HIV-1 infections have caused a worldwide pandemic that has claimed the lives of over 20 million people and infected over 40 million more. Our long term goal is to understand how HIV-1 assembles into a viral particle and what cellular machinery is utilized in this process. During HIV-1 assembly, the transmembrane surface glycoprotein (Env) has to traffic to the precise location within the host cell where viral budding occurs. Although a direct interaction between Env and the structural protein (Gag) is believed to contribute to this recruitment, it has long been recognized that many foreign viral glycoproteins are also efficiently incorporated into viral particles. Since there is no sequence similarity between these foreign viral glycoproteins and HIV-1 Env, there must be additional mechanisms, likely involving host cell machinery, that facilitate this co-assembly. We hypothesize that the mechanisms utilized by HIV-1 to recruit foreign viral glycoproteins are also utilized to recruit its native Env protein. An understanding of these mechanisms will shed light on how HIV-1 interacts with the host cell during the assembly process and could lead to new targets for antiviral therapies. We have developed a scanning electron microscopy (SEM)-based assay that allows qualitative and quantitative imaging of the distribution of Env on the cell surface while simultaneously imaging the location of individual HIV-1 budding sites. Using this assay, we can clearly show that viral Env is enriched over 50-fold at viral budding sites. Enrichment can be observed with native as well as some foreign Env proteins. Truncation of the cytoplasmic tail of Env can eliminate enrichment at budding sites, but it does not eliminate passive Env incorporation. Surprisingly, enrichment of Env at budding sites can occur even when the domain of Gag believed to interact with Env (matirx) is replaced with a non-retroviral membrane-binding domain. This novel SEM Env distribution assay, as well as other assays, will be used to study how Env acquisition by HIV-1 occurs. 1) We will first identify divergent viral glycoproteins that retain the ability to be recruited to HIV-1 budding sites. Because such foreign proteins are unlikely to directly bind to HIV-1 Gag, studies with these proteins are less likely to be hampered by multiple modes of interaction. 2) These glycoproteins will be dissected to determine the specific domains required to facilitate recruitment to budding sites. 3) The individual domains of HIV-1 Gag will be exchanged with complementing retroviral and non-retroviral domains to determine what components of HIV-1 Gag contribute to Env recognition.NARRATIVE HIV-1 infections have caused a worldwide pandemic that has claimed the lives of over 20 million people and infected over 40 million more. It has long been recognized that viruses, including HIV, have distinct mechanisms for recruiting all the viral components to the correct viral assembly site within the cell, but these mechanisms remain poorly understood. An understanding of these mechanisms will shed light on how HIV-1 interacts with the host cell and could lead to new targets for antiviral therapies.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI073098-05
Application #
8204753
Study Section
AIDS Molecular and Cellular Biology Study Section (AMCB)
Program Officer
Sharma, Opendra K
Project Start
2008-01-01
Project End
2013-09-29
Budget Start
2012-01-01
Budget End
2013-09-29
Support Year
5
Fiscal Year
2012
Total Cost
$292,272
Indirect Cost
$96,252
Name
University of Missouri-Columbia
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
153890272
City
Columbia
State
MO
Country
United States
Zip Code
65211
Gregory, Devon A; Olinger, Grace Y; Lucas, Tiffany M et al. (2014) Diverse viral glycoproteins as well as CD4 co-package into the same human immunodeficiency virus (HIV-1) particles. Retrovirology 11:28
Janaka, Sanath Kumar; Faurot, Jared; Johnson, Marc C (2013) Functional complementation of a model target to study Vpu sensitivity. PLoS One 8:e68507
Janaka, Sanath Kumar; Gregory, Devon A; Johnson, Marc C (2013) Retrovirus glycoprotein functionality requires proper alignment of the ectodomain and the membrane-proximal cytoplasmic tail. J Virol 87:12805-13
Gregory, Devon A; Lyddon, Terri D; Johnson, Marc C (2013) Multiple Gag domains contribute to selective recruitment of murine leukemia virus (MLV) Env to MLV virions. J Virol 87:1518-27
Baluyot, Mariju F; Grosse, Sarah A; Lyddon, Terri D et al. (2012) CRM1-dependent trafficking of retroviral Gag proteins revisited. J Virol 86:4696-700
Zhang, Fengwen; Zang, Trinity; Wilson, Sam J et al. (2011) Clathrin facilitates the morphogenesis of retrovirus particles. PLoS Pathog 7:e1002119
Johnson, Marc C (2011) Mechanisms for Env glycoprotein acquisition by retroviruses. AIDS Res Hum Retroviruses 27:239-47
Lucas, Tiffany M; Lyddon, Terri D; Grosse, Sarah A et al. (2010) Two distinct mechanisms regulate recruitment of murine leukemia virus envelope protein to retroviral assembly sites. Virology 405:548-55
Dilley, Kari A; Gregory, Devon; Johnson, Marc C et al. (2010) An LYPSL late domain in the gag protein contributes to the efficient release and replication of Rous sarcoma virus. J Virol 84:6276-87
Lucas, Tiffany M; Lyddon, Terri D; Cannon, Paula M et al. (2010) Pseudotyping incompatibility between HIV-1 and gibbon ape leukemia virus Env is modulated by Vpu. J Virol 84:2666-74

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