Lipid Droplets and Viral Infections - Methods in Cell Biology - page 167

upregulating the liver-X receptor and its lipogenic target genes SREBP-1, FAS, and
peroxisome proliferator-activated receptor g ( Kim et al., 2007, 2008; Na et al., 2009 ).
Liver steatosis occurs in more than 50% of individuals chronically infected with
HCV ( Asselah, Rubbia-Brandt, Marcellin, &Negro, 2006 ). In mice and cell cultures,
this condition is recapitulated by the expression of a single viral protein, the nucle-
ocapsid core ( Clement et al., 2011; Moriya et al., 1997, 1998 ). Core activates tran-
scription factors SREBP-1 ( Su et al., 2002 ) and RXR a ( Tsutsumi et al., 2002;
Yamaguchi et al., 2005 ), leading to enhanced activity of various enzymes involved
in lipid biosynthesis ( Fukasawa et al., 2006 ), and downregulates PPAR a and mito-
chondrial carnitine palmitoyl transferase-1, resulting in reduction of fatty acid oxi-
dation ( Tsutsumi et al., 2002 ). Core also inhibits the activity of microsomal
triglyceride transfer protein, a limiting factor in VLDL assembly and secretion
( Perlemuter et al., 2002 ). In addition, a small fraction of core protein can access
mitochondria and may induce peroxidation of lipids and proteins involved in VLDL
secretion by producing reactive oxygene species ( Okuda et al., 2002; Schwer et al.,
2004 ). Recent studies also pointed to a bipartite mechanism whereby the viral core
protein utilizes the host DGAT1 enzyme to gain access to LDs and, once localized to
this compartment, interfere with LD triglyceride turnover, thereby stabilizing LDs
necessary for viral assembly ( Harris et al., 2011; Herker et al., 2010 )
( Fig. 10.2 B). Collectively, these results indicate that multiple activities of the core
protein may lead to LD accumulation in chronic HCV infection.
Viruses that use LDs as a protein-storage depot
Recent reports propose that LDs serve as transient storage depots for proteins that
lack appropriate binding partners in the cell at a given time ( Li et al., 2012 ). Such
sequestration may provide a general cellular strategy for handling excess proteins,
such as histones.
The outer capsid protein m1 of the orthoreovirus (a member of the Reoviridae
family) localizes to the membranes of the ER, outer mitochondria, and LDs via a
C-terminal amphipathic helix that also mediates apoptosis in cell culture ( Coffey
et al., 2006 ). Similarly, the HCV core protein also induces apoptosis via the induction
of ER stress and mitochondrial dysfunctions ( Benali-Furet et al., 2005; Korenaga
et al., 2005 ). Thus, localization of viral capsid proteins to LDs may be a strategy
to control viral protein-induced ER stress reactions, mitochondrial dysfunction,
and cell death during viral infection.
10.1 MATERIALS
10.1.1 Tools for analysis of viral infection
1. MegaScript T7 kit (Ambion) for in vitro transcription of HCV RNA
2. Cytomix buffer (all in mM: 120 KCl, 5MgCl 2 , 0.15 CaCl 2 , 2 EGTA, 1.9 ATP, 4.7
GSH, 25 HEPES, 10 potassium phosphate buffer, pH 7.6) for electroporation of
hepatoma cells with viral RNA