Fusamichi Akita

Light-induced structural changes and the site of O=O bond formation in PSII caught by XFEL

Photosystem II (PSII) is a huge membrane-protein complex consisting of 20 different subunits with a total molecular mass of 350 kDa for a monomer. It catalyses light-driven water oxidation at its catalytic centre, the oxygen-evolving complex (OEC). The structure of PSII has been analysed at 1.9 Å resolution by synchrotron radiation X-rays, which revealed that the OEC is a Mn4CaO5 cluster organized in an asymmetric, ‘distorted-chair’ form. This structure was further analysed with femtosecond X-ray free electron lasers (XFEL), providing the ‘radiation damage-free’ structure. The mechanism of O=O bond formation, however, remains obscure owing to the lack of intermediate-state structures. Here we describe the structural changes in PSII induced by two-flash illumination at room temperature at a resolution of 2.35 Å using time-resolved serial femtosecond crystallography with an XFEL provided by the SPring-8 ångström compact free-electron laser. An isomorphous difference Fourier map between the two-flash and dark-adapted states revealed two areas of apparent changes: around the QB/non-haem iron and the Mn4CaO5 cluster. The changes around the QB/non-haem iron region reflected the electron and proton transfers induced by the two-flash illumination. In the region around the OEC, a water molecule located 3.5 Å from the Mn4CaO5 cluster disappeared from the map upon two-flash illumination. This reduced the distance between another water molecule and the oxygen atom O4, suggesting that proton transfer also occurred. Importantly, the two-flash-minus-dark isomorphous difference Fourier map showed an apparent positive peak around O5, a unique μ4-oxo-bridge located in the quasi-centre of Mn1 and Mn4 (refs 4,5). This suggests the insertion of a new oxygen atom (O6) close to O5, providing an O=O distance of 1.5 Å between these two oxygen atoms. This provides a mechanism for the O=O bond formation consistent with that proposed previously.

Crystallographic Analysis Reveals Octamerization of Viroplasm Matrix Protein P9-1 of Rice Black Streaked Dwarf Virus

ABSTRACT The P9-1 protein of Rice black streaked dwarf virus accumulates in viroplasm inclusions, which are structures that appear to play an important role in viral morphogenesis and are commonly found in viruses in the family Reoviridae. Crystallographic analysis of P9-1 revealed structural features that allow the protein to form dimers via hydrophobic interactions. Each dimer has carboxy-terminal regions, resembling arms, that extend to neighboring dimers, thereby uniting sets of four dimers via lateral hydrophobic interactions, to yield cylindrical octamers. The importance of these regions for the formation of viroplasm-like inclusions was confirmed by the absence of such inclusions when P9-1 was expressed without its carboxy-terminal arm. The octamers are vertically elongated cylinders resembling the structures formed by NSP2 of rotavirus, even though there are no significant similarities between the respective primary and secondary structures of the two proteins. Our results suggest that an octameric structure with an internal pore might be important for the functioning of the respective proteins in the events that occur in the viroplasm, which might include viral morphogenesis.

Assembly of the viroplasm by viral non-structural protein Pns10 is essential for persistent infection of rice ragged stunt virus in its insect vector.

Rice ragged stunt virus (RRSV), an oryzavirus, is transmitted by brown planthopper in a persistent propagative manner. In this study, sequential infection of RRSV in the internal organs of its insect vector after ingestion of virus was investigated by immunofluorescence microscopy. RRSV was first detected in the epithelial cells of the midgut, from where it proceeded to the visceral muscles surrounding the midgut, then throughout the visceral muscles of the midgut and hindgut, and finally into the salivary glands. Viroplasms, the sites of virus replication and assembly of progeny virions, were formed in the midgut epithelium, visceral muscles and salivary glands of infected insects and contained the non-structural protein Pns10 of RRSV, which appeared to be the major constituent of the viroplasms. Viroplasm-like structures formed in non-host insect cells following expression of Pns10 in a baculovirus system, suggesting that the viroplasms observed in RRSV-infected cells were composed basically of Pns10. RNA interference induced by ingestion of dsRNA from the Pns10 gene of RRSV strongly inhibited such viroplasm formation, preventing efficient virus infection and spread in its insect vectors. These results show that Pns10 of RRSV is essential for viroplasm formation and virus replication in the vector insect.

Immunity to Rice black streaked dwarf virus, a plant reovirus, can be achieved in rice plants by RNA silencing against the gene for the viroplasm component protein.

The nonstructural protein P9-1 of Rice black streaked dwarf virus has been confirmed to accumulate in viroplasms, the putative sites of viral replication, in infected plants and insects. We transformed rice plants by introducing an RNA interference construct against the P9-1-encoding gene. The resultant transgenic plants accumulated short interfering RNAs specific to the construct. All progenies produced by self-fertilization of these transgenic plants with induced RNA interference against the gene for P9-1 were resistant to infection by the virus. Our results demonstrated that interfering with the expression of a viroplasm component protein of plant reoviruses, which plays an important role in viral proliferation, might be a practical and effective way to control plant reovirus infection in crop plants.

Rice Dwarf Viruses with Dysfunctional Genomes Generated in Plants Are Filtered Out in Vector Insects: Implications for the Origin of the Virus

ABSTRACT Rice dwarf virus (RDV), with 12 double-stranded RNA (dsRNA) genome segments (S1 to S12), replicates in and is transmitted by vector insects. The RDV-plant host-vector insect system allows us to examine the evolution, adaptation, and population genetics of a plant virus. We compared the effects of long-term maintenance of RDV on population structures in its two hosts. The maintenance of RDV in rice plants for several years resulted in gradual accumulation of nonsense mutations in S2 and S10, absence of expression of the encoded proteins, and complete loss of transmissibility. RDV maintained in cultured insect cells for 6 years retained an intact protein-encoding genome. Thus, the structural P2 protein encoded by S2 and the nonstructural Pns10 protein encoded by S10 of RDV are subject to different selective pressures in the two hosts, and mutations accumulating in the host plant are detrimental in vector insects. However, one round of propagation in insect cells or individuals purged the populations of RDV that had accumulated deleterious mutations in host plants, with exclusive survival of fully competent RDV. Our results suggest that during the course of evolution, an ancestral form of RDV, of insect virus origin, might have acquired the ability to replicate in a host plant, given its reproducible mutations in the host plant that abolish vector transmissibility and viability in nature.

Hairpin RNA derived from the gene for Pns9, a viroplasm matrix protein of Rice gall dwarf virus, confers strong resistance to virus infection in transgenic rice plants.

The nonstructural Pns9 protein of Rice gall dwarf virus (RGDV) accumulates in viroplasm inclusions, which are structures that appear to play an important role in viral morphogenesis and are commonly found in host cells infected by viruses in the family Reoviridae. An RNA interference construct was designed to target the gene for Pns9 of RGDV, namely Trigger_G9. The resultant transgenic plants accumulated short interfering RNAs specific for the construct. All progenies from self-fertilized transgenic plants had strong and heritable resistance to RGDV infection and did not allow the propagation of RGDV. By contrast, our transgenic plants remained susceptible to Rice dwarf virus, another phytoreovirus. There were no significant changes in the morphology of our transgenic plants compared with non-inoculated wild-type rice plants, suggesting that genes critical for the growth of rice plants were unaffected. Our results demonstrate that the resistance to RGDV of our transgenic rice plants is not due to resistance to the vector insects but to specific inhibition of RGDV replication and that the designed trigger sequence is functioning normally. Thus, our strategy to target a gene for viroplasm matrix protein should be applicable to plant viruses that belong to the family Reoviridae.

Viroplasm matrix protein Pns9 from rice gall dwarf virus forms an octameric cylindrical structure

The non-structural Pns9 protein of rice gall dwarf virus (RGDV) accumulates in viroplasm inclusions, which are structures that appear to play an important role in viral morphogenesis and are commonly found in host cells infected by viruses in the family Reoviridae. Immunofluorescence and immunoelectron microscopy of RGDV-infected vector cells in monolayers, using antibodies against Pns9 of RGDV and expression of Pns9 in Spodoptera frugiperda cells, demonstrated that Pns9 is the minimal viral factor necessary for formation of viroplasm inclusion during infection by RGDV. When Pns9 in solution was observed under a conventional electron microscope, it appeared as ring-like aggregates of approximately 100 Å in diameter. Cryo-electron microscopic analysis of these aggregates revealed cylinders of octameric Pns9, whose dimensions were similar to those observed under the conventional electron microscope. Octamerization of Pns9 in solution was confirmed by the results of size-exclusion chromatography. Among proteins of viruses that belong to the family Reoviridae whose three-dimensional structures are available, a matrix protein of the viroplasm of rotavirus, NSP2, forms similar octamers, an observation that suggests similar roles for Pns9 and NSP2 in morphogenesis in animal-infecting and in plant-infecting reoviruses.

The protruding domain of the coat protein of Melon necrotic spot virus is involved in compatibility with and transmission by the fungal vector Olpidium bornovanus

The Chi and W strains of Melon necrotic spot virus (MNSV) are efficiently transmitted by isolates Y1 and NW1, respectively, of the fungal vector Olpidium bornovanus. Analysis of chimeric viruses constructed by switching the coat protein (CP) gene between the two strains unveiled the involvement of the CP in the attachment of MNSV to zoospores of a compatible isolate of O. bornovanus and in the fungal transmission of the virus. Furthermore, analysis of the chimeric virus based on the Chi strain with the protruding domain of the CP from strain W suggested the involvement of the domain in compatibility with zoospore. Comparison of the three-dimensional structures between the CP of the two MNSV strains showed that many of the differences in these amino acid residues are present on the surface of the virus particles, suggesting that these affects the recognition of fungal vectors by the virus.

Optical identification of the long-wavelength (700-1700 nm) electronic excitations of the native reaction centre, Mn4CaO5 cluster and cytochromes of photosystem II in plants and cyanobacteria

Visible/UV absorption in PS II core complexes is dominated by the chl-a absorptions, which extend to ~700 nm. A broad 700-730 nm PS II core complex absorption in spinach has been assigned to a charge transfer excitation between ChlD1 and ChlD2. Emission from this state, which peaks at 780 nm, has been seen for both plant and cyanobacterial samples. We show that Thermosynechococcus vulcanus PS II core complexes have parallel absorbance in the 700-730 nm region and similar photochemical behaviour to that seen in spinach. This establishes the low energy charge transfer state as intrinsic to the native PS II reaction centre. High-sensitivity MCD measurements made in the 700-1700 nm region reveal additional electronic excitations at ~770 nm and ~1550 nm. The temperature and field dependence of MCD spectra establish that the system peaking near 1550 nm is a heme-to-Fe(III) charge transfer excitation. These transitions have not previously been observed for cyt b559 or cyt c550. The distinctive characteristics of the MCD signals seen at 770 nm allow us to assign absorption in this region to a dz(2)→d(x2-y2) transition of Mn(III) in the Ca-Mn4O5 cluster of the oxygen evolving centre. Current measurements were performed in the S1 state. Detailed analyses of this spectral region, especially in higher S states, promise to provide a new window on models of water oxidation.

Electron microscopic imaging revealed the flexible filamentous structure of the cell attachment protein P2 of Rice dwarf virus located around the icosahedral 5-fold axes

The minor outer capsid protein P2 of Rice dwarf virus (RDV), a member of the genus Phytoreovirus in the family Reoviridae, is essential for viral cell entry. Here, we clarified the structure of P2 and the interactions to host insect cells. Negative stain electron microscopy (EM) showed that P2 proteins are monomeric and flexible L-shaped filamentous structures of ∼20 nm in length. Cryo-EM structure revealed the spatial arrangement of P2 in the capsid, which was prescribed by the characteristic virion structure. The P2 proteins were visualized as partial rod-shaped structures of ∼10 nm in length in the cryo-EM map and accommodated in crevasses on the viral surface around icosahedral 5-fold axes with hydrophobic interactions. The remaining disordered region of P2 assumed to be extended to the radial direction towards exterior. Electron tomography clearly showed that RDV particles were away from the cellular membrane at a uniform distance and several spike-like densities, probably corresponding to P2, connecting a viral particle to the host cellular membrane during cell entry. By combining the in vitro and in vivo structural information, we could gain new insights into the detailed mechanism of the cell entry of RDV.