Sindbis virus (SINV) is an enveloped, single-stranded RNA virus, which is transmitted via mosquitos to a wide range of vertebrate hosts. SINV produced by vertebrate, baby hamster kidney (BHK) cells is more than an order of magnitude less infectious than SINV produced from mosquito (C6/36) cells. The cause of this difference is poorly understood. In this study, charge detection mass spectrometry was used to determine the masses of intact SINV particles isolated from BHK and C6/36 cells. The measured masses are substantially different: 52.88 MDa for BHK derived SINV and 50.69 MDa for C6/36 derived. Further analysis using several mass spectrometry-based methods and biophysical approaches indicates that BHK derived SINV has a substantially higher mass than C6/36 derived because in the lipid bilayer, there is a higher portion of lipids containing long chain fatty acids. The difference in lipid composition could influence the organization of the lipid bilayer. As a result, multiple stages of the viral lifecycle may be affected *
There are many techniques
for monitoring and measuring the interactions
between proteins and ligands. Most of these techniques are ensemble
methods that can provide association constants and in some cases stoichiometry.
Here we use charge detection mass spectrometry (CDMS), a single particle
technique, to probe the interactions of antigen binding fragments
(Fabs) from a series of antibodies with the canine parvovirus (CPV)
capsid. In addition to providing the average number of bound Fabs
as a function of Fab concentration (i.e., the binding curve), CDMS
measurements provide information about the distribution of bound Fabs.
We show that the distribution of bound ligands is much better at distinguishing
between different binding models than the binding curve. The binding
of Fab E to CPV is a textbook example. A maximum of 60 Fabs bind and
the results are consistent with a model where all sites have the same
binding affinity. However, for Fabs B, F, and 14, the distributions
can only be fit by a model where there are distinct virus subpopulations
with different binding affinities. This behavior can be distinguished
from a situation where all CPV particles are identical, and each particle
has the same distribution of sites with different binding affinities.
The different responses to viral heterogeneity can be traced to the
Fab binding sites. A comparison of Fab binding to new and aged CPV
capsids reveals that a post-translational modification at the binding
site for Fab E (M569) probably reduces the binding affinity.
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