Mode of virus transmission into the brain in a mouse model of HIV-infecion

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DESCRIPTION (provided by applicant): In response to the need to understand the neurological complications of HIV infection of human beings, we have constructed a mouse model of systemic HIV infection based upon a new chimeric HIV, EcoHIV. EcoHIV infection of mice is particularly appropriate to study of virus replication and disease in the brain because the virus infects macrophages as well as lymphocytes, enters the brain early after inoculation, induces changes in brain cell gene expression, and neuropathology in some mice. Our primary goal in this revised application is to employ EcoHIV infection of mice to determine conclusively whether HIV-1 establishes infection in the brain through entry of infected monocytes or through other mechanisms. The Specific Aims are 1) To optimize EcoHIV infection in different cell types in the monocyte lineage; 2) To compare cell-free virus to infected monocyte/macrophages for the efficiency of establishment of EcoHIV infection in the brain; and 3) To test T cell-deficient and monocyte-depleted mice for EcoHIV entry and load in the brain. The methods will include efficient systems for monocyte differentiation and microglial cell isolation, optimized approaches for isolation of DNA and RNA from the brain, quantitative PCR for viral DNA and selected brain cell transcripts, two color immunofluorescence microscopy to unambiguously identify infected cells, infection of mice by inoculation of infected isogenic monocytes; and quantitative determination of monocyte migration into the brain using QPCR for a unique cellular marker. These studies meet the challenge of PAS 05078 by testing a key hypothesis of HIV-1 neuroinvasiveness in an animal model. Clear identification of the mode(s) of virus transmission into the brain will build the foundation for a major study to investigate the optimal route to inhibit brain infection either by depletion of cellular reservoirs of HIV or by control of overall burden of free virus. Because HIV-1 infected people continue to develop problems in movement and memory there is a need to control and ultimately block virus infection in the brain. The study proposed here will employ laboratory mice and a new form of HIV to clearly determine how the virus enters the brain to establish infection to develop better methods to block this facet of HIV disease and improve health for the infected population.