HIV-1 enters cells by using the CD4 receptor, and one of two co-receptors, CCR5 or CXCR4. For those that harbor only CCR5-using viruses, there is evidence that the relative efficiency by which HIV-1 uses CD4 and CCR5 may correlate with the pathogenic potential of the virus. However, our ability to quantify the efficiency of CD4 and CCR5 usage has been limited by indirect and non-standardized measures for how well a virus uses CD4 and/or CCR5. Therefore, our driving hypothesis is that there are real underlying associations between strains of HIV-1 and their relative efficiencies of CD4 and CCR5 usage that account for specific aspects of viral pathogenesis, but that these associations have not been revealed due to the limitations of current methods. Our objective is to derive a comprehensive and quantitative way of characterizing the CD4/CCR5 usage efficiencies of any given viral isolate in order to facilitate comparative studies that will explore our hypothesis. We propose a multidisciplinary project that takes advantage of the unique expertise and resources of the PI (Dr. Benhur Lee), co-PI (Dr. Tom Chou, Biomathematics), and collaborators with access to precious cohorts of primary patient isolates with defined pathogenic characteristics. Dr. Lee has developed a dual-inducible cell line where CD4 and CCR5 can be simultaneously and independently regulated. For any given isolate, viral infectivity can be monitored at up to 48 distinct levels of CD4 and CCR5 expression. Dr. Chou has devised a method to transform the multi-dimensional data obtained into quantifiable metrics that describes the overall CD4/CCR5 usage efficiency of a particular isolate. This system for profiling the receptor usage efficiencies allows for large-scale intra- and inter-cohort comparisons of CD4 and CCR5 usage efficiencies. Thus, my Specific Aims are: (1) To develop higher throughput and more sophisticated methods for quantifying the relative CD4 and CCR5 usage efficiencies of various HIV isolates in our CD4/CCR5 dual-inducible cell line, We propose (a) engineering a tat/rev dependent Gaussia luciferase reporter vector that will allow for a higher throughput and kinetic analysis of the efficiency of CD4 and CCR5 usage of primary viral isolates, (b) optimizing an ultra-sensitive real-time fusion kinetics assay to complement the infection data, and (c) automating and optimizing the computational analysis via a web-based interface, and (2) To characterize the relative infectivity of primary HIV-1 strains, and to determine if viral pathogenicity or tropism correlates with the efficiency of CD4 and CCR5 usage. We will test the correlates of pathogenicity with viruses from various cohorts. Our underlying hypothesis is that the differential efficiency of CD4 and/or CCR5 usage are associated with specific aspects of viral pathogenesis, and that these differences may be better revealed by the system optimized in Aim 1.
Our dual inducible cells can directly measure and profile the CD4/CCR5 usage efficiency of any given viral isolate, and provide a quantitative metric that can be used for multiple comparisons. Our system is a valuable tool not only for better understanding the relationship between pathogenesis and the efficiency of CD4/CCR5 usage, but may also have clinical implications for guiding entry inhibitor use.
|Webb, Nicholas E; Montefiori, David C; Lee, Benhur (2015) Dose-response curve slope helps predict therapeutic potency and breadth of HIV broadly neutralizing antibodies. Nat Commun 6:8443|
|Joseph, Sarah B; Arrildt, Kathryn T; Swanstrom, Adrienne E et al. (2014) Quantification of entry phenotypes of macrophage-tropic HIV-1 across a wide range of CD4 densities. J Virol 88:1858-69|
|Chikere, Kelechi; Webb, Nicholas E; Chou, Tom et al. (2014) Distinct HIV-1 entry phenotypes are associated with transmission, subtype specificity, and resistance to broadly neutralizing antibodies. Retrovirology 11:48|
|Salimi, Hamid; Roche, Michael; Webb, Nicholas et al. (2013) Macrophage-tropic HIV-1 variants from brain demonstrate alterations in the way gp120 engages both CD4 and CCR5. J Leukoc Biol 93:113-26|
|Ping, Li-Hua; Joseph, Sarah B; Anderson, Jeffrey A et al. (2013) Comparison of viral Env proteins from acute and chronic infections with subtype C human immunodeficiency virus type 1 identifies differences in glycosylation and CCR5 utilization and suggests a new strategy for immunogen design. J Virol 87:7218-33|
|Chikere, Kelechi; Chou, Tom; Gorry, Paul R et al. (2013) Affinofile profiling: how efficiency of CD4/CCR5 usage impacts the biological and pathogenic phenotype of HIV. Virology 435:81-91|
|Roche, Michael; Jakobsen, Martin R; Ellett, Anne et al. (2011) HIV-1 predisposed to acquiring resistance to maraviroc (MVC) and other CCR5 antagonists in vitro has an inherent, low-level ability to utilize MVC-bound CCR5 for entry. Retrovirology 8:89|
|Bi, Shuguang; Hong, Patrick W; Lee, Benhur et al. (2011) Galectin-9 binding to cell surface protein disulfide isomerase regulates the redox environment to enhance T-cell migration and HIV entry. Proc Natl Acad Sci U S A 108:10650-5|
|Roche, Michael; Jakobsen, Martin R; Sterjovski, Jasminka et al. (2011) HIV-1 escape from the CCR5 antagonist maraviroc associated with an altered and less-efficient mechanism of gp120-CCR5 engagement that attenuates macrophage tropism. J Virol 85:4330-42|
|Pfaff, Jennifer M; Wilen, Craig B; Harrison, Jessamina E et al. (2010) HIV-1 resistance to CCR5 antagonists associated with highly efficient use of CCR5 and altered tropism on primary CD4+ T cells. J Virol 84:6505-14|
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