TY  - GEN
ID  - heidok10683
Y1  - 2010///
TI  - Quantitative analysis of the early steps of virus host cell interaction of human immunodeficiency virus type 1 and hepatitis C virus
KW  - Viruseintritt 
KW  -  Virusbindung 
KW  -  Eintrittsinhibitorvirus entry 
KW  -  quantitative analysis 
KW  -  virus receptor 
KW  -  co-receptor 
KW  -  entry inhibitor
UR  - https://archiv.ub.uni-heidelberg.de/volltextserver/10683/
AV  - public
N2  - Viruses are obligatory intracellular pathogens; hence an essential step of their replication cycle is the entry into a host cell. Enveloped viruses like the human immunodeficiency virus type 1 (HIV-1) and the hepatitis C virus (HCV) enter cells by fusion with cellular membranes. The current knowledge of this process relies mostly on bulk measurements, which often comprise the outcome of several distinct replication steps in a non-synchronized manner. The recent development of novel quantitative approaches has opened the door for deeper understanding of the process by analyses on a single cell and single particle level.  Fluorescently labelled viruses allow studying single steps in the interaction of individual virions with the host cell. A strategy for labelling HIV-1 with organic dyes using the recently described SNAP-tag was established and evaluated in this thesis. Introduction of the SNAP-tag did not significantly interfere with virus entry and infectivity and allowed specific labelling of the Gag structural polyprotein in the context of virions and virus producing cells. Combining fluorescently labelled HIV-1 with the HCV pseudoparticle system (HCVpp) allowed the analysis of virion attachment and entry dependent on the envelope protein of HCV on a single particle level. In addition, the ?-Lactamase virion fusion assay was adapted to and optimized for HCVpp, allowing the detection of virus-cell fusion. The dependency of virus particle binding, endocytic uptake and fusion on various stimulating and inhibiting agents was investigated. The virus binding assay developed for HCVpp was subsequently adapted to HIV-1 and revealed cell-type specific kinetics of virion attachment. Interestingly, the expression level of the cellular virion tethering factor CD317 was shown to have no effect on exogenous virus binding.  The main part of this thesis aimed at the acquisition and analysis of quantitative multi-parameter data of HIV-1 entry. Besides the receptor CD4, HIV-1 entry depends on the presence of either one of the two major co-receptors, CXCR4 or CCR5. The ability of a virus variant to use a co-receptor defines the tropism of the virus and is at least in part determined by the sequence of the third variable loop (V3-loop) of the viral envelope protein (Env). In summary, HIV entry efficiency is determined by a complex interplay between Env sequence, receptor and co-receptor densities. A deeper insight into the interdependencies of these critical parameters provides a basis for the understanding of the mechanism of action of HIV entry inhibitors as well as of pathways of resistance development against these compounds. Mathematical models describing the interdependencies will also aid in the refinement of algorithms for the genotypic prediction of co-receptor tropism, which is essential for the use of co-receptor antagonists in antiretroviral therapy. Here, experimental systems were developed which allow acquisition of detailed quantitative data on the interdependencies between these parameters as a basis for a mathematical model of the HIV-1 entry process. This comprised the selection and characterization of suitable model cell lines and experimental conditions, the generation and characterization of defined isogenic virus variants and the establishment and calibration of virological assay systems. Multivariant data sets were acquired under standard conditions and algorithms for multivariant data analysis which have been developed in collaboration with bioinformaticians were evaluated. A comprehensive data set was obtained by studying a subset of viruses carrying patient-derived Env variants which revealed quantitative differences in receptor and co-receptor dependency as well as sensitivity to prototype entry inhibitors beyond the overall results of common phenotyping/genotyping methods.
A1  - Eckhardt, Manon
ER  -