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Investigation of the cellular tropism and in vivo distribution of HIV-1

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NewmanK_2005redux.pdf (39.27Mb)
Date
2005
Author
Newman, Kirsty
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Abstract
 
 
Abstract Since its discovery in 1983, HIV-1 has spread rapidly resulting in a global pandemic. Currently an estimated 40 million people are infected with the virus. Infection leads eventually to the acquired immunodeficiency syndrome (AIDS) and death. The symptoms of the syndrome are largely due to the devastating effect HIV has on the immune system. HIV has been the focus of an unprecedented amount of research however there are still many aspects of HIV pathogenesis which remain incompletely characterized. One area which continues to attract attention is HIV tropism. HIV requires two receptors- CD4 and a co-receptor- for infection. In vivo, HIV variants mainly use either CCR5 or CXCR4 as co-receptors and thus variants can be classified as either R5 (CCR5 using), X4 (CXCR4 using) or (R5X4) (dual tropic). However these classifications do not fully explain the differing abilities of variants to infect particular cell types. It is thought that other factors, such as receptor density or alternative receptors, may underlie observed differences in tropism. In addition to studying viral variants to investigate determinants of tropism, it is also possible to investigate post-mortem material from HIV infected individuals to characterize nature and cellular location of HIV variants. HIV can infect cells in the brain and this leads in some cases to HIV associated dementia (HAD). The presence of HIV in the brain poses a challenge to attempts to eradicate the virus as many drugs have poor CNS penetration. There have been concerns that sub-optimal levels of drugs in the CNS may support the evolution of drug resistant variants. Alternatively, low levels of drugs may allow growth of drug sensitive variants which could reseed the periphery if the drug regime was halted. Characterisation of the provirus present in post mortem brain material could lead to a better understanding of this issue. The virus in the brain appears to mainly infect microglia and macrophages however it is not clear to what extent other brain cells might be infected. Conflicting reports have emerged regarding the infection of astrocytes and neurons. Thus a method for separating the various brain cells which allows them to be tested for HIV could be valuable.
 
In the first part of this project, methods to characterise the cellular tropism of HIV were investigated. A problem with many previous methods is that manipulation or amplification of the HIV genome has resulted in artefactual changes in the sequence. Thus any results obtained regarding the cellular tropism of variants may not accurately reflect the in vivo situation. A vector was produced which could be used to produce recombinant HIV expressing study subject derived env. The vector incorporated the EGFP to allow easy identification of infected cells. Three methods for production of virus without the need to amplify the genome in E. coli were designed. All methods were capable of producing infectious virus expressing various env genes.
 
The second part of the project sought to investigate the distribution of drug resistance variants in vivo. DNA was extracted from post mortem brain and lymphoid tissues from HIV infected study subjects. Limiting dilution PCR was used to obtain PCR products each derived from a single template molecule. No differences were found in the distribution of drug-resistance mutations between the two compartments for three out of four study subjects. In one study subject there were considerably more drug resistance mutations in the lymphoid derived sequences than in those amplified from brain tissue. This is probably due to low levels of anti-HIV drugs in the CNS of this subject.
 
In the final part of the project, a method for the bulk isolation of microglia and astrocytes from post-mortem HIV-infected brains was optimised. Cellular debris was first removed using a density gradient. The cells were then fixed using a gentle ethanol fixation technique. This inactivated the HIV, fixed and permeabilised the cells while maintaining DNA integrity. FACS sorting was carried out using CD68 and GFAP as markers of microglia and astrocytes respectively. Separation of cells was carried out using two HIV-infected brains. No provirus was detected in the separated cells however the brain material used was from asymptomatic study subjects and had extremely low proviral loads. It is hoped that in the future the technique could be used to separate brain material which has a higher proviral load. This would allow quantification of the level of HIV infection of these two cell types.
 
URI
http://hdl.handle.net/1842/29916
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