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G W Witherell

Publications and source records attributed to G W Witherell.

11 recordsLinked to original sources
PubMed2001

Statistical analysis of combined substitutions in nonstructural 5A region of hepatitis C virus and interferon response.

Interferon (IFN) therapy (+/- ribavirin) is the only currently available treatment for chronic hepatitis C virus (HCV), but is not effective for a majority of patients. Several studies have correlated IFN response with substitutions in a small region of the nonstructural 5A (NS5A) gene product of HCV, termed the interferon sensitivity determining region (ISDR). Many other studies, however, have been unable to verify this correlation. To address this issue, available data from published studies was used to create a database of 675 individual ISDR sequences. The database was used to analyze substitutions in the ISDR with regard to IFN response. Combined data was analyzed by the chi-square independence test and by logistic regression analysis. Each statistical analysis demonstrated a strong correlation between IFN response and substitutions in the ISDR. A statistically significant correlation was also found between IFN-response and substitutions in ISDRs of combined studies that independently were unable to detect a correlation. The failure of these individual studies to verify a correlation seems to be at least partially due to inadequate sample size. A new model for chi-square analysis is proposed that could allow a correlation between IFN-response and ISDR sequence to be detected for data sets with less statistical power than that required for the current model. The ISDR database was also used to analyze individual substitutions in the ISDR. The results show that IFN-sensitive viruses contain a larger number and more diverse collection of substitutions than IFN-resistant viruses. Substitutions that are most likely to be tolerated or detrimental to NS5A function in the IFN-response mechanism were identified as a first step toward site directed mutagenesis of the ISDR.

Amino Acid Substitution↗
PubMed2001

ISIS-14803 (Isis Pharmaceuticals).

Isis and Merck are developing an antisense oligonucleotide for the potential treatment of hepatitis C virus (HCV) infection [305306]. As of March 2000, the lead from this program, ISI-14803, was being studied in a phase I/II trial where it was administered in three weekly 2-h infusions for 4 weeks [327913], [357823]. Based on these results Isis plans to evaluate the potential of ISIS-14803 in single-agent trials and in combination studies with PEGylated interferon and ribavirin, and in studies that will examine longer-term dosing with both i.v. and s.c. administration [412920]. In January 2000, Isis signed a binding letter of agreement with Elan to form a new subsidiary of Isis to develop ISIS-14803 [351881], [362727]. In October 2001, Isis reported that it had earned a $1.5 million research milestone payment from Merck for progress in this collaboration [426015].

Animals↗
PubMed1995

Cis-acting elements of the encephalomyocarditis virus internal ribosomal entry site.

Translation initiation of encephalomyocarditis virus (EMCV) mRNA occurs by ribosomal entry into the 5' untranslated region. Internal ribosome binding to EMCV mRNA requires a viral cis-acting element, termed the internal ribosomal entry site (IRES), and cellular trans-acting factors. The polypyrimidine tract binding protein (PTB) has been identified as one such trans-acting factor required for EMCV IRES-dependent translation. Using a dicistronic mRNA and an in vitro translation system, we have identified cis-acting elements of the EMCV IRES required for IRES-dependent translation. The results identify several regions of the IRES that are required for efficient IRES-dependent translation, including the PTB binding site. Other regions of the IRES may act only as spacer sequences, analogous to the spacer sequences found in rhinovirus and enterovirus IRES elements, to link essential regions of the IRES together. The flexibility of one region of the EMCV IRES was demonstrated by an insertion of 125 nucleotides that had little effect on IRES function while the constraint imposed on another region was demonstrated by a 3-nucleotide deletion that nearly abolished IRES-dependent translation.

Binding Sites↗
PubMed1994

Encephalomyocarditis virus internal ribosomal entry site RNA-protein interactions.

Translational initiation of encephalomyocarditis virus (EMCV) mRNA occurs by ribosomal entry into the 5' nontranslated region of the EMCV mRNA, rather than by ribosomal scanning. Internal ribosomal binding requires a cis-acting element termed the internal ribosomal entry site (IRES). IRES elements have been proposed to be involved in the translation of picornavirus mRNAs and some cellular mRNAs. Internal ribosome binding likely requires the interaction of trans-acting factors that recognize both the mRNA and the ribosomal complex. Five cellular proteins (p52, p57, p70, p72, and p100) cross-link the EMCV IRES or fragments of the IRES. For one of these proteins, p57, binding to the IRES correlates with translation. Recently, p57 was identified to be very similar, if not identical, to polypyrimidine tract-binding protein. On the basis of cross-linking results with 21 different EMCV IRES fragments and cytoplasmic HeLa extract or rabbit reticulocyte lysate as the source of polypeptides, consensus binding sites for p52, p57, p70, and p100 are proposed. It is suggested that each of these proteins recognizes primarily a structural feature of the RNA rather than a specific sequence.

Base Sequence↗
PubMed1993

Interaction of polypyrimidine tract binding protein with the encephalomyocarditis virus mRNA internal ribosomal entry site.

Translation of encephalomyocarditis virus (EMCV) mRNA occurs in a cap-independent manner, requiring instead a cis-acting element termed the internal ribosomal entry site (IRES). Binding of a 57-kDa ribosome-associated protein (p57) to the EMCV IRES has been found to correlate with cap-independent translation. p57 has recently been reported to be very similar, if not identical, to the polypyrimidine tract binding protein (pPTB), a spliceosome-associated factor possibly involved in U2 snRNP/pre-mRNA complex formation of 3'-splice-site recognition. The interaction between purified pPTB and the EMCV IRES was characterized in this study using nitrocellulose filter binding and UV cross-linking assays. pPTB bound the EMCV IRES with high affinity (Kd = 40 nM at 25 degrees C, pH 5.5, 80 mM ionic strength). pPTB also bound strongly to RNA fragments containing either the 5'-end, 3'-end, or an internal stem-loop of the IRES. The binding properties of 16 RNA variants derived from the IRES revealed however that purified pPTB bound with less specificity than pPTB in a mixture of cytoplasmic HeLa cell polypeptides. The addition of HeLa extract to purified pPTB increased the binding specificity, suggesting that factors within the extract alter the binding specificity of pPTB. The binding of pPTB to the full-length IRES and three IRES-derived fragments was studied in detail. Complex formation was optimal at low pH and was driven entirely by entropy. As many as four ion pairs are formed upon binding, with electrostatic interactions accounting for approximately 35% of the total free energy of complex formation.

Base Sequence↗
PubMed1993

A cytoplasmic 57-kDa protein that is required for translation of picornavirus RNA by internal ribosomal entry is identical to the nuclear pyrimidine tract-binding protein.

Initiation of translation of the RNA genomes of picornaviruses such as poliovirus and encephalomyocarditis virus is cap-independent and results from interaction of ribosomes with a segment of the 5' noncoding region of these mRNAs termed the internal ribosomal entry site. Genetic and biochemical studies have previously shown that a 57-kDa cytoplasmic RNA-binding protein (p57) plays an essential role in this translation mechanism. We have now found that p57 shares physical, biochemical, and antigenic properties with the pyrimidine tract-binding protein (PTB), a nuclear protein that has been implicated in various processes involving pre-mRNA. These data indicate that p57 and PTB are the same protein. Purified recombinant PTB bound specifically to a bulged hairpin within the internal ribosomal entry site of encephalomyocarditis virus and had a much lower affinity for a mutated derivative of this hairpin and for unrelated RNAs. Immunodepletion of p57/PTB from a HeLa cell-free lysate inhibited translation of poliovirus and encephalomyocarditis virus mRNAs but had no effect on translation of beta-globin mRNA, confirming the essential role of p57 in translation by internal ribosomal entry.

Base Sequence↗
PubMed1990

Cooperative binding of R17 coat protein to RNA.

The binding of the R17 coat protein to synthetic RNAs containing one or two coat protein binding sites was characterized by using nitrocellulose filter and gel-retention assays. RNAs with two available sites bound coat protein in a cooperative manner, resulting in a higher affinity and reduced sensitivity to pH, ionic strength, and temperature when compared with RNAs containing only a single site. The cooperativity can contribute up to -5 kcal/mol to the overall binding affinity with the greatest cooperativity found at low pH, high ionic strength, and high temperatures. Similar solution properties for the encapsidation of the related fr and f2 phage suggest that the cooperativity is due to favorable interactions between the two coat proteins bound to the RNA. This system therefore resembles an intermediate state of phage assembly. No cooperative binding was observed for RNAs containing a single site and a 5' or 3' extension of nonspecific sequence, indicating that R17 coat protein has a very low nonspecific binding affinity. Unexpectedly weak binding was observed for several RNAs due to the presence of alternative conformational states of the RNA.

Base Sequence↗
PubMed1990

Cap-independent translation of picornavirus RNAs: structure and function of the internal ribosomal entry site.

Picornaviruses are mammalian plus-strand RNA viruses whose genomes serve as mRNA. A study of the structure and function of these viral mRNAs has revealed differences among them in events leading to the initiation of protein synthesis. A large segment of the 5' nontranslated region, approximately 400 nucleotides in length, promotes 'internal' entry of ribosomes independent of the non-capped 5' end of the mRNA. This segment, which we have called the internal ribosome entry site (IRES), maps approximately 200 nt down-stream from the 5' end and is highly structured. IRES elements of different picornaviruses, although functionally similar in vitro and in vivo, are not identical in sequence or structure. However, IRES elements of the genera entero- and rhinoviruses, on the one hand, and cardio- and aphthoviruses, on the other hand, reveal similarities corresponding to phylogenetic kinship. All IRES elements contain a conserved Yn-Xm-AUG unit (Y, pyrimidine; X, nucleotide) which appears essential for IRES function. The IRES elements of cardio-, entero- and aphthoviruses bind a cellular protein, p57. In the case of cardioviruses, the interaction between a specific stem-loop of the IREs is essential for translation in vitro. The IRES elements of entero- and cardioviruses also bind the cellular protein, p52, but the significance of this interaction remains to be shown. The function of p57 or p52 in cellular metabolism is unknown. Since picornaviral IRES elements function in vivo in the absence of any viral gene products, we speculate that IRES-like elements may also occur in specific cellular mRNAs releasing them from cap-dependent translation. IRES elements are useful tools in the construction of high yield expression vectors, or for tagging cellular genetic elements.

Animals↗
PubMed1989

Specific RNA binding by Q beta coat protein.

The interaction between the bacteriophage Q beta coat protein and its specific binding site on Q beta genomic RNA was characterized by using a nitrocellulose filter binding assay. Q beta coat protein bound to a synthetic 29-nucleotide RNA hairpin with an association constant of 400 microM-1 at 4 degrees C, 0.2 M ionic strength, pH 6.0. Complex formation had a broad pH optimum centered around pH 6.0 and was favored by both enthalpy and entropy. The salt dependence of Ka revealed that four to five ion pairs may be formed in the complex although approximately 80% of the free energy of complex formation is contributed by nonelectrostatic interactions. Truncation experiments revealed that coat protein binding required only the presence of a hairpin with an eight base pair stem and a three-base loop. Analysis of the binding properties of hairpin variants showed that the sequence of the stem was not important for coat protein recognition and only one of the three loop residues was essential. A bulged adenosine present in the coat protein binding site was not required for coat protein binding. Q beta coat protein binding specific is therefore primarily achieved by the structure and not by the sequence of the operator.

Base Sequence↗
PubMed1987

Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates.

A method is described to synthesize small RNAs of defined length and sequence using T7 RNA polymerase and templates of synthetic DNA which contain the T7 promoter. Partially single stranded templates which are base paired only in the -17 to +1 promoter region are just as active in transcription as linear plasmid DNA. Runoff transcripts initiate at a unique, predictable position, but may have one nucleotide more or less on the 3' terminus. In addition to the full length products, the reactions also yield a large amount of smaller oligoribonucleotides in the range from 2 to 6 nucleotides which appear to be the result of abortive initiation events. Variants in the +1 to +6 region of the promoter are transcribed with reduced efficiency but increase the variety of RNAs which can be made. Transcription reaction conditions have been optimized to allow the synthesis of milligram amounts of virtually any RNA from 12 to 35 nucleotides in length.

DNA↗