No description available.

No description available.

The drug, N,N bis (ethylene)-P (1-adamantyl) phosphonic diamide inhibits focus formation of Rous Sarcoma Virus in tissue culture. Transformation of chick cells was inhibited when the drug was added to chick cells prior to infection. The drug did not inhibit the transformation of Normal Rat Kidney Cells infected with RSV, when the cells were grown at non-permissive temperatures and shifted to permissive temperatures upon addition of the drug. Nor did the drug revert cells transformed at permissive temperatures. These studies indicated that the inhibition of RSV is in the early stage of viral growth, possible penetration or uncoating.

The basis of this investigation is to characterize growth property differences in normal versus virally transformed cells. Using a temperature-sensitive mutant of Rous sarcoma virus, the cells' transformation state is regulated by the growth temperature; at 33°C the cells are transformed, while at 39°C the cells have normal characteristics. The morphology of NRK cells is elongated and fibroblastic; when transformed the cells are rounded. Normal cells grow to a monolayer and stop, while transformed cells grow to saturation densities greater than just a monolayer amount. Transformed cells can form foci when grown in mixture with normal cells. Normal cells must be in contact with the culture vessel in order to grow, but transformed cells lack anchorage dependence for growth.

LA31-NRK and B77-NRK are established cell lines that were normal rat kidney cells transformed with temperature-sensitive mutant (LA31) and wild-type Bratislava 77 (B77) of Rous sarcoma virus. It is recognized that many transformation-induced changes differentiate between normal and transformed cells. Morphology and four parameters of transformed cells such as saturation density, anchorage independence, plasminogen activator, and colony stimulating factor were used as indicators to observe the effect of human alpha interferon on the growth of NRK, LA31-NRK and B77-NRK. The results show that interferon could neither reverse the transformed cells to normal fashion nor change their behaviors or cause release of protease.

Solid State Nuclear Magnetic Resonance (ssNMR) spectroscopy can be a powerful tool for investigating the atomic-level structures and dynamics of biological macromolecules, including proteins. In this dissertation, I present an ssNMR study of three diverse proteins, revealing insights into their respective secondary structures, conformational variations, and intermolecular interactions. Additionally, I introduce novel computational methods to facilitate the assignment of chemical shifts of ssNMR spectra. The first of the proteins is the capsid protein of the Rous Sarcoma Virus. In previous research, the structure of the hexameric lattice of the in-vitro tubular assembly of the capsid protein was determined. In this study, chemical shift assignments were completed and the structure of the T=1 capsid assembly (comprising entirely of a pentameric lattice) of the I190V mutant variant of the capsid was determined, providing the missing component of the in-vivo capsid structure. The second protein studied was amyloid-beta 42, a particularly cytotoxic variant of the main component of amyloid plaques in the brains of Alzheimer's disease patients. Chemical shift assignments were made on ssNMR data from samples aggregated in cholesterol-containing phosphatidylcholine (POPC) lipid vesicles, and secondary structure and molecular distance information was obtained. Lastly, preliminary chemical shift assignments, statistics, and structural analysis was done on the polypeptide Ref-2Cx4, derived from the conserved domain of the Hawaiian bobtail squid reflectin protein. The reflectin protein, used in the squid's camouflage mechanism, possesses optically reflective and proton-conductive properties. The final part of the dissertation addresses a major bottleneck in ssNMR studies—the assignment of chemical shifts. I introduce Visual Assist, a suite of computational tools designed to streamline and expedite the assignment process. The developed computational methods are validated on the diverse set of proteins above, demonstrating their general applicability and efficiency.

Most viruses can infect only a reduced range of organisms and an effective replication is possible only in selected hosts. These hosts are called permissive for the virus. Molecular principles of a nonpermissiveness and viral mechanisms of overcoming replication obstacles are still not clearly elucidated. This thesis discusses the molecular causes of the cellular nonpermissiveness against a model retrovirus - Rous sarcoma virus. The research is conducted on duck cells which are semipermis- sive to the subgroup C of Rous sarcoma virus. The virus can enter those cells, but it is not able to produce enough infectious viral progeny. Two blocks of the viral replication cycle in the duck cells are described in the thesis. The first one is the probably not optimal cellular receptor recognition. The second one is in the late phase of the replication cycle when the viral proteins are synthesized. The amount of the envelope glyco- protein coding mRNA is reduced due to the altered splicing ratios, and the virions produced from the duck cells are less infectious. This block is recessive and can be partially omitted by cell fusions with permissive chicken cells; therefore, the block is not caused by specific restriction fac- tors in sensu stricto. Additionally, the influence of mutations in duck adapted Rous...

One of the important tasks of virology and immunology is to explore the species- and cell-barriers preventing virus horizontal transmission and reveal the ways how viruses overcome these barriers and "adapt" to different species. This work is based on a well- established retroviral model - avian Rous sarcoma virus (RSV) and studies virus replication blocks in mammalian cells at both pre- and post-integration level. Interaction of the viral envelope glycoprotein (Env) with a specific cellular receptor mediates virus entry into cells. Although mammalian orthologues of specific chicken receptors do not support RSV entry, it was observed that some RSV strains are able to enter mammalian cells. Several RSV-transformed rodent cells lines were described and analysis of provirus H20- RSV in one these cells lines (hamster H-20 tumor cell line) showed multiple mutations including two crucial amino acid substitutions in different regions of Env. Substitutions D32G and L378S confer virus transmission to hamster, human and also chicken cells lacking the appropriate receptor. Altered conformation of H20-RSV Env is similar to a receptor-primed (activated) state of Env. This observation indicates that virus can circumvent the need of original cell receptor because of spontaneous Env activation caused by single...