Lorraine Everitt

Initial Cleavage of the Human Immunodeficiency Virus Type 1 GagPol Precursor by Its Activated Protease Occurs by an Intramolecular Mechanism

ABSTRACT Processing of the GagPol polyprotein precursor of human immunodeficiency virus type 1 (HIV-1) is a critical step in viral assembly and replication. The HIV-1 protease (PR) is translated as part of GagPol and is both necessary and sufficient for precursor processing. The PR is active only as a dimer; enzyme activation is initiated when the PR domains in two GagPol precursors dimerize. The precise mechanism by which the PR becomes activated and the subsequent initial steps in precursor processing are not well understood. However, it is clear that processing is initiated by the PR domain that is embedded within the precursor itself. We have examined the earliest events in precursor processing using an in vitro assay in which full-length GagPol is cleaved by its embedded PR. We demonstrate that the embedded, immature PR is as much as 10,000-fold less sensitive to inhibition by an active-site PR inhibitor than is the mature, free enzyme. Further, we find that different concentrations of the active-site inhibitor are required to inhibit the processing of different cleavage sites within GagPol. Finally, our results indicate that the first cleavages carried out by the activated PR within GagPol are intramolecular. Overall, our data support a model of virus assembly in which the first cleavages occur in GagPol upstream of the PR. These intramolecular cleavages produce an extended form of PR that completes the final processing steps accompanying the final stages of particle assembly by an intermolecular mechanism.

Pharmacogenomic characterization of US FDA-approved cytotoxic drugs

AIMS Individualization of cancer chemotherapy based on the patients genetic makeup holds promise for reducing side effects and improving efficacy. However, the relative contribution of genetics to drug response is unknown. MATERIALS & METHODS In this study, we investigated the cytotoxic effect of 29 commonly prescribed chemotherapeutic agents from diverse drug classes on 125 lymphoblastoid cell lines derived from 14 extended families. RESULTS The results of this systematic study highlight the variable role that genetics plays in response to cytotoxic drugs, ranging from a heritability of <0.15 for gemcitabine to >0.60 for epirubicin. CONCLUSION Putative quantitative trait loci for cytotoxic response were identified, as well as drug class-specific signatures, which could indicate possible shared genetic mechanisms. In addition to the identification of putative quantitative trait locis, the results of this study inform the prioritization of chemotherapeutic drugs with a sizable genetic response component for future investigation.

A Comparison of Association Methods for Cytotoxicity Mapping in Pharmacogenomics

Cytotoxicity assays of immortalized lymphoblastoid cell lines (LCLs) represent a promising new in vitro approach in pharmacogenomics research. However, previous studies employing LCLs in gene mapping have used simple association methods, which may not adequately capture the true differences in non-linear response profiles between genotypes. Two common approaches summarize each dose-response curve with either the IC50 or the slope parameter estimates from a hill slope fit and treat these estimates as the response in a linear model. The current study investigates these two methods, as well as four novel methods, and compares their power to detect differences between the response profiles of genotypes under a variety of different alternatives. The four novel methods include two methods that summarize each dose-response by its area under the curve, one method based off of an analysis of variance (ANOVA) design, and one method that compares hill slope fits for all individuals of each genotype. The power of each method was found to depend not only on the choice of alternative, but also on the choice for the set of dosages used in cytotoxicity measurements. The ANOVA-based method was found to be the most robust across alternatives and dosage sets for power in detecting differences between genotypes.

ANALYSIS OF HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 PROTEASE ACTIVITY IN EUKARYOTIC AND BACTERIAL CELLS

Publisher Summary This chapter describes several methods for evaluating the activity of the HIV-1 protease during virus assembly when the gag and gag-pol precursors are cleaved to give rise to its constituent proteins. The proteins that make up the core are translated as part of either the gag or gag-pol polyproteins. The individual proteins that make up the precursors are subsequently released by proteolytic cleavage by a virally encoded protease. An experiment is discussed in the chapter that design to follow a group of labeled precursor proteins through the processing pathway. The chapter concludes this experiment: (1) the rate of precursor processing may be determined, (2) one can obtain a comparison of the relative rate of cleavage at the different processing sites, (3) it is possible to locate the cellular compartment in which various processing steps occur, and (4) one may identify alternative processing pathways.

Analysis of temperature-sensitive mutants of the HIV-1 protease

Human Immunodeficiency Virus Type-1, like other retroviruses, encodes an aspartic proteinase whose activity is required for the production of infectious virions.1–6 The protease (PR) is encoded at the 5′ end of the viral pol gene and is responsible for cleavage of the viral gag and gag/pol precursor proteins to their mature forms.7,8 Viruses with inactivating mutations in their protease domain yield immature, noninfectious particles containing unprocessed gag and gag/pol polyproteins;1 for this reason the enzyme has been a prime target for structural and biochemical studies leading to the design of inhibitors of virus replication.