Identification of Genes Involved in Replication and Movement of Peanut Clump Virus

Three 'triple gene block' proteins known as TGBp1, TGBp2 and TGBp3 are required for cell-tocell movement of plant viruses belonging to a number of genera including Hordeivirus. Hordeiviral TGBp1 interacts with viral genomic RNAs to form ribonucleoprotein (RNP) complexes competent for translocation between cells through plasmodesmata and over long distances via the phloem. Binding of hordeivirus TGBp1 to RNA involves two protein regions, the C-terminal NTPase/ helicase domain and the N-terminal extension region. This study demonstrated that the extension region of hordeivirus TGBp1 consists of two structurally and functionally distinct domains called the N-terminal domain (NTD) and the internal domain (ID). In agreement with secondary structure predictions, analysis of circular dichroism spectra of the isolated NTD and ID demonstrated that the NTD represents a natively unfolded protein domain, whereas the ID has a pronounced secondary structure. Both the NTD and ID were able to bind ssRNA non-specifically. However, whilst the NTD interacted with ssRNA non-cooperatively, the ID bound ssRNA in a cooperative manner. Additionally, both domains bound dsRNA. The NTD and ID formed low-molecular-mass oligomers, whereas the ID also gave rise to high-molecular-mass complexes. The isolated ID was able to interact with both the NTD and the C-terminal NTPase/helicase domain in solution. These data demonstrate that the hordeivirus TGBp1 has three RNA-binding domains and that interaction between these structural units can provide a basis for remodelling of viral RNP complexes at different steps of cell-to-cell and long-distance transport of virus infection. INTRODUCTIONTransport of plant viruses from cell to cell occurs through plasmodesmata and requires virus-encoded movement proteins (Boevink & Oparka, 2005;Lucas, 2006;Epel, 2009). In the genus Hordeivirus, viral movement proteins are represented by three proteins encoded by a 'triple gene block' (TGB) and referred to as TGBp1, TGBp2 and TGBp3 (Solovyev et al., 1996). The TGB is a conserved module of overlapping genes found in a number of virus groups (Morozov & Solovyev, 2003). Hordeivirus TGBp1 binds viral genomic RNA forming a cell-to-cell transportcompetent complex (Brakke et al., 1988;Kalinina et al., 2001;Lim et al., 2008;Jackson et al., 2009). TGBp2 and TGBp3 are small membrane proteins necessary for intracellular transport of complexes containing TGBp1 and viral RNA to plasmodesmata (Morozov & Solovyev, 2003;Zamyatnin et al., 2004; Haupt et al., 2005;Jackson et al., 2009 Lim et al., 2008;Jackson et al., 2009). Such TGBp1-RNA complexes are considered to be a form of viral genome capable of cell-to-cell and long-distance transport in plants (Morozov & Solovyev, 2003;Lim et al., 2008). In potex-like TGBs, the whole TGBp1 sequence is represented by the NTPase/helicase domain (HELD) with seven conserved motifs of the superfamily 1 NTPases/helicases (Gorbalenya et al., 1989), whereas the hordei-like TGBp1 has an additional N-terminal 'extension region' (Morozov & Solovyev, ...

Section: Discussionmentioning

confidence: 99%

Domain organization of the N-terminal portion of hordeivirus movement protein TGBp1

Makarov

Rybakova

Ефимов

et al. 2009

“…Unless otherwise stated, plasmids pPC1 and pPC2, corresponding to full-length cDNA clones of PCV RNA-1 and RNA-2 (Herzog et al, 1998), or Rep-15, a chimera containing 59-terminal cDNA sequence from RNA-2 and 39-terminal sequence from RNA-1 (Dunoyer et al, 2001), were used as starting material for mutant constructs. For some mutants, plasmid 2MAUGS (Herzog et al, 1995), a pPC2 derivative consisting of the first 2223 nt of RNA-2 and containing a novel NcoI site, introduced at the position of the CP initiation codon by PCR-mediated overlap extension mutagenesis (Ho et al, 1989), was employed in the construction.…”

Section: Methodsmentioning

confidence: 99%

“…Inoculation of Chenopodium quinoa with virion RNA (1?25 mg in 50 ml for each of two leaves) was as described (Herzog et al, 1998). Protoplasts (10 6 protoplasts in 0?5 ml) from BY-2 tobacco cells (Nagata et al, 1992) were prepared and inoculated with transcripts (Dunoyer et al, 2001).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Mapping of viral RNA sequences required for assembly of peanut clump virus particles

Hemmer

Dunoyer

Richards

et al. 2003

RNA sequences required for assembly into rod-shaped virions of RNA-1 and RNA-2 of Peanut clump virus (PCV) were mapped by testing the ability of different RNA-1 and -2 deletion mutants to be encapsidated in vivo in an RNase-resistant form. Encapsidation of RNA-1 was found to require a sequence domain in the 59-proximal part of the P15 gene, the 39-proximal gene of RNA-1. On the other hand, the subgenomic RNA which encodes P15 was not encapsidated, suggesting that other features of RNA-1 are important as well. Two sequences which could drive encapsidation of RNA-2 deletion mutants were located. One was in the 59-proximal coat protein gene and the other in the P14 gene near the RNA 39 terminus. There were no obvious sequence homologies between the different assembly initiation sequences.

“…In contrast to filamentous viruses translocated through plasmodesmata as virions or virion-like structures, the CP of rodshaped viruses is not required for viral cell-to-cell movement (Herzog et al, 1998;Petty & Jackson, 1990;Saito et al, 1990;Savenkov et al, 2003;Schmitt et al, 1992;Tamada et al, 1996;Ziegler-Graff et al, 1991). In these viruses, the transport form of the virus genome is assumed to be a non-virion RNP, as has been reported for well-studied TMV and BSMV (Brakke et al, 1988;Citovsky et al, 1990Citovsky et al, , 1992.…”

Section: Role Of Virions In Cell-to-cell Transport Of Filamentous Virmentioning

confidence: 97%

Helical capsids of plant viruses: architecture with structural lability

Solovyev

Makarov

2016

Capsids of numerous filamentous and rod-shaped plant viruses possess helical symmetry. In positive-stranded RNA viruses, helical capsids are typically composed of many identical subunits of the viral capsid protein (CP), encapsidating a molecule of viral genomic RNA. Current progress in structural studies of helical plant viruses has revealed differences between filamentous and rod-shaped viruses, both in structural folds of their CPs and in the interactions of CP molecules in their capsids. Many filamentous and rod-shaped viruses have functionally similar lateral inter-subunit contacts on the outer virion surface. Additionally, the extreme Nterminal CP region in filamentous viruses is intrinsically disordered. Taken together, the available data establish a link between the structural features of molecular interactions of CP molecules and the physical properties of helical virions ranging from rigidity to flexibility. Overall, the structure of helical plant viruses is significantly more labile than previously thought, often allowing structural transitions, remodelling and the existence of alternative structural forms of virions. These properties of virions are believed to be functionally significant at certain stages of the viral life cycle, such as during translational activation and cell-to-cell transport. In this review, we discuss structural and functional features of filamentous and rod-shaped virions, highlight their shared features and differences, and lay emphasis on the relationships between the molecular structure of viral capsids and their properties including virion shape, lability and capability of structural remodelling.