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A loosely helical, non-rigid nucleocapsid formed by a close association of a virus
encoded protein, commonly referred to as N protein, with the genomic RNA is a common
feature in many of the enveloped ssRNA viruses including coronaviruses. Structural
information on the N protein and the molecular understanding of how this protein
facilitates the formation of the nucleocapsid is limited. From the biochemical
characterization of the N protein of IBV, a prototype coronavirus, presented here, and
from similar characterization of the N protein from other coronoviruses, it is apparent that
this protein has two major protease-resistant domains. Our X-ray crystallographic
analysis of these two domains, NTD and CTD, provides some insights into how the two
domain organization of the N protein may coordinate nucleocapsid assembly.
NTD and CTD interact with RNA
Several biochemical studies have shown that determinants for RNA binding
reside in the N-terminal region with the minimal region being mapped to residues 177 to
231 in MHV (corresponding to 136 to 190 in IBV). In addition to NTD, the involvement
of CTD in the RNA binding has been shown by Fan et. al. using gel-shift assays{Fan,
2005 #51}. Based on their recent structure of the NTD (IBV Beaudette strain), Fan et. al.
have proposed that the arms of the “U” shaped monomer, which are quite basic in nature
are likely the regions of the N-protein binding to RNA. This is also consistent with
NMR–NOE analysis of NTD-RNA interactions in the SARS-coronavirus N-protein.
A novel finding in our crystal structure analysis of the NTD (IBV-Gray strain) is
that it can form a strong interlocking dimer, in contrast to the weak dimeric interaction
observed in the NTD of the IBV Beaudette strain reported by Fan et. al., . These
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interlocking dimers associate to form a linear fiber with the basic tethers exposed along
the surface. Such a fiber could provide for closely packed interactions of NTD with the
genomic RNA. Analysis of N protein-RNA interactions in MHV at different stages of
the virus life cycle revealed that these interactions progress from an RNAse sensitive
complex involving subgenomic RNA to an RNAse resistant complex involving genomic
RNA{Narayanan, 2003 #5}. The strong and weak NTD dimer interactions seen in the
two structures possibly correspond to these different states of N protein-RNA
associations. The electrostatic potential surface of the CTD dimer is significantly
polarized with one of its phases being acidic and the other basic. This basic phase made
up of the -helix lined groove is a likely candidate for its interactions with RNA.
Plausible model for nucleocapsid formation.
In the formation of the nucleocapsid, the N-protein has to self associate tightly and
interact with RNA so that the resulting structure is RNAse resistant. Our crystal structure
analysis of CTD indicates a tight dimer mediated by domain swapped interaction and
thus the full length N-protein very likely functions as a dimer, with CTD providing a
structural scaffold while the NTD serves as a module for RNA interaction. The
orientation of the NTD with respect to the CTD in the N-protein is not certain from our
crystal structure analysis of these two domains because they are connected by a 47
residues long protease sensitive region. It is possible that the RNA binding regions of
these two domains face each engulfing the RNA between them conferring resistance to
RNAse.
In the various crystal forms of the CTD, we have seen the ability of CTD to self-associate
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with significant BSA of greater than 1000 Å . Thus self association of the full length Nprotein is very likely nucleated by the CTD. As seen in our analysis, CTD can selfassociate in multiple modes. A relevant question is which of these interdimeric
interactions is used in the formation of the nucleocapsid. Both type S and F interactions
are conducive to form fibril structures. Propagation of any single type of interactions,
however, would lead to rigid strictly helical nucleocapsid. Considering that the
nucleocapsid is not a rigid rod-like structure in coronaviruses, nucleocapsid assembly
may involve a combination of various inter-dimeric interactions observed in our studies.
In such an assembly, one possibility is that the type S interaction is primarily used,
because it is observed over a wider range of pH and in non-crystallographic environment
(as in CTD2 crystals), along with other interactions such as type L and type F to
appropriately modulate the curvature and change the direction of the nucleocapsid in the
virion (Figure 5b).
N-protein interactions with M-protein.
In addition to its interactions with RNA, N protein is also known to interact with M
protein which is an integral part of the viral membrane. Based on reverse genetic
complementation assays, the interaction region between these two proteins has been
mapped to their C-termini {Kuo, 2002 #33}. The C-terminus of M protein is significantly
basic and recent mutational studies in M protein have demonstrated that its interaction
with N-protein is predominantly electrostatic in nature {Luo, 2005 #50}. The exposed
acidic β-sheet floor, on the opposite side of the proposed RNA-binding region, in the
CTD dimer may serve as a suitable site for its interaction with the M protein.
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Thus the CTD may serve a dual purpose of not only mediating the self-association of the
N-protein in nucleocapsid formation but also in providing a complementary surface for
intermittent interactions with the M protein in the virus envelope.
Similarity with other coronaviral N proteins
Coronaviruses are classified into four groups, with SARS-CoV being an independent
group. The N-protein sequences are more similar within each group (~40%) than across
groups (20-30%). The only X-ray structure of a coronaviral N protein available to date is
that of the IBV N protein as described here and by Hui et al., who determined the first
structure of the NTD. However, NMR structures of the N- and C-terminal domains of
the SARS-N protein have been reported (REFS). Despite very low sequence similarity
between IBV and SARS N-proteins, their NTD and CTD structures show same general
polypeptide fold perhaps indicating these folds are conserved across the Coronaviridae N
proteins.
The polypeptide fold of the NTD is novel and is observed only in the coronavirus N
protein. A DALI search using the IBV-CTD structure revealed a very striking similarity
to the 73 amino acid capsid forming domain of PRRSV (porcine reproductive and
respiratory syndrome virus), a corona-like virus, which is a member of the Arteriviridae
family. In the PRRSV N-protein structure, this fragment also forms a very similar
domain swapped dimer as seen in our CTD structure and exhibits self-association
involving a salt bridge like in the type S interdimeric interactions (ref). Based on this
similarity between IBV-CTD and a distantly related arterivirus N-protein, it is tempting
to speculate that this type of domain-swapped dimer capable of self association may
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indeed be common in other enveloped viruses with non-rigid helical nucleocapsid such as
orthomyxovirus, paramyxovirus, bunyavirus and arenavirus, all of which contain
genomic ssRNA associated with their respective nucleocapsid proteins.
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