Biology Reference
In-Depth Information
One common challenge when studying viral proteins is their multifunctionality.
Each viral protein usually interacts with several host factors at different stages of the
viral replication cycle as the virus hijacks cellular pathways for its own needs. Thus,
expression of a viral protein often results in a variety of secondary consequences
in the cell. This effect is accentuated during viral infection when many proteins
are simultaneously expressed.
Most Flaviridae proteins are associated with ER membranes that sometimes sur-
round LDs. It can thus be difficult to decipher by microscopy whether a protein
localizes at the LDs or at the ER membranes associated with LDs. For this reason,
biochemical methods should be used in conjunction with microscopy analysis to
define the precise location of viral components.
A major limitation for the study of pathogens, such as HCV or DENV, is the lack
of a proper physiological model to study their replication. For HCV, only one spe-
cific cell line isolated from a hepatocellular carcinoma is capable of replicating one
HCV genotype (2a). This cell line (Huh7.5 and its sister Huh7-Lunets), however, is
defective for the RIGI-mediated immune-response pathway as well as the VLDL
release pathway, which is essential for the release of new viral particles. Primary
hepatocytes are extremely difficult to infect with HCV, and only few laboratories
have succeeded to do so consistently. Similarly, although the primary targets of
DENV infection in humans are macrophages, most DENV studies have been per-
formed in human hepatoma Huh7, baby hamster kidney bhk-21, or insect C64/36
cell lines. The role of liver infection in dengue propagation and pathogenesis is
yet to be fully uncovered.
In summary, the protocols that have helped to uncover LDs as important dynamic
organelles, combined with standard experimental procedures to study viral infection,
have identified the vital role that LDs play in viral infections. New studies on the
importance of LDs in viral infection are constantly emerging. This indicates that
LDs functioning as crucial “hubs” in viral life cycles might be a more common fea-
ture for viruses and other pathogens than originally believed. Studies on HCV have
been leading the way in identifying mechanistic details of how LDs are important
components of the HCV assembly machinery, similar insight into the role of LDs
in other viral infections can be expected to follow.
Acknowledgments
G. C. would like to thank the American Liver Foundation for their fellowship award. D. A. V.
was supported by the Training Grant (T32 DK060414) from the US National Institute of
Health. We thank Eva Herker for her instrumental contribution in developing some of these
protocols and for helpful discussion. Thanks to Brian Webster, Robert Farese, Charles Harris,
Joel Haas, and all members of the Ott laboratory for helpful discussion and support. Some of
the work described in this chapter was supported by funds from the Gladstone Institutes,
and the National Institutes of Health (R056 AI069090 (M. O.); P30 DK026743 (University
of California, San Francisco Liver Center)).
