Julia Chen

http://www.biophysj.org/cgi/content/full/74/1/54/F2


FIGURE 2  


Cryo-TEM of influenza PR/8 virions at 30°C.
(A) Influenza PR/8 virus at pH 7.4. (B)
Influenza virus incubated for 2 min at pH 4.9. Note that the spikes on
the virions are still distinct and well-defined, as in
A. (C) Virions incubated for 10 min at pH
4.9. Spikes are still well defined and distinct. (D)
Virions incubated for 68 min at pH 4.9. Note that the spikes on the
virions have become disorganized and are no longer well defined or
distinct. This field was chosen because it contains a rare sample of a
virion with organized spikes like those in A
(arrowhead), which shows that the focus conditions were
appropriate for detecting spikes. (E) Another example of
virions incubated at pH 4.9 for 68 min. Many of the virions incubated
under these conditions had blebs and discontinuities visible on their
surfaces. Examples of such features are shown in the membrane of small
(arrow) and large (arrowhead) virions.
Scale bars = 200 nm in all figures.







http://www.biophysj.org/cgi/content/full/74/1/54

Fluorescence lipid mixing assay

Lipid mixing between unlabeled influenza PR/8 virus and
CPT/DABS-labeled liposomes was measured in 10 mM HBS buffer by
the resonance energy transfer (RET) assay (Silvius et al., 1987). This
RET pair is less susceptible to protein conformational changes than
other RET pairs tested (Alford et al., 1994; Shangguan et al., 1996a).
CPT fluorescence was recorded on a PTI Alphascan fluorometer (South
Brunswick, NJ) in a thermostatted cuvette with continuous stirring. The
excitation and emission wavelengths were 395 and 477 nm, respectively.
For the inactivation assay, 80 µl virus was incubated at pH 4.9, 30°C, for the designated period of time and injected into a cuvette
containing 1.92 ml 10 mM HEPES buffer (pH 7.5) and liposomes. The final
concentration was 10 µM viral phospholipid and 10 µM liposomal
lipid. This fluorescence level was set as 0% lipid mixing. Lipid
mixing was initiated after 50 s by injecting 20 µl 1.67 M acetic
acid/acetate solution to reach pH 4.9. The fluorescence level at
infinite probe dilution obtained by C₁₂E₈ (0.75 mM) lysis was considered as 100% lipid mixing.

TABLE 1   Morphological changes in virions as a function of time at pH 4.9 and 30°C
Condition	Tubular virions		Blebs or membrane discontinuities	Sample size
	(R > 2)	R > 10
pH 7.4	10%	1%	0.3%	399
pH 4.9, 2 min	1.4%	0.3%	0%	660
pH 4.9, 10 min	0.2%	0.05%	3%	2190
pH 4.9, 68 min	0%	0%	>20%	1201
R, Ratio of shortest to longest diameter.
At 60-68 min, most virions were in very large aggregates, and it was impossible to image the entire periphery of all of the virions. Thus many more than 20% of the virions may have had blebs or discontinuities.