Biology Reference
In-Depth Information
mediated by HIV pseudotyped with the Ebola EnvGP (Bhattacharyya
.,
2010). The use of control pseudoparticles (e.g., pseudotyped with Vesicular
Stomatitis Virus EnvGP (VSV G)) can be used as controls to assess cell viability
and specificity of inhibition. Inhibition of clathrin function traditionally relied
on three principal approaches: drugs that inhibit acidification of endosomes
(such as BafA1, a specific, nonreversible endosomal proton pump inhibitor), as
well as commonly used lysosomotropic agents (such as ammonium chloride
(NH
4
Cl) and chloroquine); potassium depletion; and finally, treatment of cells
with brefeldin A (BFA) or chlorpromazine (Sieczkarski and Whittaker, 2002).
As such, these drugs have multiple effects on cell function, and their use to
inhibit virus infection should be treated with some caution. Thanks to pseudo-
types, some other studies have implicated the actin cytoskeleton in Ebola virus
entry, where agents such as cytochalasin D and swinholide A that impair
microfilament
et al
function were
shown to inhibit EnvGP-mediated entry
(Yonezawa
., 2005). Ebola enters cells through a low-pH-dependent, endo-
cytosis-mediated process. A large body of evidence indicates that Ebola viruses
enter cells by clathrin-mediated endocytosis (Sanchez, 2007), but lipid raft-
associated, caveolin-mediated endocytosis has also been proposed as an alterna-
tive mechanism of Ebola virus uptake (Empig and Goldsmith, 2002). Low-pH
events lead to cathepsin-dependent cleavage of Ebola virus EnvGP that is
required for productive uptake of the virus (Chandran
et al
et al
., 2005; Kaletsky
et al
., 2006). Other low-pH-dependent events have
been postulated to be required as well (Schornberg
., 2007; Schornberg
et al
., 2006). Furthermore,
Ebola virus likely uses a Rho-mediated pathway, as is seen in VSV virus,
suggesting that this may be a route of entry utilized by many different viruses
(Quinn
et al
., 2009).
More recently, proteins interfering with endocytosis, such as the use of
dominant-negative Eps15, or RNA interference (RNAi) have been developed,
and such approaches target the different pathways with higher specificity
(Mercer and Helenius, 2009; Mercer
et al
., 2010b). The RNAi approach allows
researchers to perform screens to identify previously unrecognized host factors
that are required for viral replication. RNAi screens rely on either short inter-
fering RNAs (siRNAs) or short hairpin RNAs (shRNAs) to knock down the
function of a particular gene in a cell. Researchers can then infect the cells with
specific viruses and monitor levels of viral replication. If viral replication is
reduced, then the knocked-down gene might be necessary for the virus to
replicate itself or function within the host cell.
Some small-scale screens have been developed using wild-type viruses
(Kolokoltsov
et al
., 2009). Pseudoparti-
cles can be exploited to focus a siRNA screen specifically on the entry step of a
virus. Indeed, only the entry steps are governed by the EnvGP, whereas all the
uncoating,
et al
., 2007) or pseudoparticles (Trotard
et al
integration, and expression steps of the transgene depend on
