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and in directing RNPs to the site of virus assembly and budding. The
tail of the Gn protein located on the cytoplasmic side of the Golgi
membrane could also determine the RNPs that are included in the
virion. It has been suggested that the cytoplasmic tail of Gn interacts
with the N protein.
25
This could be an important step in virus assem-
bly, which would secure the incorporation of RNP into virus particles.
The hantaviral N protein seems to interact with a variety of cellu-
lar proteins (for a review, see Kaukinen
et al.
, 2005
8
), but the discus-
sion of these activities would go beyond the scope of this chapter.
3D-Structure of the N Protein
Crystallization of hantavirus N protein is pending. Purification of
even modest quantities of the protein suitable for crystallization (i.e.
water-soluble) is difficult to accomplish from purified virus particles
because hantaviruses grow to low titers in cell cultures. As for recom-
binant N protein produced in heterologous expression systems (e.g. in
the baculovirus-driven system), it appears to be “sticky” and tends to
form aggregates that can be solubilized in 6-7 M urea, for example, but
not directly in water or aquatic buffers.
26
Luckily, a substantial portion
of the hantavirus N protein expressed in a baculovirus-based system
exists in the form of a stable trimer, in which monomers are covalently
linked to each other via S-S-bridges between cystein residues.
16
This
trimer presented a useful model for our initial structural studies.
First, cryo-EM analysis of recombinant N protein was performed.
27
It confirmed the existence of the N-trimer and suggested a curved
shape for the N protein monomers (with dimensions of approximately
8 nm by 3 nm), which resemble influenza and rabies virus N protein
monomers in the reconstructed 3D models.
28,29
Importantly, contacts
on both N- and C-termini of the monomers were clearly seen. In
agreement with these data, the 3D reconstruction of the N-trimer
using EM after negative staining revealed curve-shaped monomers
attached to each other at both ends.
19
These observations, together
with results of
in silico
docking of monomers into a trimer, were
helpful in the mapping of oligomerization domains.
19,21
To reconstruct the N-protein trimer in greater detail, the single-
particle reconstruction was done assuming the symmetry of three.
