicating that the most stable base pairing occurs at the base of the elongated hairpin (stem section I). Mutations that destabilize stem section I decrease both the melting temperature of the leader and the expression in vitro and in vivo of the downstream CAT-reporter gene. Compensatory mutations restoring the stable elongated hairpin upregulate the translation efficiency. Our results demonstrate that the regulation of translation of the CaMV 35 S RNA is mediated by a stable hairpin in the leader.

Base Sequence↗
PubMed1997

Plant ribosome shunting in vitro.

It has been proposed that cauliflower mosaic virus 35S RNA with its 600 nt long leader uses an unusual translation process (the translational shunt). A wheat germ in vitro translation assay was used to improve the study of this mechanism. Deletions, the introduction of stable stem-loop structures, and the inhibitory effect of antisense oligonucleotides on gene expression were used to determine the roles of various parts of the leader. It was found that the 5'- and 3'-ends of the leader are absolutely required for translation whereas the middle part is apparently dispensable. These results confirm the data already reported from transient expression experiments with protoplasts. However, the in vitro data suggest in contrast to protoplast experiments that only two relatively short regions at both ends, approximately 100 nt each, are required. The in vitro system provides tools for further studying the shunt model at the molecular level and for examining the involvement of proteins in this mechanism. Shunting was also found to occur with the rice tungro bacilliform virus leader. As wheat is neither a host plant of cauliflower mosaic virus nor rice tungro bacilliform virus, the shunt seems to be host independent, a finding that deviates from earlier studies in protoplasts.

Badnavirus↗
PubMed1997

Alternative structures of the cauliflower mosaic virus 35 S RNA leader: implications for viral expression and replication.

The CaMV 35 S RNA functions as both messenger and pregenomic RNA under the control of its 600 nts leader, which contains regulatory elements involved in splicing, polyadenylation, translation, reverse transcription, and probably also packaging. The structure of the leader has been characterized theoretically and experimentally. The predicted conformation, a low-energy elongated hairpin, base-pairing the two halves of the leader, with a cross-like structure at the top, is strongly supported by enzymatic probing, chemical modification, and phylogenetic comparison. The elongated hairpin is stabilized by strong base-pairing between the ends of the leader, regions which are important in allowing translation downstream of the leader via the ribosome shunt mechanism. At high ionic strength the 35 S RNA leader exhibits additional higher order structures of low electrophoretic mobility: (1) a long-range pseudoknot connecting central and terminal parts of the leader; (2) a dimer. Alternative structures of the CaMV 35 S RNA leader may co-exist and have specialized functions. Their potential impact on CaMV life cycle regulation is discussed.

Base Sequence↗
PubMed1997

Rice tungro bacilliform virus open reading frames II and III are translated from polycistronic pregenomic RNA by leaky scanning.

Posttranscriptional components of the gene expression mechanism of rice tungro bacilliform virus (RTBV) were studied in transiently transfected protoplasts. RTBV translates several open reading frames from a polycistronic mRNA by leaky scanning. This mechanism is supported by the particular sequence features of the corresponding genome region and does not require a virus-encoded transactivator.

Badnavirus↗
PubMed1996

Cellular proteins bind to multiple sites of the leader region of cauliflower mosaic virus 35S RNA.

UV crosslinking assays were performed to characterize interactions between the leader of the cauliflower mosaic virus (CaMV) 35S RNA and cellular, cytoplasmic proteins. From turnip, a host for CaMV, three different proteins, p35, p49, and p100, interacted with multiple binding sites within the leader p35 binds to RNA nonspecifically and p49 binds with low specificity, whereas p100 interacts specifically with viral sequences. The expression of the proteins is not induced upon virus infection, as there is no difference in the protein pattern between healthy and infected cell extracts. In a cellular fractionation, p35 and p100 remain in the high-speed supernatant whereas p49 cosediments with polysomes. A possible involvement of these proteins in the translation of the CaMV 35S RNA is discussed.

Base Sequence↗