No description available.

No description available.

No description available.

The HIV/AIDS still remains a global health challenge with 42 million people infected with the virus. An alarming 70% of these people reside in sub-Saharan Africa with HIV-1 subtype C being the most prevalent subtype in this region and globally. HIV-1 protease (PR) is an obligate homodimer which plays a pivotal role in the maturation and hence propagation of the HI virus. Although successful developments on PR active site inhibitors have been achieved, the major limiting factor has been the emergence of HIV drug resistant strains. It has been postulated that disruption/dissociation of the dimer interface may lead to an inactive enzyme. The development of small molecules and peptides has been a major research area with the key target being the N- and C-termini antiparallel β-sheet. This is due to its highly conserved nature and because it consists of a cluster of amino acids that contribute most of the binding energy and stability of the dimer interface. Hence it is referred to as a ‘hot-spot’. Therefore, binding of protease inhibitors at this site could cause destabilisation and/or dissociation of the enzyme. The terminal residue, F99, was mutated to an alanine disrupting the presumed lock-and-key motif it forms and in turn creating a cavity at the N- and C-termini antiparallel β-sheet. A second mutant, W42F/F99A, was created for monitoring tertiary structural changes exclusively at the N- and C-termini antiparallel β-sheet. The F99A and W42F/F99A, compared to the wild-type, showed a higher expression yield and also migrated further when separated using tricine SDS-PAGE. Wild-type protease CD spectra showed a minimum at 214 nm and a local maximum at 230 nm, while the mutants exhibited minima at 203 nm and absence of the local maxima. A 50% higher fluorescence intensity and a 2 nm red-shift for the mutants versus the wild-type was observed. According to SE-HPLC data the relative molecular weight of the wild-type, F99A and W42F/F99A are 16.4 kDa, 20.7 kDa and 18.1 kDa, respectively. Although the thermal unfolding of all three proteases was irreversible, the unfolding transition of the wild-type was clearly defined between 55 °C and 63 °C. The F99A and W42F/F99A unfolding curves were linear without clearly defined transition states. The specific activity of the F99A (0.13 μmol/min/mg) amounted to a ten-fold reduction compared to the wild-type (1.5 μmol/min/mg). The substrate binding affinity (KM) for the F99A was 41% lower than the wild-type when 2 μM of protein was used. The Vmax and kcat values were about 30-fold and two-fold, respectively, higher for the wild-type when compared to the F99A. Therefore, the tricine SDS-PAGE analysis, secondary and tertiary structural characterisation and thermal denaturation curve showed that the F99A mutation has altered the structure causing ‘partial’ unfolding of the protein. But, the protein still maintained minute activity. The overlap between the ANS binding spectra of the wild-type and variants suggests that the dimeric form still exists.

The Human Immunodeficiency Virus (HIV) subtype C accounts for the majority of infections in Southern Africa. The HIV protease is one of the targets in HIV treatment due to its pivotal role in HIV maturation in the host cell. However, because of polymorphisms in the HIV genome, drug resistance becomes a major problem in HIV treatment. Polymorphisms in the HIV protease gene result in altered substrate cavities, and /or flap hinge modifications leading to unfavourable drug interaction with the enzyme. The most common form of drug resistant mutations is single amino acid substitutions. Although, amino acid insertions have been reported, this form of mutation in the HIV protease is rare. L38↑N↑L insertion is a unique form of HIV protease polymorphism that was isolated from a patient failing drug therapy in South Africa. The objective of this research was to assess the impact of the L38↑N↑L insertions, with accompanying background mutations, on the structure and function of this form of polymorphism in HIV-1 South African subtype C protease. The far-UV circular dichroism (CD) spectra of L38↑N↑L protease shows a trough at 203 nm, suggesting alterations in the secondary structure content of this mutant. Whereas the wild type (WTCSA-HIVPR) displays a trough at 215 nm. However, tertiary structure characterisation using fluorescence spectroscopy did not detect changes within the local tryptophan environment of L38↑N↑L protease in comparison with the wild type due to no significant shift in emission wavelength. The specific activity of L38↑N↑L protease and wild type was 28.0±1.3 μmol.min-1.mg-1 and 123.45±6.4 μmol.min-1.mg-1 respectively. The turn-over number for L38↑N↑L protease and wild type was 1.0 × 10-3 ± 6.0 × 10-5 and 7.7 × 10-3 ± 5.6 × 10-4 respectively. As much as the presence of known drug resistance mutations in L38↑N↑L can be attributed to drug resistance, it should also be noted that the insertions may have also caused local structural alterations that may have enhance drug resistance of L38↑N↑L. These changes could have lead to the decreased catalytic activity of the L38↑N↑L protease. Homology modelling studies show that the insertions in L38↑N↑L protease may have resulted in a fold similar to 2HS1 (PDB code), which has a modification on the flap hinge. In addition, the homology modelling studies suggest that L38↑N↑L protease may have a second inhibitor binding site next to one of the flap hinge regions as seen in the 2HS1 model. In conclusion, the L38↑N↑L insertions and accompanying background mutations may have contributed to the local structural modifications that lead to drug resistance in L38↑N↑L protease.

Survival for patients with advanced cancer has remained dismal, and there is a need for new treatments. In this context viral immune therapy is a promising novel strategy. Intravenous delivery confers advantages as it enables simultaneous treatment of primary tumour and any metastatic deposits but host defences limit Vaccinia virus's (VV) ability to infect tumour after systemic administration. Although Vaccinia virus can potentially be delivered systemically as it can evade both complement and neutralising antibodies, our investigations have revealed that VV cannot effectively infect tumour cells in immunocompetent mice after systemic delivery. Strikingly, we observed that if macrophages were depleted in the mice using clodronate liposomes, VV infection of tumours was dramatically enhanced. However, clodronate liposomes non-selectively deplete macrophages and potentially diminish any beneficial macrocytic activity in the tumour microenvironment unrelated to viral clearance. Consequently, a more clinically appropriate agent is needed. Macrophages recognise and ingest pathogenic microorganisms through phagocytosis, a process for which several lines of evidence have highlighted an important role for phosphatidylinositol 3-kinases. Accordingly, in these investigations I have evaluated the effect of selective PI3K inhibitors on macrophage phagocytosis in vitro and demonstrated that IC87114 (a PI3 kinase delta inhibitor) is effective at reducing uptake of VV by macrophages, confirming this finding in transgenic macrophages with a mutant of the PI3 kinase delta isoform knocked in. Subsequently, it was confirmed that IC87114 affects attachment of the virus to macrophages but plays no role in internalisation of the virus. In cancer cells cultured in isolation, the inhibitor has no direct cytotoxic effect and when combined with VV, in the same in vitro system, there is no change in the amount of cell death compared to VV alone treated controls. Biodistribution studies have established that IC87114 combined with VV results in statistically significantly higher levels of virus detected in tumours compared to the groups treated with VV alone, with similarly limited off-target effects. Finally, three different efficacy studies have demonstrated statistically significantly superior tumour responses in the VV+IC87114 group. In conclusion, PI3k delta blockade is an effective strategy for enhancing systemic delivery of VV in a preclinical model and could be a useful adjuvant in VV clinical trials.

Typescript. / Digitized by Kansas Correctional Industries

Abstract not available

Bibliography: leaves 133-141.

This report describes the partial characterization of the first cultivable calicivirus isolated from a European rabbit (Oryctolagus cuniculus), named rabbit calicivirus Oryctolagus-1 and abbreviated RaCv Ory-1. RaCv Ory-1 was isolated from juvenile feeder rabbits displaying symptoms of diarrhea. Absence of neutralization by type specific neutralizing antibodies for 40 caliciviruses and phylogenetic sequence comparisons among the caliciviruses of partial ORF1 and complete ORF2 and ORF3 sequences demonstrate that RaCv Ory-1 is a novel member of the marine calicivirus sub-group. Phylogenetic evaluation of the Caliciviridae indicates that analyses using pooled 3D-polymerase and capsid sequences are more statistically robust than identically executed analyses of single gene sequence data. Phylogenetic analysis of pooled 3D-polymerase and capsid a.a. sequences show canine calicivirus isolate 48 (CaCv-48) to be an intermediate species which forms a node approximately equidistant to the feline, marine, and Sapporo-like caliciviruses. RaCV Ory-1 is suggested as a possible cultivable model of rabbit hemorrhagic disease virus. / Graduation date: 1999