Biomedical subjects

A G Solovyev

Publications and source records attributed to A G Solovyev.

At least 19 recordsLinked to original sources

PubMed2005

Localization of Poa semilatent virus cysteine-rich protein in peroxisomes is dispensable for its ability to suppress RNA silencing.

Subcellular localization of the Poa semilatent virus cysteine-rich gammab protein was studied by using different approaches. In infected tissue, gammab was detected mainly in the P30 fraction as monomers, dimers and oligomers. Green fluorescent protein-fused gammab was found to localize in punctate bodies in the cytoplasm. Colocalization with marker proteins demonstrated that these bodies represent peroxisomes. Immunoelectron microscopy revealed that gammab was localized in the peroxisomal matrix and that localization of gammab in peroxisomes required the C-terminal signal tripeptide SKL. An SKL-deletion mutant exhibited a diffuse localization, but retained the protein's ability to suppress RNA silencing, determine infection phenotype and support virus systemic spread. These data indicate that gammab functions are not associated with the protein's localization to peroxisomes.

Cysteine↗

PubMed2005

The hydrophobic segment of Potato virus X TGBp3 is a major determinant of the protein intracellular trafficking.

Potato virus X (PVX) encodes three movement proteins, TGBp1, TGBp2 and TGBp3. The 8 kDa TGBp3 is a membrane-embedded protein that has an N-terminal hydrophobic sequence segment and a hydrophilic C terminus. TGBp3 mutants with deletions in the C-terminal hydrophilic region retain the ability to be targeted to cell peripheral structures and to support limited PVX cell-to-cell movement, suggesting that the basic TGBp3 functions are associated with its N-terminal transmembrane region. Fusion of green fluorescent protein to the TGBp3 N terminus abrogates protein activities in intracellular trafficking and virus movement. The intracellular transport of TGBp3 from sites of its synthesis in the rough endoplasmic reticulum (ER) to ER-derived peripheral bodies involves a non-conventional COPII-independent pathway. However, integrity of the C-terminal hydrophilic sequence is required for entrance to this non-canonical route.

Biological Transport↗

PubMed2005

Expression, localization and effects on virulence of the cysteine-rich 8 kDa protein of Potato mop-top virus.

Potato mop-top virus (PMTV) RNA3 contains a triple gene block (TGB) encoding viral movement proteins and an open reading frame for a putative 8 kDa cysteine-rich protein (CRP). In this study, PMTV CRP was shown to be expressed in the course of virus infection, and a PMTV CRP-specific subgenomic RNA was mapped. CRP has previously been shown to be dispensable for infection of PMTV in Nicotiana benthamiana. In this study, PMTV CRP was found to increase the severity of disease symptoms when expressed from Potato virus X or Tobacco mosaic virus in N. benthamiana and Nicotiana tabacum, suggesting that the protein affects virulence of the virus or might suppress a host defence mechanism. However, PMTV CRP did not show RNA silencing suppression activity in three assays. Host responses to the PMTV CRP expression from different viral genomes ranged from an absence of response to extreme resistance at a single cell level and were dependent on the viral genome. These findings emphasized involvement of viral proteins and/or virus-induced cell components in the plant reaction to CRP. PMTV CRP was predicted to possess a transmembrane segment. CRP fused to the green fluorescent protein was associated with endoplasmic reticulum-derived membranes and induced dramatic rearrangements of the endoplasmic reticulum structure, which might account for protein functions as a virulence factor of the virus.

Cysteine↗

PubMed2003

Immunodetection and fluorescent microscopy of transgenically expressed hordeivirus TGBp3 movement protein reveals its association with endoplasmic reticulum elements in close proximity to plasmodesmata.

The subcellular localization of the hydrophobic TGBp3 protein of Poa semilatent virus (PSLV, genus Hordeivirus) was studied in transgenic plants using fluorescent microscopy to detect green fluorescent protein (GFP)-tagged protein and immunodetection with monoclonal antibodies (mAbs) raised against the GFP-based fusion expressed in E. coli. In Western blot analysis, mAbs efficiently recognized the wild-type and GFP-fused PSLV TGBp3 proteins expressed in transgenic Nicotiana benthamiana, but failed to detect TGBp3 in hordeivirus-infected plants. It was found that PSLV TGBp3 and GFP-TGBp3 had a tendency to form large protein complexes of an unknown nature. Fractionation studies revealed that TGBp3 represented an integral membrane protein and probably co-localized with an endoplasmic reticulum-derived domain. Microscopy of epidermal cells in transgenic plants demonstrated that GFP-TGBp3 localized to cell wall-associated punctate bodies, which often formed pairs of opposing discrete structures that co-localized with callose, indicating their association with the plasmodesmata-enriched cell wall fields. After mannitol-induced plasmolysis of the leaf epidermal cells in the transgenic plants, TGBp3 appeared within the cytoplasm and not at cell walls. Although TGBp3-induced bodies were normally static, most of them became motile after plasmolysis and displayed stochastic motion in the cytoplasm.

Blotting, Western↗

PubMed2002

RNA helicase activity of the plant virus movement proteins encoded by the first gene of the triple gene block.

Cell-to-cell and long-distance transport of some plant viruses requires coordinated action of three movement proteins encoded by triple gene block (TGB). The largest of TGB proteins, TGBp1, is a member of the superfamily I of DNA/RNA helicases and possesses a set of conserved helicase sequence motifs necessary for virus movement. A recombinant His-tagged form of TGBp1 of two hordeiviruses and potato virus X, a potexvirus, produced in Escherichia coli had unwinding activity on a partially duplexed RNA, but not DNA substrate. The helicase activity of these proteins was dependent on Mg2+ and ATP. The isolated C-terminal half of the PSLV TGBp1 retaining all helicase motifs was also able to unwind RNA duplex.

Adenosine Triphosphatases↗

PubMed2002

Dual-colour imaging of membrane protein targeting directed by poa semilatent virus movement protein TGBp3 in plant and mammalian cells.

The movement function of poa semilatent hordeivirus (PSLV) is mediated by the triple gene block (TGB) proteins, of which two, TGBp2 and TGBp3, are membrane proteins. TGBp3 is localized to peripheral bodies in the vicinity of the plasma membrane and is able to re-direct TGBp2 from the endoplasmic reticulum (ER) to the peripheral bodies. For imaging of TGBp3-mediated protein targeting, PSLV TGBp3 tagged with a red fluorescent protein (DsRed) was used. Coexpression of DsRed-TGBp3 with GFP targeted to the ER lumen (ER-GFP) demonstrated that ER-GFP was contained in typical ER structures and peripheral bodies formed by TGBp3 protein, suggesting an ER origin for these bodies. In transient coexpression with viral membrane proteins tagged with GFP, DsRed-TGBp3 directed to the peripheral bodies the homologous TGBp2 protein and two unrelated membrane proteins, the 6 kDa movement protein of beet yellows closterovirus and the putative movement protein encoded by the genome component 4 of faba bean necrotic yellows nanovirus. However, coexpression of TGBp3 with GFP derivatives targeted to the ER membranes by artificial hydrophobic tail sequences suggested that targeting to the ER membranes per se was not sufficient for TGBp3-directed protein trafficking to peripheral bodies. TGBp3-induced targeting of TGBp2 also occurred in mammalian cells, indicating the universal nature of the protein trafficking signals and the cotargeting mechanism.

Animals↗

PubMed2001

RNA-binding properties of the 63 kDa protein encoded by the triple gene block of poa semilatent hordeivirus.

The 63 kDa '63K' movement protein encoded by the triple gene block of poa semilatent virus (PSLV) comprises the C-terminal NTPase/helicase domain and the N-terminal extension domain, which contains two positively charged sequence motifs, A and B. In this study, the in vitro RNA-binding properties of PSLV 63K and its mutants were analysed. Membrane-immobilized 63K and N-63K (isolated N-terminal extension domain) bound RNA at high NaCl concentrations. In contrast, C-63K (isolated NTPase/helicase domain) was able to bind RNA only at NaCl concentrations of up to 50 mM. In gel-shift assays, C-63K bound RNA to form complexes that were unable to enter an agarose gel, whereas complexes formed by N-63K could enter the gel. Full-length 63K formed both types of complexes. Visualization of the RNA-protein complexes formed by 63K, N-63K and C-63K by atomic force microscopy demonstrated that each complex had a different shape. Collectively, these data indicate that 63K has two distinct RNA-binding activities associated with the NTPase/helicase domain and the N-terminal extension domain. Mutations in either of the positively charged sequence motifs A and B had little effect on the RNA binding of the N-terminal extension domain, whereas mutations in both motifs together inhibited RNA binding. Hybrid viruses with mutations in motifs A and B were able to infect inoculated leaves of Nicotiana benthamiana plants, but were unable to move systemically to uninoculated leaves, suggesting that the RNA-binding activity of the N-terminal extension domain of PSLV 63K is associated with virus long-distance movement.

Amino Acid Sequence↗

PubMed2001

Cell-to-cell movement of potato virus X involves distinct functions of the coat protein.

Complementation of movement-deficient potato virus X (PVX) coat protein (CP) mutants, namely PVX.CP-Xho lacking the 18 C-terminal amino acid residues and PVX.DeltaCP lacking the entire CP gene, was studied by transient co-expression with heterologous proteins. These data demonstrated that the potyvirus CPs and both the major and minor CPs of beet yellows closterovirus could complement cell-to-cell movement of PVX.CP-Xho but not PVX.DeltaCP. These data also indicated that the C-terminally truncated PVX CP lacked a movement function which could be provided in trans by the CPs of other filamentous viruses, whereas another movement determinant specified by some region outside the most C-terminal part of the PVX CP could not be complemented either by potyvirus or closterovirus CPs. Surprisingly, the CP of spherical cocksfoot mottle sobemovirus rescued all of the PVX CP movement functions, complementing the spread of PVX.CP-Xho and, to a lesser extent, PVX.DeltaCP. Both these mutants were also rescued by the tobacco mosaic virus (TMV) movement protein (MP). To shed light on the movement function of PVX CP, attempts were made to complement PVX.CP-Xho by a series of TMV MP mutants. An internal deletion abolished complementation, suggesting that the internal region of TMV MP, which includes a number of overlapping functional domains important for cell-to-cell transport, provides an activity complementing movement determinant(s) specified by the C-terminal region of PVX CP.

Biological Transport↗

PubMed2000

Complementation of the movement-deficient mutations in potato virus X: potyvirus coat protein mediates cell-to-cell trafficking of C-terminal truncation but not deletion mutant of potexvirus coat protein.

The cell-to-cell movement of the GUS-tagged potato virus X (PVX) coat protein (CP) movement-deficient mutant was restored by potyviral CPs of potato virus A (PVA) and potato virus Y (PVY) in Nicotiana benthamiana leaves in transient cobombardment experiments. Viral cell-to-cell movement of PVX CP mutant was complemented in Nicotiana tabacum cv. SR1 transgenic plants expressing PVY CP: PVX RNA and polymerase were detected in the PVX CP mutant-inoculated leaves of transgenic plants. These findings demonstrated the ability of the PVX CP-deficient mutant to move from cell to cell but not long distances in the transgenic plants and suggest that CPs of potex- and potyviruses display complementary activities in the movement process. Potyviral CP alone is not able to carry out these activities, since the mutated PVX CP is indispensable for restored movement. No trans-encapsidation between potyviral CP and PVX RNA was observed. Therefore, potyviral CP facilitates the PVX CP mutant movement by the mechanism that cannot be explained by coat protein substitution. Our data also suggest that CP functioning in cell-to-cell movement is not restricted to a simple passive role in forming virions.

Biolistics↗

PubMed2000

Subcellular sorting of small membrane-associated triple gene block proteins: TGBp3-assisted targeting of TGBp2.

We studied subcellular distribution of green fluorescent protein (GFP)-tagged movement proteins encoded by the second and the third genes of poa semilatent hordeivirus (PSLV) triple gene block (TGB), 15K TGBp2 and 18K TGBp3. GFP-15K transiently expressed in Nicotiana benthamiana leaf epidermal cells was associated with the endomembrane system elements. GFP-18K appeared in the membrane bodies at cell periphery. Mutation analysis demonstrated that subcellular targeting of GFP-15K depended on the protein transmembrane segment(s), whereas the TGBp3 central hydrophilic region was responsible for targeting of GFP-18K. Coexpression of GFP-15K with the intact 18K protein induced drastic changes in the TGBp2 localization: GFP-15K appeared in the cell peripheral bodies similar to those in the cells expressing GFP-18K alone. Coexpression experiments with mutant forms of both proteins argue against involvement of direct interaction between small TGB proteins in the TGBp3-assisted targeting of TGBp2 to the cell peripheral compartments. This conclusion was further confirmed by similar effects on the PSLV 15K TGBp2 localization induced by TGBp3 proteins of PSLV and potato virus X, which have no detectable sequence similarity to each other.

Amino Acid Sequence↗

PubMed1999

Evidence for two nonoverlapping functional domains in the potato virus X 25K movement protein.

To study subdomain organization of the potato virus X (PVX) movement protein (MP) encoded by the first gene in the triple gene block (TGB), we mutated the 25-kDa TGBp1 protein. The N-terminal deletion of the helicase motifs I, IA, and II resulted in loss of the ATPase activity and RNA binding. A frameshift mutation truncating the C-terminal motifs V and VI gave rise to increase of the TGBp1 ATPase activity and had little effect on RNA binding in vitro. Fusions of the green fluorescent protein with 25-kDa MP and its derivative lacking motifs V-VI exhibited similar fluorescence patterns in epidermal cells of Nicotiana benthamiana leaves. Cell-to-cell movement of the 25K-deficient PVX genome was not complemented by the TGBp1 of Plantago asiatica mosaic potexvirus (PlAMV) but was efficiently complemented by a chimeric TGBp1 consisting of the N-terminal part of PlAMV protein (motifs I-IV) and the PVX-specific C-terminal part (motifs V-VI). These results suggest that NTP hydrolysis, RNA binding, and targeting to the specific cellular compartment(s) are associated with the N-terminal domain of the TGBp1 including the helicase motifs I-IV and that the C-terminal domain is involved in specific interactions with other virus proteins.

Adenosine Triphosphatases↗

PubMed1999

Identification and study of tobacco mosaic virus movement function by complementation tests.

The phenomenon of trans-complementation of cell-to-cell movement between plant positive-strand RNA viruses is discussed with an emphasis on tobamoviruses. Attention is focused on complementation between tobamoviruses (coding for a single movement protein, MP) and two groups of viruses that contain the triple block of MP genes and require four (potato virus X) or three (barley stripe mosaic virus) proteins for cell-to-cell movement. The highlights of complementation data obtained by different experimental approaches are given, including (i) double infections with movement-deficient (dependent) and helper viruses; (ii) infections with recombinant viral genomes bearing a heterologous MP gene; (iii) complementation of a movement-deficient virus in transgenic plants expressing the MP of a helper virus; and (iv) co-bombardment of plant tissues with the cDNAs of a movement-dependent virus genome and the MP gene of a helper virus.

Biological Transport↗

PubMed1999

Movement of hordeivirus hybrids with exchanges in the triple gene block.

The barley stripe mosaic virus (BSMV) triple gene block (TGB) coding for movement proteins (MPs) was replaced with the respective TGB genes from two other hordeiviruses, poa semilatent virus (PSLV) or lychnis ringspot virus (LRSV). The BSMV/LRSV recombinant did not exhibit infectivity on the plants tested, whereas the infection rate and host range of the BSMV/PSLV hybrid were similar to those of BSMV. In particular, the BSMV/PSLV hybrid infected Nicotiana benthamiana, a nonhost plant for PSLV, indicating a contribution of non-MP elements of BSMV genome to host specificity of virus transport. Assuming that the PSLV TGB was functional in the BSMV genome context, a further series of recombinants was constructed, in which smaller portions of the BSMV TGB were replaced by the corresponding PSLV sequences. Examination of the infectivity of the hybrid viruses suggested that the TGB-coded proteins could interact in a host-dependent manner to mediate cell-to-cell movement. Analysis of recombinants with hybrid sequences of the first gene in the TGB (beta b gene) indicated that (i) sequence-independent binding of beta b to viral RNAs could occur during formation of beta b-RNA complexes in vivo, and that (ii) the beta b MP is involved in virus long-distance movement, for which homologous N- and C-terminal beta b domains are required.

Adenosine Triphosphatases↗

PubMed1998

Genome sequences of poa semilatent and lychnis ringspot hordeiviruses.

The nucleotide sequences of the genomic RNAs of two hordeiviruses, poa semilatent virus (PSLV) and lychnis ringspot virus (LRSV), were determined. The genome organization of both viruses is similar to that of the type hordeivirus, barley stripe mosaic virus (BSMV). Comparisons of the amino acid sequences of the hordeivirus replicase components revealed a high level of sequence similarity to those of soil-borne wheat mosaic virus (SBWMV), peanut clump virus (PCV), and Indian peanut clump virus. The cysteine-rich gamma b proteins of hordeiviruses are similar to those of SBWMV, PCV, and tobraviruses. Analysis of cis-acting elements in the genomes of the three hordeiviruses revealed conservation of putative core elements of subgenomic RNA promoters. Complex patterns of conservation were also found in the 5'-untranslated regions of hordeivirus genomic RNAs alpha, beta, and gamma.

Amino Acid Sequence↗

PubMed1997

Properties of the coat protein of a new tobacco mosaic virus coat protein ts-mutant.

Amino acid substitutions in a majority of tobacco mosaic virus (TMV) coat protein (CP) ts-mutants have previously been mapped to the same region of the CP molecule tertiary structure, located at a distance of about 70 A from TMV virion axis. In the present work some properties of a new TMV CP ts-mutant ts21-66 (two substitutions I21=>T and D66=>G, both in the 70-A region) were studied. Thermal inactivation characteristics, sedimentation properties, circular dichroism spectra, and modification by a lysine-specific reagent, trinitrobenzensulfonic acid, of ts21-66 CP were compared with those of wild-type (U1) TMV CP. It is concluded that the 70-A region represents the most labile portion of the TMV CP molecule. Partial disordering of this region in the mutant CP at permissive temperatures leads to loss of the capacity to form two-layer aggregates of the cylindrical type, while further disordering induced by mild heating results also in the loss of the ability to form ordered helical aggregates.

Capsid↗

PubMed1997

Host-controlled cell-to-cell movement of a hybrid barley stripe mosaic virus expressing a dianthovirus movement protein.

The triple gene block (TGB) of barley stripe mosaic virus (BSMV), coding for viral movement proteins (MPs), was replaced by the single MP gene of red clover necrotic mosaic virus (RCNMV). Accumulation of the hybrid virus in barley plants (the selective host for BSMV) was reduced compared to BSMV. The hybrid virus induced small necrotic local lesions on Chenopodium amaranticolor leaves and did not infect Nicotiana clevelandii (the selective host for RCNMV). The hybrid virus accumulated in the inoculated leaves of Nicotiana benthamiana, but not in the upper noninoculated leaves. Thus the RCNMV MP gene substituted for the BSMV TGB in cell-to-cell movement, but not in systemic spread. Hybrid virus movement was efficient only in N. benthamiana, the common host for BSMV and RCNMV. These data point to the involvement of host-specific factors in the function of virus-coded transport determinants.

Fabaceae↗

PubMed1996

Comparisons of the genomic cis-elements and coding regions in RNA beta components of the hordeiviruses barley stripe mosaic virus, lychnis ringspot virus, and poa semilatent virus.

Nucleotide sequences of the genomic RNA beta components of hordeiviruses poa semilatent virus (PSLV) and lychnis ringspot virus (LRSV) were determined. PSLV and LRSV closely resemble barley stripe mosaic virus (BSMV), type hordeivirus, in the gene arrangement of their RNAs beta, comprising 5'-proximal beta a (coat protein) gene and downstream triple gene block (TGB) coding for the beta b, beta c, and beta d putative transport proteins. The beta a, beta b, beta c, and beta d proteins of the three hordeiviruses showed significant sequence similarity, with the respective proteins of PSLV and BSMV being closer to each other than to their counterparts of LSRV. Comparisons of the TGB-encoded proteins of hordeiviruses, potexviruses, carlaviruses, and furoviruses indicate that the first and second TGB genes belong to the monophyletic groups, whereas the third gene may have multiple ancestry. LRSV, PSLV, and BSMV showed remarkable variation in the 3'-untranslated regions of their genomic RNAs. Among the three hordeiviruses, LRSV has the shortest 3'-noncoding region that lacks tentative pseudoknot-forming elements conserved upstream of the 3'-tRNA-like structure in the BSMV and PSLV genomes. On the other hand, LRSV RNA beta, like that of BSMV, contained the internal poly(A) sequence that is absent from PSLV RNA.

Adaptation, Physiological↗

PubMed1996

Movement of a barley stripe mosaic virus chimera with a tobacco mosaic virus movement protein.

The tobacco mosaic virus (TMV) 30K movement protein (MP) gene was inserted into a full-length cDNA clone of barley stripe mosaic virus (BSMV) RNA beta replacing the triple gene block (TGB). The resulting recombinant ND-MPT genome, consisting of infectious wt transcripts of BSMV RNAs alpha and gamma, together with the hybrid RNA beta transcript, was inoculated onto test plants to study the functional compatibility between the BSMV TGB-adapted genetic system and the tobamovirus transport gene. ND-MPT infected the inoculated leaves of Nicotiana benthamiana and Chenopodium amaranticolor, which are common hosts for the parental viruses; the size, growth rate, and morphology of local lesions on C. amaranticolor were influenced by the foreign MP gene. However, the hybrid virus failed to infect barley, N. tabacum (var. Samsun), and N. clevelandii, the selective hosts. Thus, the TMV MP was able to functionally substitute for the BSMV TGB-coded MPs, i.e., the 30K MP functioned independently of any other BSMV sequences. However, the TMV MP gene promoted the cell-to-cell movement in a host-dependent manner.

Amino Acid Sequence↗