Highly active antiretroviral therapy has successfully limited HIV-1 disease progression to AIDS, but is consistently compromised by the emergence and transmission of HIV-1 drug resistant strains. As a result, a continued search for novel anti-HIV-1 agents with improved pharmacological profiles has become fundamental. Chrysotherapy has been in use since the early 1920s in treatment of rheumatoid arthritis, and has since been investigated for various ailments including HIV/AIDS. This study evaluated 45 synthetic gold compounds for drug like properties using theoretical and experimental techniques with the aim of generating sufficient data to considerably aid the rational design of new anti-HIV agents. Theoretical techniques applied included the Osiris Property Explorer and the Lipinski’s Rule of Five which assessed drug-likeness and bioavailability respectively. In vitro studies included aqueous solubility assays, cytotoxicity (PM1 cell lines and PBMCs) assays, antiviral assays in PBMCs, direct enzyme (RT and IN) inhibition, and the effect of serum protein binding and biological stability on antiviral efficacy. An overall low druglikeness score and an intermediate bioavailability were predicted by the Osiris Molecular Property Explorer. Low drug-likeness was suggested to be due to a high frequency of foreign fragments in the synthetic gold compounds, while their high molecular weight reduced bioavailability. In general gold compounds exhibited cytotoxicity properties and moderate aqueous solubility in vitro. Overall, the 45 synthetic gold compounds did not show activity against HIV-1 replication in vitro. Seven compounds (AB05-AB11) exhibited direct HIV-1 RT inhibition, and compounds AB39 and AB04 demonstrated moderate direct HIV-1 IN inhibition, but this activity was abrogated in PBMC inhibition assays. Serum binding, compound stability and cytotoxicity were all implicated in the lack of HIV-1 inhibition in PBMCs. To this end, data obtained was sufficient to aid in the future rational design of second generation HIV RT and IN inhibitors with acceptable pharmacological properties.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 103-117).

Thesis (Ph. D.)--University of Wisconsin-Madison, 1974. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Bunyamwera (BUN) virus multiplied readily in mosquitoes following intrathoracic injection and also after imbibing'-an infective blood meal. This agent also multiplied following inoculation of human and avian cells in tissue cultures, with production of cytopathic effects. Both in arthropod and vertebrate tissues, enveloped virions 84 nm diameter were visualized by electron microscopic observation of tissues collected after maximum viral proliferation was attained. Following intrathroacic injection of 10 ²•² mouse LD₅₀ of BUN virus into groups of wild-caught mosquitoes comprising both Aedes canadensis and A. vexans, increments of infectivity were first detected in salivary glands and gut at 3 days, and maximum titres of 10 ⁵•² mouse LD₅₀ per organ were attained in salivary glands at 10 days. However, 50 the virus content of legs, which provided a convenient means of sampling hemocelic fluid, increased at 2 days. Virus transmission by biting mice was demonstrated with mosquitoes injected 10 days previously, but not after shorter intervals. No virus replication was demonstrated following ingestion of 10⁴•⁰ mouse LD₅₀ of BUN virus in a blood meal. Aedes aegypti mosquitoes readily supported the replication of BUN virus following injection with 10 ³•³ mouse LD₅₀ or imbibing of 10 ⁴•⁶ mouse LD₅₀. After injection, virus titres of whole mosquitoes declined to 10 ¹•⁷ mouse LD₅₀ at 12 hours, followed by an increase to a peak amount of 10 ⁵•⁰ mouse LD₅₀ at 2 days. After feeding, virus was first detected in legs and salivary glands at 4 days, and attained maximum titres of 10 ⁵•⁰ mouse LD₅₀ in salivary glands at 10 days. Transmission of virus to mice was effected by A. aegypti following feeding and injection 10 days previously, but not at earlier intervals. Following exposure of whole gut cultures of adult A. aegypti mosquitoes to 10 ³•⁷mouse LD₅₀ maximum yields of 10 ⁶•⁰ mouse LD₅₀ per ml. were observed after k days incubation at 29°C, after an initial decline of infectivity to 10 ¹•⁸ mouse LD₅₀ at 12 hours. Enveloped virions with cores 45 nm diameter and total diameters 80 to 100 nm were observed within vacuoles and lining vacuolar membranes of salivary glands and gut cells of A. aegypti mosquitoes 10 days or more after infection with BUN virus. No particles were observed earlier, despite high virus titres 4 days or more after injection. After inoculation of continuous live tissue cultures of human epidermoid corcinoma cells (H.Ep. 2) with 10 ⁶•⁵ mouse LD₅₀, the highest amount of virus produced was 10 ⁷•⁰ mouse LD₅₀ per ml. cell suspension after 24 hours incubation at 37°C. Maximum yields of BUN virus (10 ⁶•² mouse LD₅₀ per ml. cell suspension) were attained 24 hours after inoculation of primary chick embryo fibroblast monolayers with 10 ⁵•² mouse LD₅₀ following incubation at 37 C . However, a peak titre of 10 ⁵•⁰ mouse LD₅₀ was attained 3 days after inoculation with 10 ³•⁷mouse LD₅₀ in cultures incubated at 29°C. Before an increment of virus titre was observed infectivity declined to zero during the initial 4 hours after inoculation of cultures incubated at 37°C, and a tenfold decline of infectivity was noted in cultures incubated at 29°C. Enveloped virions with total diameter 84 nm which contained electron-dense nucleoids 44 nm diameter were observed extracellularly in thin sections of chick embryo fibroblasts infected 12 hours previously with BUN virus. These particles were released by budding. Precursor particles 41 nm diameter were associated with intracellular membranes in occasional cells sectioned at 4 hours. Extracellular virions released one day after inoculation of H.Ep. 2 cultures were tagged by ferritin-labelled anti-BUN antibody. Enveloped virions with mean diameters 100 nm were observed in suspensions of suckling mouse brain infected with BUN virus and stained negatively with phosphotungstic acid. These results show clearly that BUN virus exhibits the essential biological and morphological characteristics of a mosquito-borne arbovirus. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

The interaction between Murine Cytomegalovirus (MCMV) and the cell cycle has been investigated in synchronized murine cells. Based on the following evidence it was concluded that MCMV replication depends upon the host S phase: (1) the normal latent period of viral growth in exponentially growing 3T3 cells (12 h). was protracted until the host S phase (ca. 20 to 24 h) in synchronized cells infected in early G—l; (2) G-l arrested 3T3 cells failed to support viral replication; (3) entry of the virus was equally efficient in G-l, S and exponential cells; (4) in exponential 3T3 cells viral DNA synthesis began at 10 h post infection, and in synchronized cells it began approximately 16 to 18 h after infection, or early S phase. Therefore, the replication of viral DNA requires host S phase events. Another herpesvirus, Herpes Simplex Virus type-1 (HSV-1) replicated independently of S phase. However, a mutant of HSV-1, deficient in its ability to induce thymidine kinase, demonstrated a dependency upon S phase similar to MCMV. These data indicate a key role of thymidine kinase in the ability of HSV-1 to replicate outside of S phase. However, MCMV induced neither a cellular nor a viral thymidine kinase, and thymidine kinase was not essential for normal viral replication. When G-l arrested 3T3 cells were infected with MCMV, viral DNA synthesis did not initiate and the lytic cycle was reversibly blocked. The non-replicating viral genome remained viable in G-l cells and could be activated at any time by stimulating the cells to enter S phase. The G-l non-permissive system was studied to help ascertain the cell cycle requirements of MCMV. Specifically, two approaches were pursued. In vitro endogenous MCMV DNA synthesis was first studied in G-l, S phase, and exponential 3T3 cells. Under the appropriate conditions, nuclei from infected cells synthesized viral DNA when they had the capacity to dp so in vivo. Nuclei from G—1 phase cells synthesized cell DNA only and not viral DNA. Infected G-l and S phase cells contained a new DNA polymerase which was distinguished from the host enzyme by the high salt requirement for maximal activity. The putative viral DNA polymerase was inhibited by antiserum prepared against infected cell proteins. Therefore, the novel DNA polymerase present in infected G-l and S phase cells was a viral gene product. The second approach involved a comparison of viral transcription in permissive and non-permissive 3T3 cells. The kinetics of hybridization in solution were analyzed by a computer program which evaluated the number of viral RNA classes and the fraction of the viral genome coding for each class. This study revealed the following: (1) in permissive cells by 6 h post infection (early, i.e. before viral DNA synthesis), two classes of viral transcripts were detected, differing by 7 fold in concentration. The abundant class was transcribed from approximately 7% of the viral DNA and the scarce class from approximately 20%. (2) in permissive cells at 24 h post infection (late), abundant and scarce classes (differing by 6 fold in concentration) were transcribed from approximately 10 and 33% of the viral DNA respectively. (3) in non-permissive cells at 6 h, only one class of RNA was present, representing approximately 15% of the viral DNA. (4) in non-permissive cells at 24 h post infection, a single RNA class was observed which was transcribed from approximately 24% of the viral DNA. Summation hybridization experiments indicated that in non-permissive (G-l) cells, only those regions of the DNA which code for early RNA are transcribed. A model has been proposed to describe cell cycle dependent replication of MCMV. It is concluded that MCMV does not replicate in G—l cells due to the absence of specific S phase 'helper-functions' which are required either for the initiation of viral DNA synthesis directly, or for the transcription of viral DNA sequences. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

The cytoskeleton plays an important role in trafficking proteins and other macromolecular moieties throughout the cell. Viruses have been thought to depend heavily on the cytoskeleton for their replication cycles. However, studies, including one in our lab, found that some viruses are not inhibited by anti-microtubule drugs. This study was undertaken to evaluate the replication of viruses from several families in the presence of cytoskeleton-inhibiting drugs and to examine the intracellular localization of the proteins of one of these viruses, Sindbis virus, to test the hypothesis that alternate pathways are used if the cytoskeleton is inhibited. We found that Sindbis virus (Togaviridae, positive-strand RNA), vesicular stomatitis virus (Rhabdoviridae, negative-strand RNA), and Herpes simplex virus 1 (Herpesviridae, DNA virus) were not inhibited by these drugs, contrary to expectation. Differences in the localization of the Sindbis virus were observed, suggesting the existence of alternate pathways for intracellular transport.

The cytoskeleton plays an important role in trafficking proteins and other macromolecular moieties throughout the cell. Viruses have been thought to depend heavily on the cytoskeleton for their replication cycles. However, studies, including one in our lab, found that some viruses are not inhibited by anti-microtubule drugs. This study was undertaken to evaluate the replication of viruses from several families in the presence of cytoskeleton-inhibiting drugs and to examine the intracellular localization of the proteins of one of these viruses, Sindbis virus, to test the hypothesis that alternate pathways are used if the cytoskeleton is inhibited. We found that Sindbis virus (Togaviridae, positive-strand RNA), vesicular stomatitis virus (Rhabdoviridae, negative-strand RNA), and Herpes simplex virus 1 (Herpesviridae, DNA virus) were not inhibited by these drugs, contrary to expectation. Differences in the localization of the Sindbis virus were observed, suggesting the existence of alternate pathways for intracellular transport.

Thesis (Ph. D.)--Cornell University, 1989. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 102-120).

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