Biomedical subjects

Cornelis W A Pleij

Publications and source records attributed to Cornelis W A Pleij.

16 recordsLinked to original sources

PubMed2006

The 5' non-translated region of Varroa destructor virus 1 (genus Iflavirus): structure prediction and IRES activity in Lymantria dispar cells.

Structure prediction of the 5' non-translated region (NTR) of four iflavirus RNAs revealed two types of potential internal ribosome entry site (IRES), which are discriminated by size and level of complexity, in this group of viruses. In contrast to the intergenic IRES of dicistroviruses, the potential 5' IRES structures of iflaviruses do not have pseudoknots. To test the activity of one of these, a bicistronic construct was made in which the 5' NTR of Varroa destructor virus 1 (VDV-1) containing a putative IRES was cloned in between two reporter genes, enhanced green fluorescent protein and firefly luciferase (Fluc). The presence of the 5' NTR of VDV-1 greatly enhanced the expression levels of the second reporter gene (Fluc) in Lymantria dispar Ld652Y cells. The 5' NTR was active in a host-specific manner, as it showed lower activity in Spodoptera frugiperda Sf21 cells and no activity in Drosophila melanogaster S2 cells.

5' Untranslated Regions↗

PubMed2004

Crystal structure of an empty capsid of turnip yellow mosaic virus.

Empty capsids (artificial top component) of turnip yellow mosaic virus were co-crystallized with an encapsidation initiator RNA hairpin. No clear density was observed for the RNA, but there were clear differences in the conformation of a loop of the coat protein at the opening of the pentameric capsomer (formed by five A-subunits) protruding from the capsid, compared to the corresponding loop in the intact virus. Further differences were found at the N terminus of the A-subunit. These differences have implications for the mechanism of decapsidation of the virus, required for infection.

Capsid↗

PubMed2004

Two histidines of the coat protein of turnip yellow mosaic virus at the capsid interior are crucial for viability.

RNA-coat protein interactions in turnip yellow mosaic virus (TYMV) have been shown to involve low pK proton-donating groups. Two different types of interaction have been proposed. In the so-called type I interaction, protonated C-residues interact with acidic amino acids at low pH, thereby providing a rationale for the high C-content (38%) of the genomic RNA. The type II interaction involves charged histidines interacting with phosphates of the RNA backbone. Site-directed mutagenesis of the TYMV coat protein and subsequent in vivo analysis were performed to distinguish between these two types of RNA-protein interaction. The results reveal a prominent role for the histidines H68 and H180, since mutation to an alanine residue inhibits symptom development on secondary leaves, indicating that spreading of the virus in the plant is blocked. Viral RNA and coat protein synthesis are not altered, showing that these two histidines may play a role in the process of RNA encapsidation. Overexpression of the TYMV coat protein in Escherichia coli leads to the formation of bona fide capsids, showing that the two histidines are not critical in capsid assembly. Mutagenesis of the acidic amino acids D11, E135, and D143 to alanine apparently did not interfere with virus viability. The functional role of the histidines during the infection cycle is discussed in terms of the structure of the coat protein, both at the level of amino acid sequence conservation among the members of the Tymoviridae family and as the three-dimensional structure of the coat protein.

Alanine↗

PubMed2004

Conservation of RNA structures enables TNV and BYDV 5' and 3' elements to cooperate synergistically in cap-independent translation.

The subgenomic RNA 2 of tobacco necrosis virus A (TNV sgRNA2) encodes the viral coat protein, is unpolyadenylated and presumably uncapped. Here, we show that TNV sgRNA2 is translated cap independently. This cap-independent translation requires the leader and a 140 nt element of the trailer both in wheat germ extract and in tobacco protoplasts. Similar to barley yellow dwarf virus (BYDV), the TNV 5' and 3' elements stimulate translation synergistically. Computer-aided phylogenetic analysis of the secondary structure of the TNV trailer revealed that the 3' translation element is part of a major conserved stem-loop that contains similarities to structures in the BYDV 3' translation element. These data suggest that the translation mechanisms of TNV sgRNA2 and BYDV RNA are related. To further characterize this relationship, we tested whether cooperativity exists between TNV sgRNA2 and BYDV 5' and 3' elements. We found that the TNV sgRNA2 5' element stimulates translation synergistically with the BYDV 3' element in vitro. This finding is the first evidence for conservation of structures that enable a 5'-3' interaction stimulating cap-independent translation.

3' Untranslated Regions↗

PubMed2004

Single molecule studies of RNA secondary structure: AFM of TYMV viral RNA.

Nowadays, the development of experimental procedures for the determination of the secondary structure of RNA molecules is taking advantage of the novel single-molecule probing and imaging techniques. We report a method for the mapping of the secondary structure of RNA molecules spread on a flat surface by means of the atomic force microscope. Globular domains comprising groups of RNA secondary and tertiary structure elements separated by unstructured domains can be discerned in the micrographs and their position along the molecule contour can be measured directly on unstained specimens. We have analyzed the morphology of a population of single molecules of 3' fragments of the Turnip Yellow Mosaic Virus RNA shorter than 1 kb in different temperature and electrolytic conditions. We found a satisfying agreement of the shape of the imaged structures with previously available evidence. The method we have developed can be used to map also different types of RNA molecules and has the advantage of showing the distribution of the single molecule conformations within the population.

Image Processing, Computer-Assisted↗

PubMed2004

tRNA-like structure regulates translation of Brome mosaic virus RNA.

For various groups of plant viruses, the genomic RNAs end with a tRNA-like structure (TLS) instead of the 3' poly(A) tail of common mRNAs. The actual function of these TLSs has long been enigmatic. Recently, however, it became clear that for turnip yellow mosaic virus, a tymovirus, the valylated TLS(TYMV) of the single genomic RNA functions as a bait for host ribosomes and directs them to the internal initiation site of translation (with N-terminal valine) of the second open reading frame for the polyprotein. This discovery prompted us to investigate whether the much larger TLSs of a different genus of viruses have a comparable function in translation. Brome mosaic virus (BMV), a bromovirus, has a tripartite RNA genome with a subgenomic RNA4 for coat protein expression. All four RNAs carry a highly conserved and bulky 3' TLS(BMV) (about 200 nucleotides) with determinants for tyrosylation. We discovered TLS(BMV)-catalyzed self-tyrosylation of the tyrosyl-tRNA synthetase but could not clearly detect tyrosine incorporation into any virus-encoded protein. We established that BMV proteins do not need TLS(BMV) tyrosylation for their initiation. However, disruption of the TLSs strongly reduced the translation of genomic RNA1, RNA2, and less strongly, RNA3, whereas coat protein expression from RNA4 remained unaffected. This aberrant translation could be partially restored by providing the TLS(BMV) in trans. Intriguingly, a subdomain of the TLS(BMV) could even almost fully restore translation to the original pattern. We discuss here a model with a central and dominant role for the TLS(BMV) during the BMV infection cycle.

Base Sequence↗

PubMed2003

Structural motifs in the RNA encoded by the early nodulation gene enod40 of soybean.

The plant gene enod40 is highly conserved among legumes and also present in various non-legume species. It is presumed to play a central regulatory role in the Rhizobium-legume interaction, being expressed well before the initiation of cortical cell divisions resulting in nodule formation. Two small peptides encoded by enod40 mRNA as well as its secondary structure have been shown to be key elements in the signalling processes underlying nodule organogenesis. Here results concerning the secondary structure of mRNA of enod40 in soybean are presented. This study combined a theoretical approach, involving structure prediction and comparison, as well as structure probing. Our study indicates five conserved domains in enod40 mRNA among numerous leguminous species. Structure comparison suggests that some domains are also conserved in non-leguminous species and that an additional domain exists that was found only in leguminous species developing indeterminate nodules. Enzymatic and chemical probing data support the structure for three of the domains, and partially for the remaining two. The rest of the molecule appears to be less structured. Some of the domains include motifs, such as U-containing internal loops and bulges, which seem to be conserved. Therefore, they might be involved in the regulatory role of enod40 RNA.

Base Sequence↗

PubMed2003

Entrapping ribosomes for viral translation: tRNA mimicry as a molecular Trojan horse.

Turnip yellow mosaic virus (TYMV) has a genomic plus-strand RNA with a 5' cap followed by overlapping and different reading frames for the movement protein and polyprotein, while the distal coat protein cistron is translated from a subgenomic RNA. The 3'-untranslated region harbors a tRNA-like structure (TLS) to which a valine moiety can be added and it is indispensable for virus viability. Here, we report about a surprising interaction between TYMV-RNA-programmed ribosomes and 3'-valylated TLS that yields polyprotein with the valine N terminally incorporated by a translation mechanism resistant to regular initiation inhibitors. Disruption of the TLS exclusively abolishes polyprotein synthesis, which can be restored by adding excess TLS in trans. Our observations imply a novel eukaryotic mechanism for internal initiation of mRNA translation.

3' Untranslated Regions↗

PubMed2003

The 5'-proximal hairpin of turnip yellow mosaic virus RNA: its role in translation and encapsidation.

The RNA genome of turnip yellow mosaic virus (TYMV) consists of more than 6,000 nucleotides. During a study of the roles of the two hairpins located in its 90-nucleotide 5' untranslated region, it was observed that stabilization of the 5'-proximal hairpin leads to a delay in the development of symptoms on plants. This delay in symptom development for both locally and systemically infected leaves was found to be dependent on a change in the free energy of the hairpin caused by introduced mutations. A protoplast transfection assay revealed that the accumulation of plus-strand full-length RNA and subgenomic RNA, as well as protein expression levels, was affected by hairpin stability. Stabilization of this hairpin inhibited translation. A model is proposed in which a destabilized 5'-proximal hairpin allows maximal translation of the viral proteins. It is suggested that this hairpin may exist in close proximity to the 5' cap as long as its stability is low enough to enable translation. However, at an acidic pH, the hairpin structure becomes more stable and is functionally transformed into the initiation signal for viral packaging. Slightly acidic conditions can be found in chloroplasts, where TYMV assembly is driven by a low pH generated by active photosynthesis.

Base Sequence↗

PubMed2002

Protonation of non-Watson-Crick base pairs and encapsidation of turnip yellow mosaic virus RNA.

The 5' UTR of turnip yellow mosaic virus RNA contains two conserved hairpins with internal loops consisting of C.C and C.A mismatches. In this article, evidence is presented indicating that the 5' proximal hairpin functions as an encapsidation initiation signal. Extensive mutagenesis studies on this hairpin and sequencing of virus progeny showed a clear preference for C.C and C.A mismatches within the internal loop. The importance of these mismatches lies in their pH-dependent protonation and stable base pair formation. Encapsidation efficiency was found to be severely affected for several mutants lacking the protonatable mismatches in the internal loop of the 5' proximal hairpin. Furthermore, gel mobility-shift assays were performed with various RNA hairpins and empty capsids with a hole. Protonatable hairpins containing C.C and/or C.A pairs were found to bind specifically to the interior of the protein shell under acidic conditions (pH 4.5) in the presence of spermidine. Based on these results we propose that this binding of protonated cytosines to the coat protein of turnip yellow mosaic virus may represent a new motif in RNA-protein interactions.

5' Untranslated Regions↗

PubMed2002

Structural variation and functional importance of a D-loop-T-loop interaction in valine-accepting tRNA-like structures of plant viral RNAs.

Valine-accepting tRNA-like structures (TLSs) are found at the 3' ends of the genomic RNAs of most plant viruses belonging to the genera Tymovirus, Furovirus, Pomovirus and Pecluvirus, and of one Tobamovirus species. Sequence alignment of these TLSs suggests the existence of a tertiary D-loop-T-loop interaction consisting of 2 bp, analogous to those in the elbow region of canonical tRNAs. The conserved G(18).Psi(55) pair of regular tRNAs is found to covary in these TLSs between G.U (possibly also modified to G.Psi) and A.G. We have mutated the relevant bases in turnip yellow mosaic virus (TYMV) and examined the mutants for symptom development on Chinese cabbage plants and for accumulation of genetic reversions. Development of symptoms is shown to rely on the presence of either A.G or G.U in the original mutants or in revertants. This finding supports the existence and functional importance of this tertiary interaction. The fact that only G.U and A.G are accepted at this position appears to result from steric and energetic limitations related to the highly compact nature of the elbow region. We discuss the implications of these findings for the various possible functions of the valine-accepting TLS.

Base Sequence↗

PubMed2002

Functional evidence for D- and T-loop interactions in tmRNA.

During bacterial protein synthesis, stalled ribosomes can be rescued by tmRNA, a molecule with both tRNA and mRNA features. The tRNA region of tmRNA has sequence similarity with tRNA(Ala) and also has a clover-leaf structure folded similarly as in canonical tRNAs. Here we propose the L-shape of tmRNA to be stabilized by two tertiary interactions between its D- and T-loop on the basis of phylogenetic and experimental evidence. Mutational analysis clearly demonstrates a tertiary interaction between G(13) and U(342). Strikingly, this in evolution conserved interaction is not primarily important for tmRNA alanylation and for binding to elongation factor Tu, but especially for a proper functioning of SmpB.

Alanine↗

PubMed2002

Selective pressures on RNA hairpins in vivo and in vitro.

Comparison of the most stable potential hairpins in the sequences of natural ribozymes with those in the randomized sequences has revealed that the hairpin loop energies are lower than expected by chance. Although these hairpins are not necessarily parts of functional structures, there is a selective pressure to diminish the destabilizing free energies of the hairpin loops. In contrast, no significant bias is observed in the stacking values of the most stable stems. In the ribozymes isolated in vitro the loops of potential hairpins are closer to random values, which can result in less efficient folding rates. Furthermore, the effects of kinetic traps seem to be more significant in the folding pathways of the in vitro isolates due to a potential to form stable stacks incompatible with the functional folds. Similarly to natural ribozyme sequences, the untranslated regions of viral RNAs also form hairpins with relatively low loop free energies. These evolutionary trends suggest ways for efficient engineering of improved RNA constructs on the basis of analysis of in vitro isolates and approaches for the search of regions coding for functional RNA structures in large genome sequences.

Evolution, Molecular↗

PubMed2002

Self-induced structural switches in RNA.

Many biologically active RNAs show a switch in their secondary structure, which is accompanied by changes in their function. Such changes in secondary structure often require trans-acting factors, e.g. RNA chaperones. However, several biologically active RNAs do not require trans-acting factors for this structural switch, which is therefore indicated here as a "self-induced switch". These self-induced structural switches have several characteristics in common. They all start from a metastable structure, which is maintained for some time allowing or blocking a particular function of the RNA. Hereafter, a structural element becomes available, e.g. during transcription, triggering a rapid transition into a stable conformation, which again is accompanied by either a gain or loss of function. A further common element of this type of switches is the involvement of a branch migration or strand displacement reaction, which lowers the energy barrier of the reaction sufficiently to allow rapid refolding. Here, we review a number of these self-induced switches in RNA secondary structure as proposed for several systems. A general model for this type of switches is presented, showing its importance in the biology of functionally active RNAs.

Animals↗

PubMed2002

The 5' and 3' extremities of the satellite tobacco necrosis virus translational enhancer domain contribute differentially to stimulation of translation.

The translational enhancer domain (TED) of satellite tobacco necrosis virus (STNV) RNA stimulates translation of uncapped RNAs autonomously. Here we set out to identify the 5' and 3' extremities of TED and features of these sequences with respect to translation. We found that both in wheat germ extract and in tobacco protoplasts, the 5' border is confined to 3 nt. Mutational analysis revealed that the autonomous function of TED is sensitive to 5' flanking sequences. At the 3' end of TED, 23 nt have a cumulative, quantitative effect on translation in wheat germ extract, whereas in tobacco protoplasts, the most 3' 14 nt of these 23 nt do not enhance translation. The 5' and 3' sequence requirements triggered the development of a new secondary structure model. In this model, TED folds into a phylogenetically conserved stem-loop structure in which the essential 5' nucleotides base-pair with the 3' nucleotides that stimulate translation both in vitro and in vivo. Importantly, the 14 3' nucleotides in TED that stimulate translation in the wheat germ extract only do not require the predicted base-pairing in order to function. The discrepancy between in vitro and in vivo sequence requirements thus correlates with potential base-pairing requirements, opening the possibility that TED contains two functional domains.

Base Sequence↗

PubMed2002

Complete genome sequence of Montana Myotis leukoencephalitis virus, phylogenetic analysis and comparative study of the 3' untranslated region of flaviviruses with no known vector.

Montana Myotis leukoencephalitis virus (MMLV), a virus isolated from bats, causes an encephalitis in small rodents reminiscent of flavivirus encephalitis in humans. The complete MMLV genome is 10690 nucleotides long and encodes a putative polyprotein of 3374 amino acids. The virus contains the same conserved motifs in genes that are believed to be interesting antiviral targets (NTPase/helicase, serine protease and RNA-dependent RNA polymerase) as flaviviruses of clinical importance. Phylogenetic analysis of the entire coding region has confirmed the classification of MMLV in the clade of the flaviviruses with no known vector (NKV) and within this clade to the Rio Bravo branch (both viruses have the bat as their vertebrate host). We have provided for the first time a comparative analysis of the RNA folding of the 3' UTR of the NKV flaviviruses (Modoc, Rio Bravo and Apoi viruses, in addition to MMLV). Structural elements in the 3' UTR that are preserved among other flaviviruses have been revealed, as well as elements that distinguish the NKV from the mosquito- and tick-borne flaviviruses. In particular, the pentanucleotide sequence 5' CACAG 3', which is conserved in all mosquito- and tick-borne flaviviruses, is replaced by the sequence 5' C(C/U)(C/U)AG 3' in the loop of the 3' long stable hairpin structure of all four NKV flaviviruses. The availability of this latter sequence motif allows us to designate a virus as either an NKV or a vector-borne flavivirus.

3' Untranslated Regions↗