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into two disulfide-linked subunits, HA
1
and HA
2
. This cleavage is a
prerequisite for HA to undergo conformational change to a fusion-
competent form in response to the low pH environment of endo-
somes. HA trimers have been shown to function cooperatively during
the process of membrane fusion.
37-39
The minimum number of HA
trimers required to initiate a fusion event was estimated to be three to
four.
38
Many enveloped viruses, including influenza virus, human
immunodeficiency virus (HIV), Ebola virus, and paramyxoviruses,
share a very similar molecular mechanism for membrane fusion.
40
For
HIV, it was also estimated that cooperation of three to six Env trimers
was required for formation of a fusion pore.
41
If these trimers of
fusion proteins (such as HA and Env) distribute randomly on a vast
field of plasma membranes, how efficiently can they find their coop-
eration partners for the process of membrane fusion? It has been
established that HA expressed on the plasma membrane by virus infec-
tion distributes in clusters at the sites of virus budding.
42
Recently we
showed that rafts have a crucial role for this clustering of HA.
17
We
found that nonraft HA mostly distributed homogeneously on the
plasma membrane.
17
We compared fusion activity of the nonraft HA
expressed on the cell surface with that of wt HA (cell-to-cell fusion)
by lipid mixing and aqueous content mixing assays (HA-expressing
BHK cells and dual-labeled human erythrocytes were used as effecter
cells and target cells, respectively).
17
At any expression level, either wt
or nonraft HA caused complete fusion, but the number of fusion
events by nonraft HA was reduced (
60%).
17
This reduced fusion activ-
ity may be due to the lowered local density of HA. Similarly, nonraft
HA virus showed a reduced virus-to-cell fusion activity in a fluores-
cent dequenching fusion assay with R18 (lipid fluorescence probe)-
labeled virions and red blood cell ghosts (target membranes).
17
Since
nonraft HA virions contained a smaller amount of HA than wt virions
(
∼
60%), the reduced fusion activity by nonraft HA virus could also be
explained by the lowered density of HA. However, other mechanisms
can be proposed, such as a need for HA trimers to be stabilized by
lipid molecules in rafts for efficient membrane fusion, or a need for
HA trimers to form a higher-order structure with lipids in rafts. When
cholesterol was depleted from purified virions by methyl-
∼
β
-cyclodextrin
(M
β
CD), virus infectivity was reduced by 10-fold to 100-fold.
17,43
