flat joint


Nodaviridae is a group 4 member of the Baltimore classification of viruses. The genome is linear, positive sense, single stranded RNA consisting of 4500 nucleotides with a 5’ terminal methylated cap and a non-polyadenylated 3’ terminal. The virus is not enveloped and has a simple 30 nm diameter round capsid. The capsid exhibits iscosahedral symmetry with a triangulation number of 3, constructed of 32 capsomers.

Members of the Nodaviridae virus family include Nodamura virus (NV), Flock House virus (FHV), Beetle virus (BBV), Boolarra virus (BoV), Striped jack nervous necrosis virus (SJNNV), Gypsy moth virus (GMV) and Manawatu virus (MwV). The Flock House virus (FHV) is the best studied of the Nodaviruses.

The FHV coat protein, also known as the capsid protein, is described by Dasgupta, R. et al., Nucleic Acids Res. 17(18):7525-7526 (1989). The core of the structure is made up of eight stranded anti-parallel beta-barrels, as seen in many other viral capsid proteins. The helical domain is made up of 3 alpha-helices, formed by the polypeptide chain located sequentially, `N` and `C` terminal to the beta-barrel. The helix is the gamma-peptide, one of the cleavage products. A region between amino acids 205-209 from the "N" terminus forms a loop that is exposed on the exterior surface of the assembled capsid. The formed loop is between the beta E-beta F strands of a beta barrel.

The FHV genome contained within the capsid includes two messenger-sense RNA molecules, RNA 1 and RNA 2 (Schneemann, A., et al. 1993. In W. Doerfler and P. Bhm (eds.), Viral Strategies, Verlag Chemie, Weinheim, Germany. p. 167-176). RNA 1 (3.1 kb) directs synthesis of protein A (102 kDa) the putative RNA-dependent RNA polymerase (Fisher, A. J. and J. E. Johnson. 1993. Nature (London) 361:176-179). RNA 2 (1.4 kb) encodes protein alpha (43 kDa), the precursor of the coat protein (Gallagher, T. M. and R. R. Rueckert. 1988. J. Virol. 62:3399-3406). In addition to the genomic RNAS, infected cells also contain a subgenomic RNA 3 (0.4 kb) derived from the 3' end of RNA 1. It encodes protein B (10 kDa), whose function is to modulate replication (Ball, L. A. (1994) PNAS 91:12443-12447; Ball, L. A. (1995) J. Virol. 69:720-727).

A specific region of FHV RNA 2 (bases 186-217) has a predicted stem-loop structure, which serves as the packaging signal for in vivo encapsidation of RNA 2 (Zhong, W., Dasgupta, R., and Rueckert, R. 1992. Proc. Natl. Acad. Sci. USA 89:11146-11150). Similar regions of the other Nodaviral RNA 2 sequences are also shown in FIG. 2 and serve an identical function. The initial step is believed to involve the formation of a nucleating complex, in which a coat protein substructure interacts with this encapsidation signal on the viral RNA. The initiation complex may then be propagated into a spherical particle by addition of coat protein subunits, which are guided into the growing shell by binding to the viral RNA.

Flock House virus (FHV) can be grown to high titers in Drosophila cell culture with synthesis of proteins A and B peaking at about 5 hours and 8 hours, respectively (Schneemann, A., et al. 1993. In W. Doerfler and P. Bhm (eds.), Viral Strategies, Verlag Chemie, Weinheim, Germany. p. 167-176). In contrast, synthesis of protein alpha remains low during the first 12 hours but rises rapidly thereafter with peak production at about 15 hours. The newly synthesized alpha chains are assembled within minutes into labile precursor particles, called provirions. Provirions contain 180 alpha subunits, arranged with icosahedral symmetry, as well as a copy of each of the genomic RNA molecules. The assembly process triggers an autoproteolytic reaction in the 407 amino acid alpha chain which results in cleavage between asparagine 363 and alanine 364 (Hosur, M. V. et al. 1987. Proteins: Struc. Funct. Genet. 2:167-176; Fisher, A. J. and J. E. Johnson. 1993. Nature (London) 361:176-179).

The newly formed polypeptides, beta (38 kDa, 363 amino acids) and gamma (5 kDa, 44 amino acids), remain associated with the mature virion.

Following injection of wild-type FHV, antibody formation occurs without symptoms or disease in pigs, adult mice, rabbits, guinea pigs, Syrian hamsters, and chickens. In addition, there is no cytopathology in mammalian cell culture lines including primate kidney and human amino cells (Hendry, D. A. 1991. In Viruses of Invertebrates (ed. E. Kurstak) Marcel Dekker, Inc.: New York).

The structure of FHV was solved to atomic resolution and shows that the viral capsid is composed of 60 triangular units, which consist of three identical polypeptides related by icosahedral symmetry (Hosur, M. V. et al., 1987. Struc. Funct. Genet. 2:167-176). All three subunits, designated A, B and C, contain a central beta-barrel motif similar to that observed in many other virus structures (Rossmann, M. G. and J. E. Johnson. 1989. Ann. Rev. Biochem. 58:533-573). The exterior surface consists of elaborate loops between the beta-strands and the inner surface is made up of helical domains from the amino- and carboxy-terminal ends of the protein. The helical domain formed by the amino-terminal end of the protein is only visible for the C subunits, in which amino acid residues 20 through 30 constitute an ordered peptide "arm". In the A and B subunits, the amino terminal-end is disordered and not visible in the electron density map. This variation results in a significant difference in the subunit contacts across the icosahedral twofold and quasi-twofold axes. While the interaction between the protein subunits across the quasi-twofold axes (A/A.sub.2 and C/B.sub.5) are bent, the interactions across the twofold axes (C/B.sub.2 and C.sub.2 /B contacts are flat. This is because the peptide arm in the C subunits folds into the hinge between the subunits, preventing them from forming the dihedral angle seen at the quasi-twofold axes. In addition, the flat joint at the twofold axes is stabilized by a segment of genomic RNA that forms a wedge between the C/C.sub.2 joints, but not the A/B.sub.5 joints.

The alpha protein cleavage site is located deep inside the virion near the RNA core, explaining its inaccessibility to proteinase inhibitors and virus precipitating antibodies. The cleavage product gamma (carboxy-terminal residues 364-407) is located in the particle interior and forms an amphipathic-helix. At the twofold axes of symmetry, the gamma helices interact with the ordered duplex RNA whereas at the fivefold axes they form a pentameric helical bundle that is stabilized by interactions among the hydrophilic surfaces of the helices (Cheng, H. R. et al. 1994. Structure 2:271-282).


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