The productive site of HIV-1 assembly is the plasma membrane, both in cultured cell lines and primary cells. This conclusion is based both on the observation that newly synthesized Gag first appears at the plasma membrane, and is only detected later in endosomes, and the demonstration that inhibiting endocytosis blocks the appearance of virions in the internal organelles without affecting the yield of extracellular particles This assembly of HIV-1 at the plasma membrane make the assembly accessible to imaging using total internal reflection fluorescence microscopy (TIR-FM) a technique on which a large fraction of the experiments in this proposal are based. This technique has been used to visualize individual virions of HIV-1 as they assemble as well as the movement and packaging of individual molecules or dimers of HIV-1 genome. The technique has been used to demonstrate that the virions accumulate at the plasma membrane over a period of 6-20 minutes. The genome is recruited to the plasma membrane immediately before recruitment of Gag. In contrast, ESCRTs are recruited for only tens of seconds and tens of molecules transiently at the very end of the recruitment of Gag. The AAA-ATPase, Vps4, is recruited just seconds later. Super-resolution optical microscopy has added the information that ESCRT recruitment is to the neck that links the nascent virion to the mother cell. The long-term goal of this project is to identify, characterize and ultimately understand the steps in the biogenesis of HIV-1 and related retroviruses. We want to understand the dynamics of viral components as they interact with each other and with host components. Imaging based approaches allow the examination of both the dynamics and localization of molecules during which may be inaccessible through biochemical techniques. The main foci will be on the dynamics and localization of the viral protein Gag, the dynamics and localization of host molecules and then the relative dynamics and localization of the viral and host molecules.

Public Health Relevance

Some of the most powerful anti-viral agents interfere with the ability of the viruses to assemble. This project will examine the assembly if single viruses of HIV-1 with the goal of understanding the steps in the assembly of the virus.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM119585-01
Application #
9136384
Study Section
AIDS Molecular and Cellular Biology Study Section (AMCB)
Program Officer
Sakalian, Michael
Project Start
2016-05-05
Project End
2020-03-31
Budget Start
2016-05-05
Budget End
2017-03-31
Support Year
1
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Rockefeller University
Department
Biology
Type
Graduate Schools
DUNS #
071037113
City
New York
State
NY
Country
United States
Zip Code
10065
Simon, Sanford (2018) Günter Blobel (1936-?2018). Nature 556:32
Simon, Sanford M (2018) Günter Blobel (1936–2018) Cell 173:278-280
Tomasini, Michael D; Johnson, Daniel S; Mincer, Joshua S et al. (2018) Modeling the dynamics and kinetics of HIV-1 Gag during viral assembly. PLoS One 13:e0196133
Takacs, Constantin N; Andreo, Ursula; Dao Thi, Viet Loan et al. (2017) Differential Regulation of Lipoprotein and Hepatitis C Virus Secretion by Rab1b. Cell Rep 21:431-441
Takacs, Constantin N; Andreo, Ursula; Belote, Rachel L et al. (2017) Green fluorescent protein-tagged apolipoprotein E: A useful marker for the study of hepatic lipoprotein egress. Traffic 18:192-204
Johnson, Daniel S; Jaiswal, Jyoti K; Simon, Sanford (2012) Total internal reflection fluorescence (TIRF) microscopy illuminator for improved imaging of cell surface events. Curr Protoc Cytom Chapter 12:Unit 12.29