This is an application for four years of support to determine the role of chemokine receptors in HIV infection of the central nervous system. The long-term objective of this application is to identify mechanisms of HIV entry, which can then be interrupted in the therapy of HIV dementia. Specifically, the Investigator wishes to test the hypotheses that: (1) the ability of viruses in the brain to use CCR3 as, perhaps, another co-receptor may be important in infection and pathogenesis of the CNS; (2) the ability of viruses to use CCR3 as well as CCR5 as co-receptors may enhance viral tropism for microglia; (3) the propensity of HIV-1 to replicate in the brain may select for genetic variants that use CCR3 and perhaps other co-receptors more efficiently; and, finally, (4) the chemokine co-receptors--specifically CCR3 and CCR5--are potential therapeutic targets for inhibition of viral replication in the brain. To test these hypotheses, the Investigator and collaborators in the Aim #1 will clone envelope sequences from brain and spleen, determine whether any variation exists in clones obtained from the brains and spleens of the same individuals, and then test these envelopes in a number of assays, the chief being the formation of pseudotypes, which can be tested in a panel of cells expressing single chemokine receptors in conjunction with CD4.
In Aim #2 they will associate the use of specific chemokines receptors with replication in microglia having first analyzed the chemokine receptor profile of microglia using a quantitative real-time RT-PCR and immunohistochemical staining.
In Aim #3, the researchers will prepare chimeric recombinant viruses containing HIV env genes from the spleen and select for brain adaptation in vitro. The passaged viruses will then be analyzed for co-receptor use and specific changes associated with varying co-receptor usage analyzed using the same technology described for Aim #1.
In Aim #4, the researchers will determine the sensitivity of brain viruses to a range of concentrations of chemokines. Also, they will use monoclonal antibodies against CCR3 and CCR5 to inhibit virus entry in primary brain cultures and purified microglial cultures. Any small molecule inhibitors of CCR3 or CCR5 that are developed by pharmaceutical companies will be requested for use in these types of assays.
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