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a 20% sucrose cushion. SDS-polyacrylamide gel electrophoresis showed
that the re-suspended pellet contained large amounts of a 37 kDa cap-
sid protein that was not observed in mock-infected cells, presumably
VLPs. The re-suspended pellet was centrifuged through a 10-40%
(wt/wt) sucrose gradient followed by fractionation of the gradient
with continuous absorbance at 254 nm. The absorbance profile
showed three peaks, but gel electrophoresis of the corresponding frac-
tions indicated that only the material in the two fastest peaks, labeled
I and II, contained protein. This protein migrated as a single band and
had an apparent molecular mass of approximately 37 kDa. Electron
microscopy of negatively stained material taken from peak I showed
the presence of irregularly assembled particles, many of which were
stain-permeable, whereas peak II showed intact, non-permeable
particles that had a diameter of approximately 31 nm. A comparison of
these particles with authentic MGNNV virions confirmed that the
VLPs were closely similar in size and geometry to native virus. Interest-
ingly, the surface of both native particles and VLPs was distinctly
rougher than that of the insect nodavirus Flock house virus (FHV).
There was no evidence for the presence of coat protein cleavage prod-
ucts as observed for the insect nodaviruses. Neither of such cleavage
products was detected by employing the more sensitive technique of
mass spectrometry using purified VLPs (Lin, unpublished data). It was
shown for DlEV that the protein doublet is not derived from an
assembly-dependent maturation cleavage of the capsid coat protein as
it is in the insect nodaviruses. Rather, it was proposed that the doublet
might arise from cotranslational modification.
Piscine nodavirus VLPs package random cellular RNAs when
assembled in Sf 21 cells.
3
The particles in peak I contained a mixture
of RNAs ranging in size from approximately 100 bases, presumably
tRNAs, to 1300 bases, whereas particles in peak II contained a broader
mixture of RNAs ranging in size from about 100 to 4500 bases. The
observation was essentially identical to what has been seen for the insect
nodavirus VLPs. The sharp cutoff in size probably reflects the largest
single RNA molecule that can be packaged in this size of particle.
The promiscuity of RNA packaging is likely to reflect the electrostatic
requirements of assembly and the ability of the capsid protein to
