Vpu is an HIV accessory gene that induces degradation of CD4 in the endoplasmic reticulum and enhances viral particle release. This project seeks to define the mechanism by which Vpu enhances viral particle release. Preliminary studies in our laboratory show that human cells are restricted for particle assembly and require Vpu for efficient particle production (restrictive/Vpu-responsive). Other cells, notably simian cells, are permissive for efficient assembly and do not respond to Vpu (permissive/Vpu-unresponsive). In heterokaryons, the restrictive phenotype dominates, suggesting strongly that a cellular inhibitor of assembly is present in restrictive cell types. Experiments in Aim 1 of this proposal will further characterize this cellular inhibitor. The Vpu-responsive cellular factor will be measured in cell lines and in human primary T cells and macrophages, and a survey of simian cells will determine if indeed this is a species-specific factor.
In Aim 2, the site of the assembly block in restrictive cells will be determined. Biochemical fractionation of pulse-labeled cells will define the kinetics of Gag trafficking in the presence and absence of Vpu. Quantitative live cell imaging techniques will be employed to identify the subcellular location where Gag accumulates in restrictive cells, and to define the subcellular site of action of Vpu. A novel Gag-Gag interaction assay based upon fluorescence resonance energy transfer (FRET) will allow the identification of intracellular Gag multimers in living cells; the influence of Vpu on these assembly intermediates will be assessed. Experiments in Aim 3 will identify the cellular inhibitor of assembly that is overcome by Vpu. An expression cloning strategy will be utilized to identify cellular factor(s) in restrictive cells that inhibit HIV assembly and are overcome by Vpu. In parallel, a subtractive hybridization approach utilizing restrictive and permissive Jurkat cell clones will identify candidate cellular cDNAs that restrict assembly. These studies will define the mechanism by which Vpu enhances HIV particle release and will characterize a novel host cell restriction to HIV replication that is present in human cells.
|Hammonds, Jason E; Beeman, Neal; Ding, Lingmei et al. (2017) Siglec-1 initiates formation of the virus-containing compartment and enhances macrophage-to-T cell transmission of HIV-1. PLoS Pathog 13:e1006181|
|Hampton, Cheri M; Strauss, Joshua D; Ke, Zunlong et al. (2017) Correlated fluorescence microscopy and cryo-electron tomography of virus-infected or transfected mammalian cells. Nat Protoc 12:150-167|
|Strauss, Joshua D; Hammonds, Jason E; Yi, Hong et al. (2016) Three-Dimensional Structural Characterization of HIV-1 Tethered to Human Cells. J Virol 90:1507-21|
|Giroud, Charline; Marin, Mariana; Hammonds, Jason et al. (2015) P2X1 Receptor Antagonists Inhibit HIV-1 Fusion by Blocking Virus-Coreceptor Interactions. J Virol 89:9368-82|
|Johnson, Erica L; Chu, Hin; Byrareddy, Siddappa Nagadenahalli et al. (2015) Placental Hofbauer cells assemble and sequester HIV-1 in tetraspanin-positive compartments that are accessible to broadly neutralizing antibodies. J Int AIDS Soc 18:19385|
|Yi, Hong; Strauss, Joshua D; Ke, Zunlong et al. (2015) Native immunogold labeling of cell surface proteins and viral glycoproteins for cryo-electron microscopy and cryo-electron tomography applications. J Histochem Cytochem 63:780-92|
|Wen, Xiaoyun; Ding, Lingmei; Wang, Jaang-Jiun et al. (2014) ROCK1 and LIM kinase modulate retrovirus particle release and cell-cell transmission events. J Virol 88:6906-21|
|Wen, Xiaoyun; Ding, Lingmei; Hunter, Eric et al. (2014) An siRNA screen of membrane trafficking genes highlights pathways common to HIV-1 and M-PMV virus assembly and release. PLoS One 9:e106151|
|Kiss, Gabriella; Chen, Xuemin; Brindley, Melinda A et al. (2014) Capturing enveloped viruses on affinity grids for downstream cryo-electron microscopy applications. Microsc Microanal 20:164-74|
|Chu, Hin; Wang, Jaang-Jiun; Qi, Mingli et al. (2012) The intracellular virus-containing compartments in primary human macrophages are largely inaccessible to antibodies and small molecules. PLoS One 7:e35297|
Showing the most recent 10 out of 21 publications