In addition to studies of coreceptor active sites, we have previously focused on the roles of CD4 and CCR5 cell surface concentrations in controlling HIV-1 infections. These investigations have implied that a critical complex containing 4-6 CCR5s may be essential, and that infections of cells with suboptimal receptor/coreceptor concentrations and/or mutant coreceptors therefore becomes slow, which results in inefficiency due to a poorly understood competing innate viral death pathway. Consistent with this kinetic interpretation, entry inhibitors also function more efficiently when assembly of critical viral-receptor complexes becomes slow. These results raise the possibility that the innate viral decay pathway may be a common final mechanism for viral inactivation by entry inhibitors and neutralizing antibodies. We propose to extend our mechanistic analyses of HIV-1 infections by: (1) We have isolated variants of the R5 HIV-1 strain JRCSF that efficiently utilize CCR5(delta18), a coreceptor mutant that lacks the amino terminal sulfated region previously believed to be essential for HIV-1 infections. Analyze the adaptive gp120 mutations, including a surprisingly potent N-glycan loss mutation in the V4 domain. (2) Similarly, isolate HIV-1 variants that require only the amino terminal domain of CCR5, using in part an N-glycan addition approach. (3) Using our target cells that have specific concentrations of CD4 and coreceptors, analyze the kinetics of viral entry and of the competing viral death pathway. Learn whether entry inhibitors and neutralizing monoclonal antibodies function by competitive, noncompetitive, or suicide inhibitor mechanisms, a distinction likely to be critical for optimizing drug and vaccine developments. Thereby, learn whether these agents inactivate HIV-1 or whether they merely slow entry and thereby enable the virus death pathway to predominate. (4) Using this kinetic and mechanistic information, investigate the hypothesis that the innate viral death pathway implied by the above studies involves endocytosis. Determine whether inhibition of endocytosis can in some circumstances enhance HIV-1 titers and reduce efficacies of entry inhibitors and neutralizing antibodies. This program will provide important cell lines and viruses, methods and information for improving the development of HIV-1 entry inhibitors and vaccine strategies for AIDS.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA067358-11
Application #
6884879
Study Section
AIDS Molecular and Cellular Biology Study Section (AMCB)
Program Officer
Read-Connole, Elizabeth Lee
Project Start
1995-03-15
Project End
2009-03-31
Budget Start
2005-04-07
Budget End
2006-03-31
Support Year
11
Fiscal Year
2005
Total Cost
$339,750
Indirect Cost
Name
Oregon Health and Science University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
096997515
City
Portland
State
OR
Country
United States
Zip Code
97239
Marin, Mariana; Golem, Sheetal; Kozak, Susan L et al. (2016) Movements of HIV-1 genomic RNA-APOBEC3F complexes and PKR reveal cytoplasmic and nuclear PKR defenses and HIV-1 evasion strategies. Virus Res 213:124-139
Platt, Emily J; Durnin, James P; Kabat, David (2015) Short Communication: HIV-1 Variants That Use Mouse CCR5 Reveal Critical Interactions of gp120's V3 Crown with CCR5 Extracellular Loop 1. AIDS Res Hum Retroviruses 31:992-8
Platt, Emily J; Gomes, Michelle M; Kabat, David (2014) Reversible and efficient activation of HIV-1 cell entry by a tyrosine-sulfated peptide dissects endocytic entry and inhibitor mechanisms. J Virol 88:4304-18
López, Claudia S; Sloan, Rachel; Cylinder, Isabel et al. (2014) RRE-dependent HIV-1 Env RNA effects on Gag protein expression, assembly and release. Virology 462-463:126-34
Platt, Emily J; Gomes, Michelle M; Kabat, David (2012) Kinetic mechanism for HIV-1 neutralization by antibody 2G12 entails reversible glycan binding that slows cell entry. Proc Natl Acad Sci U S A 109:7829-34
Platt, Emily J; Kozak, Susan L; Durnin, James P et al. (2010) Rapid dissociation of HIV-1 from cultured cells severely limits infectivity assays, causes the inactivation ascribed to entry inhibitors, and masks the inherently high level of infectivity of virions. J Virol 84:3106-10
Platt, Emily J; Bilska, Miroslawa; Kozak, Susan L et al. (2009) Evidence that ecotropic murine leukemia virus contamination in TZM-bl cells does not affect the outcome of neutralizing antibody assays with human immunodeficiency virus type 1. J Virol 83:8289-92
Platt, Emily J; Durnin, James P; Shinde, Ujwal et al. (2007) An allosteric rheostat in HIV-1 gp120 reduces CCR5 stoichiometry required for membrane fusion and overcomes diverse entry limitations. J Mol Biol 374:64-79
Melikyan, Gregory B; Platt, Emily J; Kabat, David (2007) The role of the N-terminal segment of CCR5 in HIV-1 Env-mediated membrane fusion and the mechanism of virus adaptation to CCR5 lacking this segment. Retrovirology 4:55
Platt, Emily J; Durnin, James P; Kabat, David (2005) Kinetic factors control efficiencies of cell entry, efficacies of entry inhibitors, and mechanisms of adaptation of human immunodeficiency virus. J Virol 79:4347-56

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