HIV-1 Replication and Host Cellular Membranes
My laboratory studies the interactions between cellular structures and enveloped viruses. As obligatory parasites, viruses in general utilize a large number of host cell factors at various stages of their life cycle. Therefore, analyses of virus life cycle promise to increase our knowledge of cellular machinery with which viral proteins interact. We are particularly interested in the roles played by cellular membranes during replication of retroviruses including HIV-1. Three major areas of research in this laboratory are: i) molecular mechanisms that determine the sites of virus assembly, ii) relationships between virus assembly and organization of plasma membrane microdomains, and iii) roles played by cell polarity in cell-to-cell virus transmission.
i) Gag, a multidomain and multifunctional protein, is the major building block of retrovirus particles. Targeting of Gag to the location at which virus particle formation occurs, usually the plasma membrane, is a major aspect of retrovirus particle production. Various cellular proteins are targeted to the plasma membrane through the interaction between their basic domains (e.g., pleckstrin homology domain) and the plasma membrane-enriched lipid, phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2]. Intriguingly, we found that PI(4,5)P2 perturbation severely reduced virus particle production by reducing Gag-membrne binding and by mislocalizing Gag to other compartments. Ongoing studies in our lab focus on the mechanisms by which PI(4,5)P2 directs Gag to the plasma membrane.
ii) Advances in cell biology revealed that the plasma membrane is not a homogenous sea of lipid, but consists of various types of microdomains with specific sets of protein and lipid components. Microdomains such as lipid rafts and tetraspanin-enriched microdomains (TEMs) are implicated in multiple cellular functions including signaling and trafficking, often as delivery platforms. Notably, HIV-1 assembly occurs at the plasma membrane region enriched in these microdomains. Previous studies showed that these microdomains play important role(s) in both virus assembly and particle infectivity. Furthermore, these microdomains are implicated in the function of the cellular antiviral factor BST-2/tetherin that restricts release of nascent virus particles from the plasma membrane. We aim to fully understand the relationships between plasma membrane microdmains and HIV-1 assembly.
iii) Virus transmission at the interface between two cells is a markedly more efficient mechanism of virus spread than cell-free transmission and likely represents the major mode of transmission for HIV-1 in infected individuals. Cell-to-cell HIV-1 transmission is thought to occur at the virological synapse (VS). VS is a raft- and TEM-enriched contact structure formed between a virus-producing T cell and a non-infected T cell. Although Gag and Env as well as cellular adhesion and cytoskeletal proteins have been found to localize to the VS, it is unknown what drives accumulation of Gag and virus particles to the VS and whether there is a precursor for the VS. We observed that in a motile, polarized T cells, Gag accumulates to a rear-end protrusion known as the uropod in a multimerization-dependent manner and that the Gag-laden uropod mediates frequent contacts with other T cells. Therefore, it is possible that the uropod serves as a preformed precursor of the VS. On this front, we seek to understand the mechanistic basis of the polarized Gag localization to the uropod.
Ono, A.*, S.D. Ablan, S. J. Lockett, K. Nagashima and E.O. Freed. 2004. Phosphatidylinositol (4,5) bisphosphate regulates HIV-1 Gag targeting to the plasma membrane. Proc. Natl. Acad. Sci. USA. 41:14889-14894.
Ono, A.*, A.A. Waheed, A. Joshi, and E.O. Freed. 2005. Association of human immunodeficiency virus type 1 Gag with membrane does not require highly basic sequences in nucleocapsid: Use of a novel Gag multimerization assay. J. Virol. 79:14131-14140.
Ono, A.*, A.A. Waheed, and E.O. Freed. 2007. Depletion of cellular cholesterol inhibits membrane binding and higher-order multimerization of human immunodeficiency virus type 1. Virology 360(1):27-35.
Chukkapalli, V, I.B. Hogue, V. Boyko, W.-S. Hu, and A. Ono.* 2008. Interaction between HIV-1 Gag matrix domain and phosphatidylinositol-(4.5)-bisphosphate is essential for efficient Gag-membrane binding. J. Virol. 82:2405-2417
Kawada, S., T. Goto, H. Haraguchi, A. Ono, Y. Morikawa. 2008. Dominant negative inhibition of human immunodeficiency virus particle production by the nonmyristoylated form of gag. J. Virol. 82:4384-4399.
Ono, A.* 2009. HIV-1 assembly at the plasma membrane: Gag trafficking and localization. Future Virology. 4:241-257 (review).
Hogue, I. B., A. Hoppe, and A. Ono* 2009. Quantitative fluorescence resonance energy transfer microscopy analysis of the human immunodeficiency virus type 1 Gag-Gag interaction: relative contributions of the CA and NC domains and membrane binding. J. Virol. 83, 7322-7336.
Chukkapalli, V., S.J. Oh, and A. Ono.* 2010. Opposing mechanisms involving RNA and lipids regulate HIV-1 Gag membrane binding through the highly basic region of the matrix domain. Proc. Natl. Acad. Sci. USA. 107, 1600-1605.
Ono, A.* 2010. Relationships between plasma membrane microdomains and HIV-1 assembly. Biol. Cell. 102, 335-350 (review).
Llewellyn, G.N., I.B. Hogue, J.R. Grover, and A. Ono.* 2010. Nucleocapsid promotes localization of HIV-1 Gag to uropods that participate in virological synapses between T cells. PLoS Pathogens 6(10): e1001167. doi:10.1371/journal.ppat.1001167
* corresponding author