The HIV-1 negative regulatory factor (Nef) is important for efficient virus production from astrocytes

The HIV-1 negative regulatory factor (Nef) is important for efficient virus production from astrocytes Suresh Kandel, MS and Johnny J. He, PhD Email: Johnny.He@unthsc.edu; Suresh.kandel@my.unthsc.edu Department of Microbiology, Immunology, and Genetics, University of North Texas Health Science Center, Fort Worth, TX 76107 Purpose: The HIV-1 negative regulatory factor (Nef) is a membrane associated myristoylated protein with a molecular weight of 27-32 KDa. It downregulates immune molecules such as MHC-I/CD4 receptors on CD4+ T lymphocytes and is indispensable for AIDS progression and high level of viremia. Additionally, Nef plays important roles in the virus production from immune cells and the infectivity of viruses released from these cells. However, much remained undefined about the involvement of Nef for virus production and infectivity in the context of CNS, particularly in astrocytes. Astrocytes are the most abundant long- lived cell types in the brain. HIV-1 infection leads to restrictive virus replication and establishes latency in these cells. Therefore, the main objective of this study is to determine the role of HIV-1 Nef in the virus production from astrocytes and the infectivity of viruses released from astrocytes. Methods: We transfected pNL4-3 and pNL4-3Nef- plasmids in astrocytic cell line LN299. Non-astrocytic cell line HEK 293T was used as a control. The virus production was determined using reverse transcriptase assay, while the infectivity of viruses was determined using a LTR-driven luciferase stably expressing cell line, TZM-BL. Results: In HEK 293T, the virus production was high and showed no difference between NL4-3 and NL4-3Nef-. In comparison to HEK 293T, the virus production was lower in LN299. There was more virus production in pNL4-3 transfected cells than pNL4-3Nef- transfected cells. There were no differences in infectivity of the viruses produced between pNL4-3-transfected cells and pNL4-3Nef- transfected cells in both HEK 293T and LN299. Conclusion: The results demonstrated that Nef expression gave rise to differences in virus production in astrocytes and suggest that Nef plays an important regulatory role in HIV gene expression in astrocytes. Further studies are underway to investigate the underlying mechanisms. Keywords: Nef, HIV-1, Astrocytes, budding, Infectivity