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1.
Open Biol ; 2(10): 120109, 2012 Oct.
Article in English | MEDLINE | ID: mdl-23155484

ABSTRACT

Programmed ribosomal frameshifting is used in the expression of many virus genes and some cellular genes. In eukaryotic systems, the most well-characterized mechanism involves -1 tandem tRNA slippage on an X_XXY_YYZ motif. By contrast, the mechanisms involved in programmed +1 (or -2) slippage are more varied and often poorly characterized. Recently, a novel gene, PA-X, was discovered in influenza A virus and found to be expressed via a shift to the +1 reading frame. Here, we identify, by mass spectrometric analysis, both the site (UCC_UUU_CGU) and direction (+1) of the frameshifting that is involved in PA-X expression. Related sites are identified in other virus genes that have previously been proposed to be expressed via +1 frameshifting. As these viruses infect insects (chronic bee paralysis virus), plants (fijiviruses and amalgamaviruses) and vertebrates (influenza A virus), such motifs may form a new class of +1 frameshift-inducing sequences that are active in diverse eukaryotes.


Subject(s)
Frameshifting, Ribosomal/physiology , Gene Expression Regulation, Viral/physiology , Influenza A virus/metabolism , Repressor Proteins/metabolism , Viral Nonstructural Proteins/metabolism , Influenza A virus/genetics , Repressor Proteins/genetics , Viral Nonstructural Proteins/genetics
2.
Mol Cell Biol ; 21(24): 8657-70, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11713298

ABSTRACT

Here we investigated ribosomal pausing at sites of programmed -1 ribosomal frameshifting, using translational elongation and ribosome heelprint assays. The site of pausing at the frameshift signal of infectious bronchitis virus (IBV) was determined and was consistent with an RNA pseudoknot-induced pause that placed the ribosomal P- and A-sites over the slippery sequence. Similarly, pausing at the simian retrovirus 1 gag/pol signal, which contains a different kind of frameshifter pseudoknot, also placed the ribosome over the slippery sequence, supporting a role for pausing in frameshifting. However, a simple correlation between pausing and frameshifting was lacking. Firstly, a stem-loop structure closely related to the IBV pseudoknot, although unable to stimulate efficient frameshifting, paused ribosomes to a similar extent and at the same place on the mRNA as a parental pseudoknot. Secondly, an identical pausing pattern was induced by two pseudoknots differing only by a single loop 2 nucleotide yet with different functionalities in frameshifting. The final observation arose from an assessment of the impact of reading phase on pausing. Given that ribosomes advance in triplet fashion, we tested whether the reading frame in which ribosomes encounter an RNA structure (the reading phase) would influence pausing. We found that the reading phase did influence pausing but unexpectedly, the mRNA with the pseudoknot in the phase which gave the least pausing was found to promote frameshifting more efficiently than the other variants. Overall, these experiments support the view that pausing alone is insufficient to mediate frameshifting and additional events are required. The phase dependence of pausing may be indicative of an activity in the ribosome that requires an optimal contact with mRNA secondary structures for efficient unwinding.


Subject(s)
Nucleic Acid Conformation , RNA/chemistry , Ribosomes/metabolism , Ribosomes/physiology , Animals , Base Sequence , Frameshift Mutation , Molecular Sequence Data , Mutagenesis, Site-Directed , Plasmids/metabolism , Protein Biosynthesis , RNA/metabolism , RNA, Messenger/metabolism , Rabbits , Reticulocytes/metabolism , Time Factors
3.
RNA ; 7(5): 765-73, 2001 May.
Article in English | MEDLINE | ID: mdl-11350040

ABSTRACT

A method is described for depleting rabbit reticulocyte lysates and wheat germ extracts of endogenous tRNAs by affinity chromatography using a matrix generated by coupling ethanolamine to epoxy-activated Sepharose 6B. Greater than 90% depletion of tRNA is achieved with the result that translation becomes in effect absolutely dependent on added tRNA. This depletion procedure should prove very useful for studying the influence of tRNA concentration, and the spectrum of the tRNA population, on recoding events such as programmed frameshifting and readthrough of termination codons.


Subject(s)
Protein Biosynthesis , RNA, Transfer , Animals , RNA, Messenger , RNA, Transfer/isolation & purification , Rabbits , Reticulocytes , Triticum/genetics
4.
J Virol ; 74(21): 10081-95, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11024137

ABSTRACT

Human papillomavirus type 16 (HPV16) infects cervical epithelium and is associated with the majority of cervical cancers. The E1E4 protein of HPV16 but not those of HPV1 or HPV6 was found to associate with a novel member of the DEAD box protein family of RNA helicases through sequences in its C terminus. This protein, termed E4-DBP (E4-DEAD box protein), has a molecular weight of 66,000 (66K) and can shuttle between the nucleus and the cytoplasm. It binds to RNA in vitro, including the major HPV16 late transcript (E1E4. L1), and has an RNA-independent ATPase activity which can be partially inhibited by E1E4. E4-DBP was detectable in the cytoplasm of cells expressing HPV16 E1E4 (in vivo and in vitro) and could be immunoprecipitated as an E1E4 complex from cervical epithelial cell lines. In cell lines lacking cytoplasmic intermediate filaments, loss of the leucine cluster-cytoplasmic anchor region of HPV16 E1wedgeE4 resulted in both proteins colocalizing exclusively to the nucleoli. Two additional HPV16 E1E4-binding proteins, of 80K and 50K, were identified in pull-down experiments but were not recognized by antibodies to E4-DBP or the conserved DEAD box motif. Sequence analysis of E4-DBP revealed homology in its E4-binding region with three Escherichia coli DEAD box proteins involved in the regulation of mRNA stability and degradation (RhlB, SrmB, and DeaD) and with the Rrp3 protein of Saccharomyces cerevisiae, which is involved in ribosome biogenesis. The synthesis of HPV16 coat proteins occurs after E1E4 expression and genome amplification and is regulated at the level of mRNA stability and translation. Identification of E4-DBP as an HPV16 E1E4-associated protein indicates a possible role for E1E4 in virus synthesis.


Subject(s)
Oncogene Proteins, Fusion/chemistry , Oncogene Proteins, Fusion/metabolism , Papillomaviridae/metabolism , RNA Helicases/metabolism , Viral Proteins , Cell Nucleus/metabolism , Cytoplasm/metabolism , DEAD-box RNA Helicases , Gene Expression Regulation, Viral , HeLa Cells , Humans , Molecular Sequence Data , Nuclear Proteins/metabolism , Oncogene Proteins, Fusion/genetics , Papillomaviridae/genetics , Papillomaviridae/pathogenicity , Papillomavirus Infections/virology , RNA Helicases/chemistry , RNA Helicases/genetics , RNA, Messenger/metabolism , Ribosomes/metabolism , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Tumor Cells, Cultured , Tumor Virus Infections/virology , Two-Hybrid System Techniques
5.
J Mol Biol ; 288(3): 305-20, 1999 May 07.
Article in English | MEDLINE | ID: mdl-10329144

ABSTRACT

The ribosomal frameshifting signal present in the genomic RNA of the coronavirus infectious bronchitis virus (IBV) contains a classic hairpin-type RNA pseudoknot that is believed to possess coaxially stacked stems of 11 bp (stem 1) and 6 bp (stem 2). We investigated the influence of stem 1 length on the frameshift process by measuring the frameshift efficiency in vitro of a series of IBV-based pseudoknots whose stem 1 length was varied from 4 to 13 bp in single base-pair increments. Efficient frameshifting depended upon the presence of a minimum of 11 bp; pseudoknots with a shorter stem 1 were either non-functional or had reduced frameshift efficiency, despite the fact that a number of them had a stem 1 with a predicted stability equal to or greater than that of the wild-type IBV pseudoknot. An upper limit for stem 1 length was not determined, but pseudoknots containing a 12 or 13 bp stem 1 were fully functional. Structure probing analysis was carried out on RNAs containing either a ten or 11 bp stem 1; these experiments confirmed that both RNAs formed pseudoknots and appeared to be indistinguishable in conformation. Thus the difference in frameshifting efficiency seen with the two structures was not simply due to an inability of the 10 bp stem 1 construct to fold into a pseudoknot. In an attempt to identify other parameters which could account for the poor functionality of the shorter stem 1-containing pseudoknots, we investigated, in the context of the 10 bp stem 1 construct, the influence on frameshifting of altering the slippery sequence-pseudoknot spacing distance, loop 2 length, and the number of G residues at the bottom of the 5'-arm of stem 1. For each parameter, it was possible to find a condition where a modest stimulation of frameshifting was observable (about twofold, from seven to a maximal 17 %), but we were unable to find a situation where frameshifting approached the levels seen with 11 bp stem 1 constructs (48-57 %). Furthermore, in the next smaller construct (9 bp stem 1), changing the bottom four base-pairs to G.C (the optimal base composition) only stimulated frameshifting from 3 to 6 %, an efficiency about tenfold lower than seen with the 11 bp construct. Thus stem 1 length is a major factor in determining the functionality of this class of pseudoknot and this has implications for models of the frameshift process.


Subject(s)
Frameshifting, Ribosomal , Nucleic Acid Conformation , RNA/metabolism , Base Sequence , Mutagenesis, Site-Directed , RNA/chemistry , RNA/genetics , RNA Probes
6.
J Mol Biol ; 288(3): 321-35, 1999 May 07.
Article in English | MEDLINE | ID: mdl-10329145

ABSTRACT

RNA pseudoknots are structural elements that participate in a variety of biological processes. At -1 ribosomal frameshifting sites, several types of pseudoknot have been identified which differ in their organisation and functionality. The pseudoknot found in infectious bronchitis virus (IBV) is typical of those that possess a long stem 1 of 11-12 bp and a long loop 2 (30-164 nt). A second group of pseudoknots are distinguishable that contain stems of only 5 to 7 bp and shorter loops. The NMR structure of one such pseudoknot, that of mouse mammary tumor virus (MMTV), has revealed that it is kinked at the stem 1-stem 2 junction, and that this kinked conformation is essential for efficient frameshifting. We recently investigated the effect on frameshifting of modulating stem 1 length and stability in IBV-based pseudoknots, and found that a stem 1 with at least 11 bp was needed for efficient frameshifting. Here, we describe the sequence manipulations that are necessary to bypass the requirement for an 11 bp stem 1 and to convert a short non-functional IBV-derived pseudoknot into a highly efficient, kinked frameshifter pseudoknot. Simple insertion of an adenine residue at the stem 1-stem 2 junction (an essential feature of a kinked pseudoknot) was not sufficient to create a functional pseudoknot. An additional change was needed: efficient frameshifting was recovered only when the last nucleotide of loop 2 was changed from a G to an A. The requirement for an A at the end of loop 2 is consistent with a loop-helix contact similar to those described in other RNA tertiary structures. A mutational analysis of both partners of the proposed interaction, the loop 2 terminal adenine residue and two G.C pairs near the top of stem 1, revealed that the interaction was essential for efficient frameshifting. The specific requirement for a 3'-terminal A residue was lost when loop 2 was increased from 8 to 14 nt, suggesting that the loop-helix contact may be required only in those pseudoknots with a short loop 2.


Subject(s)
Frameshifting, Ribosomal , Nucleic Acid Conformation , RNA, Viral/metabolism , Base Sequence , Mammary Tumor Virus, Mouse/genetics , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , RNA Probes , RNA, Viral/chemistry , RNA, Viral/genetics
7.
J Gen Virol ; 79 ( Pt 2): 371-4, 1998 Feb.
Article in English | MEDLINE | ID: mdl-9472622

ABSTRACT

A 16-mer peptide library was screened using the yeast two-hybrid system to identify peptides which specifically interact with the human papillomavirus type 16 (HPV-16) E6 protein. Four different peptides were identified, three of which contained an E-L-L/V-G motif. A fifth E6 binding peptide, derived from the putative tumour suppressor protein tuberin, was identified during a two-hybrid screen of a HeLa cDNA expression library. This peptide contained a D-I-L-G motif. Homology to the peptides was found within the E6 binding proteins E6-AP and E6-BP. A synthetic peptide containing the ELLG motif blocked the interaction of E6 with both E6-AP and E6-BP. The data suggest that E6 interacts through a structurally similar binding domain present within a number of cellular proteins.


Subject(s)
Oncogene Proteins, Viral/metabolism , Papillomaviridae/physiology , Repressor Proteins/metabolism , Amino Acid Sequence , Conserved Sequence , HeLa Cells , Humans , Molecular Sequence Data , Oncogene Proteins, Viral/chemistry , Peptide Fragments/chemical synthesis , Peptide Fragments/chemistry , Sequence Alignment , Sequence Homology, Amino Acid
8.
Virology ; 238(1): 40-52, 1997 Nov 10.
Article in English | MEDLINE | ID: mdl-9375007

ABSTRACT

HPV late gene expression is initiated as an infected basal cell migrates through the differentiating layers of the epidermis, resulting in the onset of vegetative viral DNA replication and the expression of viral late proteins. We have used a large synthetic immunoglobulin library displayed on phage (diversity 6.5 x 10(10) phage) to isolate three Fabs (TVG405, 406, and 407) which recognize distinct epitopes on the E4 late protein of HPV16. A C-terminal monoclonal (TVG404) was generated by hybridoma technology, and N-terminal polyclonal antiserum was prepared by peptide immunization (alpha N-term). The most potent antibody (TVG405) had an affinity for E4 of approximately 1.0 nM. All antibodies recognized the protein in paraffin-embedded archival material, allowing us to map events in the late stages of virus infection. Expression of E4 in vivo does not coincide with synthesis of the major virus coat protein L1, but precedes it by 1 or 2 cell layers in premalignant lesions caused by HPV16 and by up to 20 cell layers in HPV63-induced warts. In higher grade lesions associated with HPV16, E4 is produced in the absence of L1. By contrast, vegetative viral DNA replication and E4 expression correlate exactly and in some lesions begin as the infected epithelial cell leaves the basal layer. Differentiation markers such as filaggrin, loricrin, and certain keratins are not detectable in E4-positive cells, and nuclear degeneration is delayed. HPV16 E4 has a filamentous distribution in the lower epithelial layers, but associates with solitary perinuclear structures in more differentiated cells. Antibodies to the N-terminus of the protein stained these structures poorly. Our findings are compatible with a role for the HPV16 E4 protein in vegetative DNA replication or in modifying the phenotype of the infected cell to favor virus synthesis or virus release. The Fabs will be of value in the evaluation of model systems for mimicking HPV infection in vitro.


Subject(s)
Oncogene Proteins, Viral/biosynthesis , Papillomaviridae/physiology , Amino Acid Sequence , Antibodies, Monoclonal , Binding Sites, Antibody , DNA Replication , DNA, Viral/analysis , Epithelial Cells/cytology , Epithelial Cells/virology , Epitopes/analysis , Filaggrin Proteins , Humans , Immunoglobulin Fab Fragments , Immunoglobulin Fragments/chemistry , Immunoglobulin Fragments/immunology , Kinetics , Molecular Sequence Data , Oncogene Proteins, Viral/analysis , Oncogene Proteins, Viral/immunology , Papillomaviridae/immunology , Papillomaviridae/pathogenicity , Precancerous Conditions/pathology , Virus Replication
9.
Virology ; 218(1): 114-26, 1996 Apr 01.
Article in English | MEDLINE | ID: mdl-8615013

ABSTRACT

The HPV1 E4 gene encodes a family of abundant nonstructural late proteins whose role in the virus life cycle is unknown. Their localization to keratin filaments when expressed in cultured epithelial cells has suggested a possible involvement in virus release by disturbing the integrity of the infected cell. Here we show that in naturally occurring HPV1-induced tumors, the majority of the E4 protein (>95%) exists as complexes which do not contain keratins. The identification of discrete species of 34K, 58K, 70K, 88K, and 105K suggests that these are simple multimers of the 17K monomer, with very little of the soluble E4 being present in complexes larger than 140K. The truncated 10/11K E4 species, which comprise the C-terminal domain of E4, exist as trimers and dimers in vivo. Less than 5% of the E4 was present as complexes greater than 140K, and these were found to be insoluble. The 34K (dimer) and 58K (putative trimer) E4 complexes were components of these larger structures. Neither E4 monomers nor E4 complexes could be shown to interact directly with keratins or with keratin filaments although formation of the 105K E4 complex was abolished (and formation of the 58K species enhanced) when keratins were present during E4 synthesis in vitro. We conclude that while E1-E4 may transiently associate with keratins during synthesis, the two proteins do not stably associate via a direct interaction. The majority of the HPV1 E4 protein in established tumors in vivo is neither filament associated nor contained in inclusion granules, but exists predominantly as soluble cytoplasmic multimers.


Subject(s)
Keratins/metabolism , Oncogene Proteins, Fusion/metabolism , Papillomaviridae/metabolism , Viral Proteins , Base Sequence , Cell Line , DNA Primers , Humans , Molecular Sequence Data , Molecular Weight , Papillomavirus Infections/metabolism , Papillomavirus Infections/virology , Protein Binding , Tumor Virus Infections/metabolism , Tumor Virus Infections/virology
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