Friday, November 22, 2024
11/22/2024
7. Project Summary/Abstract To develop new HIV-1 drugs or therapies it is important to understand the fundamental biology involved with infection of target cells. After the viral membrane fuses with the target cell membrane a series of early steps of replication must occur to facilitate infection. Viral fusion releases the conical capsid into the cytoplasm and the capsid structure disassembles by a process called uncoating. The viral RNA is reverse transcribed into DNA. The viral complex must also traffic through the cytoplasm to enter the nucleus through a nuclear pore. HIV-1 only encodes 15 proteins, so host cell proteins are needed to facilitate many steps in viral replication. One cellular factor is the actin associated protein spectrin β non-erythrocytic 1 (SPTBN1), which was found by three studies to facilitate early steps of HIV replication. However, the mechanism by which SPTBN1 aids in HIV infection remains unknown. Preliminary studies revealed that siRNA knockdown of SPTBN1 decreased HIV infection and delayed uncoating in the human microglial cell line CHME3. Uncoating is at the crossroads of the early steps of replication, so it is possible that SPTBN1 is needed for reverse transcription or intracellular trafficking which could have a secondary effect on uncoating. Therefore, the goal of this project is to determine the role of SPTBN1 in early steps HIV replication. The effect of SPTBN1 on HIV infection will be studied in cultured microglial cell lines, a natural target of HIV infection in humans. The goal of Aim 1 is to assess the effect of SPTBN1 on HIV infection in a second human microglial cell line. In addition, the siRNA transfection procedure will be optimized to achieve efficient knockdown of SPTBN1 RNA and protein levels. In Aim 2 cultured cell based and fluorescence microscopy based uncoating assays will be used to characterize the effect of SPTBN1 on uncoating kinetics. The kinetics of viral fusion will also be determined using a cultured cell-based viral fusion assay. The effect of SPTBN1 on reverse transcription will be assessed in Aim 3 using two assays. A cultured cell-based inhibitor addition assay will determine if SPTBN1 knockdown alters completion of reverse transcription. Quantitative PCR will be used to determine if SPTBN1 knockdown changes the relative amounts of different reverse transcription products at various times post-infection. In Aim 4 the localization of virions in relation to microtubules, actin, and the nucleus will be detected by confocal fluorescence microscopy in SPTBN1 and control knockdown cells. Colocalization between virus and endogenous SPTBN1 at different time post-infection will also be examined using confocal fluorescence microscopy. In summary, this project seeks to determine which step(s) of HIV replication are facilitated by SPTBN1 and how this interaction affects viral replication. The association of SPTBN1 and HIV may provide new targets for antiviral drugs or therapies that would block this interaction.
HHS’ Tracking Accountability in Government Grants System (TAGGS) website provides access to detailed descriptions of grants, loans, aggregated direct payments and other types of financial assistance awarded by the HHS Operating Divisions (OPDIVs) and the Office of the Secretary Staff Divisions (STAFFDIVs).
