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in many laboratories. However, a poor overlap between results from different
screens published on the same viruses has been observed. The differences are
probably linked not only to technical variations but also to the inherent risk of
false positives through off-target effects and false negatives due to inefficient
silencing. Cell toxicity is a common complication, as well as cell-type specificity.
Following-up such screens with pseudoparticle studies is useful to validate the
cell proteins that are involved in the entry step only and facilitate the analysis,
compared to the use of a replication competent virus.
D. Screening and development of entry inhibitors using pseudoparticles
Resistance to individual antivirals is likely to develop for most specific therapeu-
tics targeting particular viral proteins, thus making therapy consisting of a
combination of drugs targeting different stages of the viral life cycle highly
desirable. The entry process represents another series of potential targets for
therapeutic intervention. It has not been extensively explored due to high-
throughput experimental limitations for some viruses that require biosafety
level 3 or 4 or for the viruses with no robust infection system.
Pseudoparticle infection systems, utilizing different reporter genes or
proteins (i.e., firefly luciferase or GFP), can be developed for HTS of small
molecule libraries, peptide libraries, or neutralizing antibodies for their entry
inhibitor properties. In order to facilitate the screen, assay performance can be
improved by modifying the properties of both the parental host cell line and the
pseudovirus. For example, cells with improved pseudoparticle entry and cell
spreading can be selected. Pseudoparticles can be improved by using EnvGP
that increase infectivity either by selecting a specific variant or by expressing a
human codon-optimized envelope glycoprotein sequence. Finally, the pseudo-
viruses can be engineered to express a human codon-optimized reporter to
improve the sensitivity of the assay. Using these modifications, HTS can be
developed using 96- or 384-well plates. Compounds found to inhibit pseudopar-
ticle infection can then be counterscreened against pseudoparticles containing
other variants to characterize the cross-reactivity of the molecules in a virus
family. Moreover, molecules can then be counterscreened against pseudoparti-
cles harboring EnvGP from other virus families to evaluate the specificity of the
inhibitor or its large spectrum.
A wide variety of entry inhibitors, namely, peptides, chemical com-
pounds, or antibodies, exist. They can interfere with the different steps of the
entry process, such as the binding to receptors or the conformational changes of
the fusion proteins, by acting on the envelope protein itself, on the acidification
of the endosomes, if necessary, or on the endocytosis.
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