It is well documented that shortly after infection with poliovirus, host protein synthesis is inhibited. This defect is at the level of initiation and is thought to be caused by a virally-induced alteration of the initiation factor CBPII. This factor is needed for binding and recognition of capped host mRNA to the 40S ribosomal subunit. Consequently, it has been postulated that poliovirus RNA itself is able to initiate protein synthesis via a "cap-independent" mechanism. In order to define the concept of a "cap-independent" mechanism more fully, the importance of the 5' terminus and 5' noncoding region were investigated. Consequently, restriction fragments from the poliovirus clones, pVR104 and pVR105, were selected which hybridized in four regions of the poliovirus genome: (a) a site within the coding sequence; (b) the area covering the AUG at the beginning of the NCVPOO reading frame; (c) a region in the middle of the 5' untranslated region of the genome; (d) the 5' terminal region of the genome. Hybrid molecules were used to program a micrococcal nuclease treated, PV1-infected cell-free system. Message function was measured by the synthesis of protein products, analyzed by SDS-PAGE, formation of polyribosomes and initiation complexes. As expected, the hybrid formed within the coding region allowed the synthesis of the predicted truncated protein. The hybrid covering the AUG codon at position N743 prevented translation. Interestingly, the hybrid in the untranslated 5' region and the hybrid formed at the 5' terminal sequence, inhibited translation and prevented formation of an initiation complex and polyribosomes. This suggested that poliovirus mRNA, despite its lack of a 5' cap, interacts with ribosomes in an manner similar to capped eucaryotic mRNAs and in agreement with a proposed scanning mechanism. Virus specific RNA binding proteins were also characterized in a preliminary investigation to determine which viral proteins exhibit RNA binding capability and through this interaction direct the functional role that the RNA plays within the cell. It was determined that four virus specific proteins do exhibit RNA binding properties. Due to the known functions of these viral proteins, it is unlikely that any of these proteins are responsible for the translational advantage of PV1 RNA in infected cells.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1981. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 156-163).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1980. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 134-140).

No description available.

Poliovirus is a small, structurally simple virus that can serve as a powerful model system for the elucidation of the basic processes involved in the genetic control of macromolecular synthesis. The physical and biochemical characterization of temperature-sensitive and drug-resistant mutants of this virus can provide insight into the normal sequence of events during the replication and assembly of the wild-type (wt) virus. The specific interference of the infecting virus with the major pathways of macromolecular synthesis in the host cell offers a possible inroad to the exploration of the relevant control systems. This research is divided into two major segments: (1) a temperature-sensitive mutant of poliovirus type 1, tsB9, and a guanidine-resistant mutant, gʳH, were characterized physiologically; (2) a physiological screening procedure that quickly and efficiently reveals the phenotype of a large number of poliovirus mutants in a short period of time was developed and validated. Two guanidine-resistant and twelve temperature-sensitive mutants of poliovirus type 1, Mahoney, were generated and compared with wt, defective-interfering particles, gʳH, and tsB9 using the screening procedure herein developed. The temperature-sensitive mutant, tsB9, appears to be a structural protein mutant bearing a related defect in ribonucleic acid synthesis at the restrictive temperature. The mutant gʳH was found to differ from the wt virus only in the guanidine resistance of its growth and ribonucleic acid synthesis, although a detailed electrophoretic analysis of its proteins was not done. Among the newly isolated mutants, strains were found with defects in each of the major viral functions except one. No mutant was found to be defective in the ability to inhibit host-cell protein synthesis. The screening procedure developed met all of the criteria set for it mentioned above. It has the potential of being adapted to many virus-cell systems for the rapid determination of mutant phenotype.

Thesis (Ph. D., Microbiology, Molecular Biology and Biochemistry)--University of Idaho, August 2008. / Major professor: Kurt E. Gustin. Includes bibliographical references. Also available online (PDF file) by subscription or by purchasing the individual file.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1984. / Typescript. Vita. Description based on print version record. Includes bibliographical references (leaves 548-584).

Complete nucleotide sequences have been derived for the Sabin type 3 vaccine, P3/Leon/l2a1b, and its neurovirulent progenitor, P3/Leon/37 (Stanway et al., 1983, 1984). These studies revealed 10 base substitution mutations which must account for the attenuated and temperature sensitive phenotypes of the vaccine. Complete DNA copies of the genomes of P3/Leon/12a1b and P3/Leon/37 were constructed, each within the prokaryote vector, pAT 153. The full-length cDNA clones were shown to be infectious following transfection of human epithelial cells. Virus rescued from the cDNA clone of P3/Leon/12a1b could not be distinguished from reference Preparations of the Sabin type 3 vaccine in standard assays for neurovirulence and temperature sensitivity. By the same criteria, virus rescued from the cDNA clone of P3/Leon/37 was shown to be identical to the parental strain. To determine the genetic basis for the attenuated phenotype, a series of inter-strain poliovirus recombinants were constructed via cDNA in-vitro. Attenuation results from the concerted effect of two independent point mutations. The first is a C-U substitution at position 472 in the 5' non-coding region of the viral genome. The second is a C-U substitution at position 2034 which results in a serine to phenylalanine substitution in VP3. The VP3 mutation confers a temperature sensitive phenotype. This appears to be the only temperature sensitive mutation in the vaccine.

Viruses are obligate intracellular parasites that replicate utilizing the resources of host cells. The replication of positive sense RNA viruses is coupled with alterations to host cell membranes. These viruses are believed to replicate efficiently by using co-opted membrane structures assembled from viral and host cell proteins and lipids. Poliovirus is a prototypical positive-sense RNA virus, however the topological details of viral RNA replication are not well understood. In this work we use electron cryotomography, among other methods, to examine the ultrastructure of fractionated poliovirus RNA replication factories that were formed within infected cells, and to investigate oligomeric interactions within a three dimensional crystal formed by a poliovirus polymerase point mutant. Investigation of the ultrastructure of isolated viral RNA replication factories shows that the low resolution features of cryopreserved membrane structures are essentially identical to previously observed structures within plastic sections of infected cells. Furthermore, greater detail visible using electron cryotomography reveals pore-like structures and other high energy membrane conformations within the replication factories. We see a mix of single, double, and multi-membrane structures that are arranged with openings that connect their interior lumenal space to the exterior environment. The lumen of some of these membranous structures contains a linear polymeric density thought to be RNA. We conclude that the RNA replication of poliovirus may occur on the lumenal surface of vesicular membranes with an opening to the cytoplasm for metabolite and product exchange. Within the poliovirus replication machinery, the principal component is the RNA polymerase 3Dpol. This prototypical RNA-dependent RNA-polymerase forms homo-oligomeric interactions that are key to its functions. To investigate these interactions, previous studies focused on hollow helical structures formed by wild-type polymerase. Here, we investigate the structure of small three-dimensional crystals formed by 3Dpol with a mutation of a single residue, lysine 314, to alanine. Using electron cryotomography and volume averaging, we demonstrate that the crystal packing within this point mutant does not include physiological polymerase-polymerase interactions. Elucidation of the topology of poliovirus replication machinery provides a basis for future development of antiviral therapeutics.

A microtiter, solid-phase, indirect radioimmunoassay (RIA) has been developed and evaluated as a method for detecting poliovirus in water samples. Antiserum to poliovirus Type I, LSc2ab, was prepared in rabbits, and high titer, high avidity antiserum to rabbit globulins were radio- actively labeled with 125I by a modification of the Bolton-Hunter and chloramine T methods to increasing specific activities. The immunoreactivity of the labeled antibodies was assessed. After the preparation and standardization of all components of the assay. The optimum assay conditions were determined. These conditions included the method of coating the microtiter wells with antigen, the time and temperature of incubation of each antiserum, the dilution of each antiserum, and means of reducing non-specific binding. The RIA was conducted with varying numbers of viral plaque forming units. In replicate experiments, average binding ratios of 784% were obtained when different numbers of virus were first reacted with antiserum. This technique shows increased sensitivity over the direct method. These results indicate that the use of RIA to detect viruses in water is indeed feasible.