Session 18Oral Abstract Presentations Viral Neuropathogenesis Session Day and Time: Wednesday 10 am - 12:30 pm Presentation Time: 10:30 Room: Ballroom C
88 Adaptation of Neurotropic Primary HIV-1 Isolates for CD4-independent Replication R. Dunfee*1,2, P. Gorry1,2, J. Taylor3, K. Kunstman3, J. Bell4, S. Wolinsky3, D. Gabuzda1,2 1Dana-Farber Cancer Inst, Boston, MA; 2Harvard Med Sch, Boston, MA; 3Northwestern Univ Med Sch, Chicago, IL; and 4Univ of Edinburgh, Scotland
Background: Microglia, a major target for HIV-1 in the CNS, express lower levels of CD4 and CCR5 than target cells in peripheral blood. Reduced dependency on CD4 and/or CCR5 levels may be properties of the HIV-1 Env that increase neurotropism or neurovirulence. We previously demonstrated that a brain isolate from HIV-1 associated dementia (HAD) patient (pt) UK1 had reduced dependency on CD4 and CCR5 levels for entry and CD4-independent binding to CCR5. Here we screened additional primary brain isolates for reduced dependency on CD4 and CCR5, and adapted 3 neurotropic viruses for CD4-independent replication.
Methods: Cf2-Luc cells were co-transfected with 0.05 or 0.5 mug CD4- and 0, 0.05, 0.5, and 5 mug CCR5-expressing plasmid to generate 8 populations of cells expressing different levels of each receptor. Transfected cells were infected with equivalent amounts of virus stock. Cells were lysed 72 hrs post-infection and assayed for luciferase activity. Viruses chosen for adaptation for CD4-independent replication were used to infect Cf2Th cells expressing CD4 and CCR5. Supernatants from infected cultures were then passaged on fresh cultures expressing decreasing levels of CD4 followed by CD4-negative cells.
Results: We tested 6 primary brain isolates for decreased dependency on CD4 and CCR5 levels for virus entry. Of these, 3 viruses isolated from HAD pts UK1 and UK7 and other neurotropic viruses previously shown to infect cells expressing low levels of CD4 or CCR5 or to bind CCR5 independently of CD4, were chosen for adaptation for CD4-independent replication. Two (2) of these viruses, the R5 variant UK7-br and R5X4 variant dBR07, were adapted to become CD4 independent, and a third virus, the R5 variant UK1-br, was adapted to grow on cultures expressing very low levels of CD4. Full-length HIV-1 env genes from the adapted viruses are being genotypically and phenotypically characterized.
Conclusions: These results suggest that reduced dependence on CD4 and CCR5 levels may increase HIV-1 neurotropism or neurovirulence. Some neurotropic viruses can be adapted for CD4-independent replication, which will allow a better understanding of detailed molecular interactions between HIV-1 Env, CD4 and CCR5 and how these interactions impact HIV-1 neuropathogenesis.
