BIOB 425 Paper 1

Andrew Major

ID#: -01958444

Assembly of endocytic machinery around individual influenza viruses during viral entry
In order to initiate replication, a virus must deliver its genome into its host cell. Most achieve this
through endocytosis, in effect using the host cells own machinery to pass through the plasma membrane and
cytoskeleton; influenza is one such virus. A glycoprotein expressed on the influenza virus, hemagglutanin, binds
to sialic acid on the host cell surface leading to the internalization of the virus. Once inside, the virus fuses with
endosomes to release its genome and initiate infection. These stages of movement are more specifically denoted
as follows: Stage I is actin-dependent movement on the cell surface or in the cell periphery, Stage II is a rapid,
unidirectional movement on the microtubule toward the perinuclear region, and Stage III is a bidirectional,
microtubule-dependent movement. For this experiment, Stage II movement is used as a marker of viral
internalization.
In this study, the hypothesis is that real-time fluorescence imaging of single influenza viruses will allow
for the clarification of influenza viral entry pathways, with little indication of what these pathways may entail
other than previous indications of an endocytic mechanism. It was shown, consequently, that a clathrindependent pathway involves the de novo formation of clathrin-coated pits (CCPs) in response to viral binding
on the cell surface, and that at least one clathrin-independent pathway works in parallel rather than as an
alternative when endocytosis is blocked.
To study viral entry, clathrin was tagged with enhanced yellow fluorescent protein (EYFP). CCPs and
CCVs were immunolabeled with an antibody against the AP2 adaptor protein complex, and additionally,
transferrin was fluorescently labeled to provide a separate measure of co-localization. Results from fluorescence
imaging concluded that the EYFP label revealed nearly all CCPs and CCVs in living cells without functional
defects to these structures. To visualize viral entry, influenza viruses were labeled with the lipophilic dye DiD.
By simultaneously visualizing EYFP-clathrin and DiD-labeled influenza viruses it was shown that for most of
the viruses, binding to the cell induced the recruitment of clathrin ultimately forming a CCP de novo. In fact,
only 6% of the viruses entered via preexisting CCPs on the cell surface. It was further shown that, because of
substantial lag before CCP formation and through CCP formation rates, clathrin was recruited most likely due to

viral binding and not due to static or mobile hot spots on the cell surface where CCP formation rate is
elevated. Finally, it was shown that neuraminidase, an antigenic determinant on the surface of influenza, is not
essential for viral entry of influenza and thus that sialic glycoproteins are not involved in the de novo formation
of CCPs through the use of neuraminidase inhibitors, but may function elsewhere during infection.
About one third of the viruses did not associate with clathrin-coated structures even though they were
internalized as marked by Stage II movement, suggesting their internalization via a parallel clathrin-independent
pathway. This clathrin-independent pathway was also shown to be caveolin-independent by marking caveolin
with green fluorescent protein and inhibiting caveolin-mediated endocytosis with filipin. These two pathways
appeared equally efficient for viral fusion after internalization and therefore likely provide the virus with
equally efficient pathways for infection.
Further research on the viral entry of influenza and other similar viruses is likely to result in therapeutic
ways of treating infected cells and organisms at large. Although this study shows that an influenza virus can
enter via clathrin-dependent and independent pathways, it fails to mechanistically provide information about
these pathways. By understanding exactly how the virus binds to the cell, induces the recruitment of clathrin,
and enters via these pathways at a molecular level, it may be possible to design drugs which more successfully
treat and/or prevent influenza and other virus which utilize similar entry mechanisms. Questions that remain
unanswered include What is the mechanism by which an influenza virus induces the formation of a CCP?;
What are the molecular features of the clathrin-/caveolin-independent endocytic pathway?; How are
influenza viruses sorted in the endocytic system, and how are they moved between different endosomes? For
the first question, a sample experiment may involve in vitro analysis of viral binding to sialic acid and then the
individual addition of clathrin, adaptor proteins, and other components to determine how binding recruits
clathrin at a molecular level due to a presumable conformational change and what other molecular components
are involved.
Work Cited:
M.J. Rust, M. Lakadyami, F. Zhang, X. Zhuang, Assembly of endocytic machinery around individual influenza
viruses during viral entry, Nat. Struct. Mol. Biol. 11 (2004) 567573.