HIV-1 particle assembly occurs on the plasma membrane of infected cells. The assembly process is directed by the Gag polyprotein, which is synthesized on cytoplasmic ribosomes, and travels to the plasma membrane assembly site by an incompletely understood pathway. Env proteins are synthesized on ribosomes of the rough endoplasmic reticulum, form trimers in the ER, and transit the secretory pathway of the cell to the plasma membrane. Despite following very different pathways, Gag and Env meet at the particle budding site, and Env is specifically concentrated at this site. New evidence presented here indicates that Env reaches the particle assembly site in a directed manner through interactions with a trafficking complex directed by Rab11- FIP1C (FIP1C) and Rab14. This project will define the mechanisms by which FIP1C and Rab14 direct Env sorting and incorporation onto HIV-1 particles. We will then dissect specific regulators of actin polymerization discovered in a unique siRNA screen that strongly affect budding or release of HIV-1 particles. Experiments in Aim 1 will first define FIP1C functional domains that specifically are required to translocate Env onto the particle, and then determine the motif(s) within the Env cytoplasmic tail involved in FIP1C-dependent trafficking and incorporation. TIRF microscopy and live cell imaging will be pursued to fully define the steps in FIP1C-Env trafficking to the particle assembly site.
In Aim 2, the contribution of the FIP1C-Rab14 trafficking pathway to polarization of HIV structural components and to cell-cell transmission of virus will be addressed. Experiments in this aim will also identify the kinesin responsible for movement of the Env-FIP1C-Rab14 complex to the particle assembly site. We will then follow up on exciting new findings indicating that FIP1C mediates MA-Env interactions, testing two relevant models for Gag trafficking and Gag-Env interaction.
Aim 3 will examine a related area, the contribution of ROCK1-LIMK1-cofilin pathway to particle budding and release, and will define the role of related actin regulatory proteins that modulate HIV particle production. This experimental plan will add significantly to the knowledge of specific host factors involved in HIV particle assembly and budding, and will provide fundamental new insights into cellular trafficking pathways utilized by enveloped viruses.
This project is relevant to public health because it follows up on a recent discovery of how the envelope protein gets on to HIV particles as they form. This observation has the potential to allow new drugs to be developed against HIV, and will contribute to the understanding of basic ways that proteins are sorted to the plasma membrane of cells. The project will also study other host factors that are important to HIV assembly, providing additional information that is useful in fighting this virus and in ending the global epidemic.
|Spearman, Paul (2018) Viral interactions with host cell Rab GTPases. Small GTPases 9:192-201|
|Kirschman, Junghwa; Qi, Mingli; Ding, Lingmei et al. (2018) HIV-1 Envelope Glycoprotein Trafficking through the Endosomal Recycling Compartment Is Required for Particle Incorporation. J Virol 92:|
|Staitieh, Bashar S; Ding, Lingmei; Neveu, Wendy A et al. (2017) HIV-1 decreases Nrf2/ARE activity and phagocytic function in alveolar macrophages. J Leukoc Biol 102:517-525|
|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|
|Spearman, Paul (2016) HIV-1 Gag as an Antiviral Target: Development of Assembly and Maturation Inhibitors. Curr Top Med Chem 16:1154-66|
|St Gelais, Corine; Kim, Sun Hee; Ding, Lingmei et al. (2016) A Putative Cyclin-binding Motif in Human SAMHD1 Contributes to Protein Phosphorylation, Localization, and Stability. J Biol Chem 291:26332-26342|
|Qi, Mingli; Chu, Hin; Chen, Xuemin et al. (2015) A tyrosine-based motif in the HIV-1 envelope glycoprotein tail mediates cell-type- and Rab11-FIP1C-dependent incorporation into virions. Proc Natl Acad Sci U S A 112:7575-80|
|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|
|Chukwuma, Valentine U; Hicar, Mark D; Chen, Xuemin et al. (2015) Association of VH4-59 Antibody Variable Gene Usage with Recognition of an Immunodominant Epitope on the HIV-1 Gag Protein. PLoS One 10:e0133509|
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