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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.

No description available.

No description available.

Thesis (DR. P.H.)--University of Michigan.

Thesis (DR. P.H.)--University of Michigan.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1983. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

A series of P4 (Cbz and t-Boc) N-protected potential substrates and inhibitors, containing the P1 to P4 substrate recognition sequence of the type 2 adenovirus proteinase (Leu-Ala-Gly-Gly) were prepared by solution phase peptide coupling techniques and tested for activity against the proteinase. The potential substrates contained the amide and ester moieties at the P1 carbonyl position and the potential inhibitors contained the alcohol, acid, bromide, aldehyde, ketone, dimethylacetal, nitrile, alkenic, malonyl and epoxysuccinate moieties at the P1 carbonyl position. The esters, the t-Boc urethane and the p-nitroanilide moieties were substrates for the proteinase and the acid and the amides did not bind to the proteinase. Preliminary results show that the other inhibitors were mostly noncompetitive inhibitors for the adenovirus proteinase with approximate Ki's between 15 and 200 μmol dm-3. The test results indicate that the amides must contain a carbonyl group at P2' to bind to the proteinase; the loss of the P1' amine product is the rate limiting step for the hydrolysis of a substrate by the adenovirus proteinase; the P acid product leaves before the P' amine product, which is in complete contrast to classical cysteine proteinases such as papain; little protonation of the P1'amide nitrogen or the P1 carbonyl oxygen of the adenovirus proteinase-substrate complex occurs before the nucleophilic attack on the P1 carbonyl carbon of the adenovirus proteinase-substrate complex.

The development of a cell-free system in which adenovirus DNA synthesis can be initiated in vitro by using the viral genome or plasmids containing the origin of replication as template has led to the identification of the sequences important for origin function and the isolation and purification of the proteins required for viral DNA replication. In vitro studies on adenovirus types 2 and 5 have shown that their replication requires the formation of a large nucleoprotein complex. This is composed of three virally encoded proteins: adenovirus DNA polymerase, precursor terminal protein and DNA binding protein, and two cellular proteins nuclear factor I and nuclear factor III. While the presence of DNA helicases in other eukaryotic DNA replication systems have been well characterised, this was not the case for adenovirus DNA replication. Initial attempts to identify DNA helicase activity associated with any of the adenovirus replication proteins were unsuccessful. However, a novel DNA unwinding activity was found associated with the DNA binding protein (Ad.DBP). We examined the interaction of DBP with partial DNA duplexes and demonstrated that it could displace oligonucleotides annealed to single-stranded M13 DNA. In addition, DBP could also unwind small fragments of fully duplex DNA. Unlike a DNA helicase, DBP promoted DNA unwinding was nucleoside-5'-triphosphate and Mg2+ independent and exhibited no directionality. The activity required saturating amounts of DBP and was both efficient and cooperative in nature. The helix-destabilising activity was shown to be situated in the C-terminal domain of the protein. These properties suggest a role for DBP in DNA replication in which DBP destabilises duplex DNA during origin unwinding and replication fork movement. The second part of the thesis dealt with the characterisation of the putative "active site" of the adenovirus DNA polymerase. This experimental approach was prompted by data from earlier studies which indicated that DBP could increase the processitivity of the polymerase as well as its sensitivity to nucleotide analogue inhibitors. The "active site" was labelled with pyridoxal-5'-phosphate (PLP), a substrate binding site directed reagent for DNA polymerases. Treatment of Ad.5 DNA polymerase with PLP followed by reduction of the enzyme-PLP adduct resulted in irreversible inactivation of the polymerase activity while the 3'-5' exonuclease associated with Ad.5 DNA polymerase was minimally affected. Substrate protection studies indicate that PLP inhibition is complex. Neither template-primer nor substrate dNTP alone showed any protective effect from PLP mediated inhibition. However, the presence of both template -primer and complementary dNTP significantly protected against PLP inhibition. Comparative tryptic mapping of labelled enzyme, modified in the presence and absence of substrates by PLP reaction, on a C-18 reverse phase column, indicated the protection of one peptide from pyridoxylation in the presence of substrates. Amino acid sequence analyses found no sequence to be present in this peak.

The development of systems in which adenovirus DNA can be replicated in vitro has led to the elucidation of the sequences essential for origin function and to the identification of the proteins required for viral DNA replication. Much of the information currently available has been derived from investigations carried out using adenovirus types 2 and 5 which in addition to the viral proteins, adenovirus DNA polymerase, precursor terminal protein and DNA binding protein, require cellular proteins nuclear factor I and nuclear factor III for efficient initiation of DNA replication. In contrast adenovirus type 4 replicates Its DNA efficiently without these cellular proteins. Correspondingly its minimal origin of replication is remarkably simple in structure, consisting of only the terminal 18bp of the adenovirus genome ('the core sequence'). The effect of point mutations in the core region on adenovirus type 4 DNA replication in vitro was investigated and it was found that mutations within two discrete domains had a married deleterious effect on initiation of DNA replication. The crude Ad4 infected cell extracts initially used for in vitro DNA replication were fractionated and it was found that only four detectable proteins, three of which were identified as viral DNA polymerase, precursor terminal protein and DNA binding protein gave efficient DNA replication in vitro and furthermore behaved similarly to unfractionated infected cell extracts in the presence of template which contained point mutations. To examine a possible role of the core region of the origin as containing sites for specific interactions with viral replication proteins, purified adenovirus type 5 precursor terminal protein and DNA polymerase were assayed for their ability to recognise the terminal 1-18 sequence. It was found that both proteins independently and as a heterodimer bound specifically to a sequence corresponding to the core origin of replication, suggesting that sequences within this region are important for localisation of DNA replication proteins at the origin via a sequence specific DNA-protein interaction.