Molecular Biology of Bluetongue Virus
Video of the assembly of the major protein components of the BTV virus particle (http://www.reoviridae.org/dsRNA_virus_proteins/BTV.htm)
The Bluetongue virus virion is a particle about 80 nm in diameter and has a double capsid composed from three protein layers. The outer capsid is formed from 60 homotrimers of the VP2 protein and 120 homotrimers of the VP5 protein. The inner capsid is formed from two protein layers:
1) Core-surface layer formed from VP7, which has T=13 icosahedral symmetry.
2) Subcore layer formed from VP3, which has T=2 icosahedral symmetry.
Inside the subcorelayer are transcriptase complexes (formed from the three proteins VP1, VP4 and VP6) and the ten double-stranded RNA segments of the genome (Mertens et al., 2004).
X-ray diffraction and cryoelectron microscopy studies have shown that that outer capsid of the Bluetongue virus virion is composed of 180 copies of the VP2 protein (arranged in 60 trimeric 'trisekllion' or 'propellor'-shaped structures) and 360 copies of the VP5 protein (arranged in 120 trimers) (Hewat et al., 1992a,b; Mertens et al., 2004).
As the viral proteins VP2 and VP5 make up the outer capsid, it is these proteins that interact most with the host immune systems. This antibody slective pressure is probably a large part of the reason why VP2 and VP5 have the most variable sequences of all the viral proteins. VP2 is particularly important and contains the neutralising epitopes that determine the specificity of virion-neutralising antibody interactions (It is this that determines the identy of the Bluetongue virus serotypes). It has been shown that, especially for VP2, nucleotide sequence variation correlates with virus serotype. This has allowed the design of reverse transcriptase Polymerase Chain Reaction assays to identify Bluetongue virus serotypes (Mertens et al., 2004; Maan et al., 2012).
Phylogenetic grouping of Bluetongue virus serotypes, based on VP2 sequence (sequences used available here)
BTV inner core with VP3 dimers (yellow) and Decamers (white) highlighted
BTV outer core with VP7 trimer 5 (yellow) and 6 (white) member rings highlighted
The core virus is made up of a pair of protein shells. The core-surface layer is made from the protein VP7 and conforms to T=13 Icosahedral symmetry. The subcore layer is made from the 120 copies of the protein VP3 and conforms to T=2 icosahedral symmetry (Grimes et al., 1998; Mertens et al., 2004)
VP3 exists in two different conformational forms. These two different conformational forms of VP3 join together to form a homodimer. Five VP3 homodimers associate to form a VP3 decamer and twelve of the VP3 decamers assemble to form the subcore layer. The subcore layer has been shown to self-assemble if synthesised independantly of other viral proteins. Once assembled the subcore layer acts as a scaffold for the assembly of the core-surface layer (made from VP7) and then the outer capsid (constructed from the proteins VP2 and VP5).
The seven hundred and eighty VP7 proteins of the core-surface layer form homotrimers, and these 260 trimers form a series of 6 and 5 member rings around the subcore layer. The interactions between VP7 and VP3 in the virus core are complex, there are 13 different orientations that the VP7 monomers interact with the subcore layer. It is these complex inteactions that allow the 5 member rings to form and the curve of the core-surface layer to be formed. In the closely related Orbivirus African horse sickness virus, VP7 (without any VP3 to interact with) has been observed to form large flat sheets of exclusively the 6 member rings (Burroughs et al., 1994).
The core virion contains ten to twelve copies of a transcriptase complex. Each transcriptase complex is constructed from a polymerase (VP1), capping enzyme (VP4) and a helicase (VP6) and are responsible for transcribing the double-stranded RNA genome into positive sense single-stranded RNA and capping the new single-stranded RNA. The newly produced single-stranded RNA can be used as either mRNA or as the template for the production of new double-stranded RNA genomes. Electron density mapping suggests that the transcriptase complexes are attached to the inner surface of the subcore layer immediately below a pore at the five-fold axis os symmetry. From analogy with cypoviruses each transcriptase complex is thought to be associated with a single genome segment, and extrude full length mRNA copies of the segment from the core surface through the pores in the core virion at which the transciptase complexes are located (Gouet et al., 1999; Mertens et al., 2004).
Electron density in the Bluetongue virus core (figure 8 from Mertens et al., 2004)
The lytic replication cycle of Bluetongue virus (from Mertens, 2002).
Bluetongue virions are taken into the cell via an endosomal route. The reduction of the pH in the endosome is thought to cause the seperation of the outer capsid (VP2 and VP5) from the virus core. The removal of the outer capsid causes the activation of the transcriptase functions of the core and casues it to be transported into the cytoplasm of the cell.
The single-stranded RNA produced by the virus core is then used as mRNA to produce viral proteins or as a template to form double-stranded RNA copies of the viral genome. Bluetongue virus core and sub-core particles are assembled in viral inclusion bodies with the cytoplasm. The outer capsid proteins are added to each virion as it is released from the viral inclusion body.
Mature Bluetongue virus virions are released from the cell by either budding or by directly penetrating the cell membrane (which casues cell lysis). Studies have shown that NS3 can mediate virus release from insect cells. As cell lysis is not seen in Bluetongue virus infected Culicoides cells, it seems likely that NS3 mediates virus release by the budding pathway (Mertens, 2002, Mertens et al., 2004)
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Gouet et al., 1999. P. Gouet, J.M. Diprose, J.M. Grimes, R. Malby, J.N. Burroughs, S. Zientara, D.I. Stuart and P.P.C. Mertens (1999). The highly ordered double-stranded RNA genome of bluetongue virus revealed by crystallography. Cell, 97, 481-490.
Grimes et al., 1998. J.M. Grimes, J.N. Burroughs, P. Gouet, J.M. Diprose, R. Malby, S. Zientara, P.P.C. Mertens and D.I. Stuart (1998). The atomic structure of the bluetongue virus core. Nature, 395, 470-478.
Hewat et al., 1992a. E.A. Hewat, T.F. Booth, P.T. Loudon and P. Roy (1992). Three-dimensional reconstruction of baculovirus expressed bluetongue virus core-like particles by cryo-electron microscopy. Virology, 189, 10-20.
Mertens et al., 2004. P.P.C. Mertens, J. Diprose, S. Maan, K.P. Singh, H. Attoui and A. Samuel (2004). Bluetongue virus replication, molecular and structural biology. Veterinaria Italiana, 40(4), 426-437.
Maan et al., 2012. N.S. Maan, S. Maan, M.N. Belaganahalli, E.N. Ostlund, D.J. Johnson, K. Nomikou, P.P.C. Mertens (2012). Identification and Differentiation of the Twenty Six Bluetongue Virus Serotypes by RT–PCR Amplification of the Serotype-Specific Genome Segment 2. PLoS ONE 7(2): e32601. DOI: 10.1371/journal.pone.0032601