Lentiviral vector expressing Nef as a tool to study HIV neuropathology

Abstract

The goal of this work is to use a lentivirus to create a simplified endogeonous expression system producing only Nef to study the unique contributions that this viral neurotoxin makes to HIV‐related neurological impairment. We used PCR to generate the Nef gene flanked by unique restriction enzymes sites(BamH1 and Sal1). Then we digested a lentiviral packaging vector (pLenti) and this PCR product with the same restriction enzymes to generate DNA fragments with complementary ends. Cloning was performed by incubating with DNA ligase and ATP to create recombinant pLenti‐Nef plasmids. Colonies were screened by PCR to identify those containing plasmids with HIV Nef. These pLenti‐Nef plasmids were recovered by plasmid prep and confirmed by sequencing. The pLenti‐Nef plasmid can be used to generate lentiviral particles capable of delivering the HIV‐Nef gene to target cells in a single round of infection a single round of infection. We will infect rat astrocytes in vitro with these viruses followed by implantation into the hippocampus of Sprague Dawley rats in vivo to mimic the levels of Nef that have been reported in HIV‐infected patients. This investigation will help us understand the HIV disease better at a molecular level by developing a system that allows us to study the role of Nef in driving immune cell chemotaxis into the brain and the loss of neuron function that accompanies HIV neuropathology in parallel. Grant support by RR003050 and RR016470.