Page 52 - TBE_Book_V2_2019

Chapter 2b: The molecular and antigenic structure of TBEV

infectivity in various flavivirus systems. These were secreted from transfected cells and had a
observations led to the concept of ‘virus density of approximately 1.14 g/cm³. 48,49 They
breathing’ as a consequence of envelope were sensitive to disintegration by the
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glycoprotein dynamics , reflecting the detergent Triton X 100, consistent with the
metastable nature of E which transiently presence of a lipid membrane carrying the two
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exposes otherwise buried protein surfaces viral envelope proteins. The formation of RSPs
within the E dimer or at the inter-dimer contact could also be achieved by the expression of prM
regions in the virion. Retrospectively, antibody- and E from separate plasmids, but was not
induced conformational changes, described for possible with a soluble form of E that lacked its
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TBEV already in 1984, are also likely due to E membrane anchor. More detailed mapping
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protein dynamics and virus breathing. studies allowed the identification of the so-
Secondly, many data indicate that virus particles called stem together with the first trans-
released from infected cells are a hetero- membrane regions of E to be essential for
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geneous mixture of immature, partially mature particle formation.
and fully mature particles. 38,39 As a specific
structural feature, partially mature and The ER was shown to be the site of assembly of
breathing particles expose the viral membrane, RSPs by biochemical and electron microscopical
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which has been shown to be a target for analyses. In addition to the rough ER, RSPs
interactions with cellular lipid receptors that were observed in the smooth ER and down-
can mediate cell entry. 40,41 stream compartments of the secretory path-
way. Approximately 75% of the particles had a
It can be hypothesized that an ensemble of diameter of 30 nm, but a number of larger
heterogeneous particles in combination with particles and tubular structures were also seen
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virus breathing may be important for flavi- in vesicular compartments of transfected cells.
viruses to infect different tissues in their
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invertebrate and vertebrate hosts. Hetero- It is an important conclusion of these studies
geneity may also be required to maintain these that the formation of prM-E heterodimers and
viruses in their natural cycles and constitute a their lateral interactions are sufficient to drive
powerful means to adapt to new environments the budding of membrane-containing virus-like
or to acquire new pathogenic properties, such particles at the ER membrane, in the absence of
as those observed in the recent Zika virus any interactions with viral RNA or a capsid.
epidemic. 42, 43
The 30 nm RSPs were the first flaviviral particles
Subviral particles for which a cryo-EM structure was determ-
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ined. The 19Å resolution map revealed an
Flavivirus-infected cells do not only secrete arrangement of E protein dimers in a T=1
complete virus particles but also subviral icosahedral surface lattice (different from that
particles that are non-infectious and smaller of the virion, Figure 1G) and allowed the
than whole viruses but have similar HA activity. definition of interaction sites between E dimers,
Because of these properties they were positions of M relative to E, and the assignment
described as ‘slowly sedimenting hemagglu- of transmembrane regions of E and M. When
tinin’ (SHA) in the flavivirus literature. 44, 45 the prM furin cleavage site was deleted in the
plasmid construct for RSP production by
Noninfectious subviral particles of TBEV were mutagenesis, a substantial number of particles
produced in recombinant form by the co- were observed that had the same size as whole
expression of the two viral glycoproteins prM immature virions (diameter 60 nm), in addition
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and E in COS-1 cells. 46,47 These particles [desig- to the 30 nm particles described before. It was
nated ‘recombinant subviral particles’ (RSPs)] therefore concluded that the primary assembly

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