- Open Access
Structural comparisons of the nucleoprotein from three negative strand RNA virus families
© Luo et al; licensee BioMed Central Ltd. 2007
- Received: 18 May 2007
- Accepted: 10 July 2007
- Published: 10 July 2007
Structures of the nucleoprotein of three negative strand RNA virus families, borna disease virus, rhabdovirus and influenza A virus, are now available. Structural comparisons showed that the topology of the RNA binding region from the three proteins is very similar. The RNA was shown to fit into a cavity formed by the two distinct domains of the RNA binding region in the rhabdovirus nucleoprotein. Two helices connecting the two domains characterize the center of the cavity. The nucleoproteins contain at least 5 conserved helices in the N-terminal domain and 3 conserved helices in the C-terminal domain. Since all negative strand RNA viruses are required to have the ribonucleoprotein complex as their active genomic templates, it is perceivable that the (5H+3H) structure is a common motif in the nucleoprotein of negative strand RNA viruses.
- Vesicular Stomatitis Virus
- Southern Bean Mosaic Virus
- Protein Data Bank Accession Code
- Satellite Tobacco Mosaic Virus
Negative strand RNA viruses are different from all other viruses because their RNA genomes are always enwrapped by a virally coded nucleoprotein (N) to form a ribonucleoprotein (RNP) complex. This complex serves as the template for viral RNA synthesis (the plus strand cRNA, the negative strand vRNA or mRNA) and form the structural core when packaged into virions. The RNP is formed concomitant with replication of viral genomic RNAs by the viral RNA-dependent RNA polymerase. The RNP structure is a unique feature of negative strand RNA viruses such that the polymerase complex can only copy the RNA sequence in the RNP, not naked RNA. In addition, virion assembly also requires the RNP structure to be packaged in the virion. These unique functions that are common among negative strand RNA viruses may require conserved structural motifs in the N protein. Such conservation has been observed in capsid proteins of spherical viruses which share a β-barrel motif . The eight strand β-barrel has the general property of encapsidating nucleotides and self-assembly into an icosahedral shell. Structural similarities have also been noticed in the coat proteins of three other icosahedral virus groups that suggested evolutionary lineages in the virus group. dsDNA virus (e.g. adenovirus), tailed dsDNA viruses (e.g. bacteriophages), and dsRNA viruses (e.g. reovirus) share common protein folds in their coat proteins and common architectures in virion assembly .
Crystal structures of the N protein from borna disease virus (BDV) , two rhabdoviruses, vesicular stomatitis virus (VSV)  and rabies virus (RABV) , and influenza A virus (FLUAV) , have been reported. The structures of the rhabdovirus N proteins were determined with RNA bound in a cavity. The cavity is located between two separated domains that accommodate the RNA with both hydrophobic and charged/polar interactions. Extended N-termini and a loop in the C-terminal domain reach over neighboring molecules to form an extended protein network along the RNA. The BDV and FLUAV N proteins were determined as a tetramer and a trimer, respectively, in the absence of RNA. The collection of N protein structures from three negative strand RNA viruses makes it possible to identify conserved structural motifs in the nucleoprotein from different virus families. We found that the RNA binding region of the N protein contains an N-terminal domain and a C-terminal domain with a similar topology in the N protein structures of all three virus families. In the RNA binding cavity, a central α-helix surrounded by four α-helices in the N-terminal domain continues to a central α-helix surrounded by two α-helices in the C-terminal domain. By superimposing the rhabdovirus N protein structure with that of BDV and FLUAV, this structural motif was also present in the other two structures. This suggests that the (5H+3H) structure may be a common motif in the nucleoprotein of negative strand RNA viruses.
Superposition of β-barrels in the viral capsid proteins
Structural alignment of the β-barrel fold
RMSD (Å) P-value Aligned residues
HRV16-VP2 (252 residues)
SBMV C-sub (222 residues)
STMV (147 residues)
HPV (233 residues≠)
HRV16-VP1 (285 residues)
Superposition of individual domains
Structural alignment of the C-terminal domains
RMSD (Å) ⇓ P-value Aligned residues
BDV N C-domain: (residues 230–346, 109 amino acids)
FLUAV N C-domain: (residues 213–271, 59 amino acids)
VSV N C-domain: (residues 224–341, 118 amino acids)
BDV N C-domain: (residues 230–346, 109 amino acids)
Structural alignment of the N-terminal domains
RMSD (Å) ⇓ P-value Aligned residues
BDV N N-domain: (residues 50–229, 180 amino acids)
FLUAV N N-domain: (residues 21–202 [56–147]*, 73 amino acids)
VSV N N-domain: (residues 46–223, 178 amino acids)
BDV N N-domain: (residues 50–229, 180 amino acids)
Each N protein has other secondary structural elements that are not aligned (Figure 2). For instance, there is a long loop between the second and the third conserved α-helices (α3 and α4 in the VSV N protein) in the N-terminal domain, with a β-hairpin at the tip of the loop. This loop is also present in the N-terminal domain of the BDV N protein as shown by the flexible superposition of the two structures , but it is pointing to the opposite direction as a result of the insertion of an α-helix (α4 in the BDV N protein). The C-terminal end of the VSV N protein beyond the C-terminal domain includes three consecutive α-helices whereas that of the BDV N protein contains only one. There are 310 helices between the third and fourth α-helices (α11 and α12 in the VSV N protein) in the C-terminal domain (three 310 helices in the BDV N protein and one in the VSV N protein). Additional α-helices can be found in the BDV N protein (Figure 2). In the case of the FLUAV N protein, the region that is superimposable with the RNA binding region of the VSV N protein contains essentially the same number of secondary structural elements except for helix α13 of the VSV N protein. The rest of the FLUAV N protein (residues 272–489) has no homologous counterpart in either VSV or BDV N proteins. These residues constitute an additional domain near the C-terminal end of the FLUAV N protein.
RNA binding cavity
The N protein polymerizes on the genomic RNA during replication. Neighboring N molecules form an extended network of interactions along the entire length of the RNA genome. In the VSV N protein, there is a 1954 A2 buried area side-by-side between two monomers while the buried area is 2680 A2 in the BDV N protein. The larger buried area in the BDV N proteins could be the result of the tetrameric oligomerization, which has a 90° angle between two neighboring molecules compared to an angle of 144° or 147° for the rhabdovirus N proteins that were crystallized as a 10 mer and 11 mer, respectively, with RNA bound. The BDV N protein molecules would have to associate through more extended side-by-side interactions in the RNP, which should have similar contact areas between the neighboring
N molecules as observed in the rhabdovirus N-RNA complexes. The extended C-terminal and N-terminal arms in both structures reach over the neighboring molecules to add further intermolecular interactions. The oligomerization arrangement of the reported FLUAV N structure  is so different that it is impossible to make a meaningful comparison of the reported FLUAV N oligomer with that of the rhabdovirus or BDV N proteins. Comparisons of how the interactions between the FLUAV N proteins contribute to encapsidation of the RNA genome would become more apparent if a structure of the FLUAV N-RNA complex becomes available.
Comparisons of the N protein structures from three virus families showed that the RNA binding region in each N protein has a similar structure containing two domains. The overall structure of the rhabdovirus N protein can be superimposed with that of the BDV N protein, whereas the FLUAV N protein could only be superimposed with the other N proteins as separate N-terminal and C-terminal domains. However, it appears that the fold of the individual domains are conserved in the N proteins to a degree similar to that of the β-barrel fold in the capsid proteins of spherical viruses. There are five helices in the N-terminal domain and three helices in the C-terminal domain that are common among the N structures of the three virus families. This motif, which we have named the (5H+3H) motif, may be a common motif responsible for encapsidating RNA by the N protein of negative strand RNA viruses (Figure 2). The helices α8 and α9 named as in the VSV N protein are at the center of the motif and connect the two domains in the motif. However, the spatial geometry of the helices in the (5H+3H) motif is variable when the structures were compared. One possible explanation for this observation is that the structures of the BDV and FLUAV N proteins were determined without RNA bound [3, 6] whereas those of the VSV and RABV N proteins were determined with a random RNA molecule bound in the RNA binding cavity [4, 5]. The orientation of the helices in the BDV or FLUAV N protein might change when the N protein binds RNA. This question could be answered when the structure of the BDV or FLUAV N protein is determined in the presence of bound RNA. An alternative explanation could be that there are intrinsic differences in the three dimensional structure of the N proteins, a likely result of evolution despite commonality of the structure and function of the N proteins among negative strand RNA viruses.
The structural alignments of the N proteins from three negative strand RNA virus families have significant predictive values in recognizing the RNA binding site and the side-by-side interactions of the BDV N protein, which was not revealed when the BDV N structure was determined alone in the absence of bound RNA. The chemical properties of the homologous cavity in the BDV N protein and the pattern of intermolecular interactions are consistent with its functions to assemble the viral RNP. It also suggested a possible conformation of the FLUAV N protein which may be more suitable for RNA binding than the conformation observed in the recent crystal structure.
This work is supported in part by NIH grants AI050066 and AI057157.
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