Biology Reference
In-Depth Information
10.2.3.4 Free Fatty Acid Enzymatic Quantification............................ 179
10.2.3.5 Radiolabeling and Analysis with TLC .................................. 180
10.2.4 Cell Transfection ........................................................................ 180
10.2.4.1 Electroporation .................................................................. 180
10.2.4.2 Chemical Transfection ....................................................... 181
10.2.5 Production of Viral Stocks and Infection ....................................... 181
10.2.6 Viral Replication Measurements................................................... 182
10.2.6.1 Viral Replication Assays ..................................................... 182
10.2.6.2 Viral Transcription and Translation Assays .......................... 182
10.2.6.3 Viral RNA Replication Assays ............................................. 183
10.2.6.4 Viral RNA Measurement at LDs.......................................... 184
10.2.6.5 Viral Assembly and Release Assays.................................... 184
10.2.6.6 Viral Titration Assays.......................................................... 185
10.2.7 Drug Treatments......................................................................... 186
10.3 Discussion......................................................................................................186
Acknowledgments ................................................................................................... 187
References ............................................................................................................. 188
Abstract
The lifecycle of several viruses is intimately tied to the lipid metabolism of their host
cells, and lipid droplets (LDs) have emerged as crucial organelles in the propagation
of these viral infections. Investigating the roles of LDs in viral infection requires
expertise in both virology and cell metabolism pertaining to LDs. In this review,
we offer an updated list and review of the multiples methods we have used in our
laboratory to study both the role of LDs in viral infection and the effect of viral
infection on cellular LDs, with a special emphasis on hepatitis C virus and other
RNA viruses.
INTRODUCTION
Long regarded as inert fat particles, lipid droplets (LDs) have long been ignored by
cell biologists. In the last decade, however, LDs have been increasingly recognized
as dynamic organelles with central roles in cell metabolism. Their metabolic role is
highlighted by the fact that many intracellular pathogens usurp LDs during their life-
cycle. Included in this ever-expanding group of pathogens is a diverse array of
human microbes, including
Chlamydia trachomatis
,
Mycobacterium tuberculosis
,
and
Plasmodium falciparum
, as well as several RNA viruses. Although much
remains to be discovered about the mechanisms involved in these host/pathogen in-
teractions, evidence suggests that the energy storage capacity of LDs is often crucial
for these diverse pathogens. Moreover, several viruses such as positive-strand RNA
viruses, hijack cellular membranes for their replication, and viral proteins are closely
