Characterization of grass carp reovirus minor core protein VP4

Virology Journal Characterization of grass carp reovirus minor core protein VP4 Liming Yan 0 1 Hong Guo 0 Xiaoyun Sun 0 Ling Shao 0 1 Qin Fang 0 0 State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences , Wuhan 430071 , China 1 Graduate School of the Chinese Academy of Sciences , Beijing 100039 , China Background: Grass Carp Reovirus (GCRV), a tentative member in the genus Aquareovirus of family Reoviridae, contains eleven segmented (double-stranded RNA) dsRNA genome which encodes 12 proteins. A low-copy core component protein VP4, encoded by the viral genome segment 5(S5), has been suggested to play a key role in viral genome transcription and replication. Results: To understand the role of minor core protein VP4 played in molecular pathogenesis during GCRV infection, the recombinant GCRV VP4 gene was constructed and expressed in both prokaryotic and mammalian cells in this investigation. The recombinant His-tag fusion VP4 products expressed in E.coli were identified by Western blotting utilizing His-tag specific monoclonal and GCRV polyclonal antibodies. In addition, the expression of VP4 in GCRV infected cells, appeared in granules structure concentrated mainly in the cytoplasm, can be detected by Immunofluorescence (IF) using prepared anti-VP4 polyclonal antibody. Meanwhile, VP4 protein in GCRV core and infected cell lysate was identified by Immunoblotting (IB) assay. Of particular note, the VP4 protein was exhibited a diffuse distribution in the cytoplasm and nucleus in transfected cells, suggesting that VP4 protein may play a partial role in the nucleus by regulating cell cycle besides its predicted cytoplasmic function in GCRV infection. Conclusions: Our results indicate the VP4 is a core component in GCRV. The cellular localization of VP4 is correlated with its predicted function. The data provide a foundation for further studies aimed at understanding the role of VP4 in viroplasmic inclusion bodies (VIB) formation during GCRV replication and assembly. - Background Double stranded (ds) RNA viruses, which affect a very wide range of host species including vertebrates, invertebrates, plants, fungi, and prokaryotes, represent a diverse group of viral pathogens [1]. According to classification of the International Committee on Taxonomy of Viruses (ICTV), eight distinct families are currently recognized [2]. The Reoviridae, one of the most complex families in dsRNA group, consists of at least 15 distinct genera reported so far. The virus particles in the family of Reoviridae appear to be icosahedral in symmetry with an overall diameter of approximately 6085 nm comprising 912 segmented dsRNA genome enclosed within multiple concentric protein capsids. Based on their structure organization, it is possible to divide the members of the family Reoviridae into two subfamilies, Spinareovirinae and Sedoreovirinae [2]. The members of the Spinareovirinae subfamily have 12 icosahedrally pentameric turrets resided on the surface of core or at the fivefold axe of intact particle (eg. orthoreoviruses or cypoviruses), while the viruses in the Sedoreovirinae subfamily possess a relatively smooth surface and core without large surface projections at their fivefold axes (eg. rotaviruses or orbiviruses). GCRV (grass carp reovirus), a tentative member of genus Aquareovirus, could be classified into Spinareovirinae subfamily of Reoviridae [2]. GCRV has been recognized as the most pathogenic amongst all the isolated aquareoviruses [3,4]. Similar to other members of Reoviridae, GCRV is a multilayer spherical particle enclosing a dsRNA genome of 11 segments, which encode 7 structural proteins (VP1-VP7) and 5 nonstructural proteins. Amongst the 7 structural proteins, VP1-VP4 and VP6 proteins are the components of viral core, and the outer capsid of GCRV is made up of 200 trimers of VP5-VP7 heterodimers organized into an incomplete T = 13 lattice. The core is a T = 1 particle with 12 VP1 pentameric turrets decorating a shell of 60 VP3 dimers, which are clamped together by 120 VP6 monomers [5-8], while the other two core proteins, VP2 and VP4, appeared in a low copy located near the VP1 turret protein. The structure and function of the viral major constitute proteins have been well resolved according to recent progress on GCRV atom image [8], however, the minor structural proteins VP2 and VP4 that are related to enzyme activity in RNA transcription and replication are poorly understood due to their low copy in mature viral particles. Previous study of genome sequences of aquareoviruses indicated that aquareoviruses have a common evolutionary origin with genus Orthoreovirus, including mammalian reoviruses(MRV) and avian reoviruses (ARV) [9-11]. According to genome alignment of GCRV with its homologous proteins, except for the similarity presented in structural proteins between Genus Aqureovirus and Orthoreovirus, some nonstructural proteins also remain conserved domain that performed similar function in virus replication cycle, indicating that both Genus Aqureovirus and Orthoreovirus share common molecular morphogenesis during virus infection [9,10]. Notablely, uNS(or uNSC), a MRV nonstructural protein, has been demonstrated that it is sufficient for forming phasedense viroplasmic inclusion bodies (VIB) in the cytoplasm of transiently transfected cells [11-17]. The VIB like structures formed by single uNS are similar in its appearance to globular inclusions formed in MRV infected cell, suggesting that uNS is able to form matrix of viral factories [17]. Different from the dominated role played by NS in VIB formation in MRV, another nonstructural protein NS is also recognized to be related to form VIB like structures by interaction with NS [18-20]. Besides, the core protein 2 and 2 were also verified to play very important roles by interacting with NS in the formation of VIB. Recent progress indicated that the core protein 2, known as a cellular microtubule associated protein, is recognized to determine the morphology of VIB (showed either globular or filamentous) in MRV [17,21]. Studies on reassortment suggest that 2 determines viral strain differences in transcriptional efficiencies of core particles [22,23], and also displays both ssRNA and dsRNA binding abilities, which demonstrates that 2 possesses both nucleoside triphosphatase (NTPase) and RNA-triphosphatase (RTPase) activities [24-26]. As a homologue of 2 protein, the VP4 protein in GCRV was presumed to have a similar function to 2 of MRV. In light of the previous investigation on GCRV genome and molecular biology characterization [9,10,27-29], nonstructural protein NS80 of GCRV, the analog of NS protein in MRV, has been previously identified to be related to viral inclusion formation during virus replication and particle assembly [28]. The protein VP4 was predicted to be a core protein possessing NTPase activity played in viral genome transcription, and identified to have interaction with NS (...truncated)