scholarly journals Complete Genome Sequence and In Planta Subcellular Localization of Maize Fine Streak Virus Proteins

2005 ◽  
Vol 79 (9) ◽  
pp. 5304-5314 ◽  
Author(s):  
Chi-Wei Tsai ◽  
Margaret G. Redinbaugh ◽  
Kristen J. Willie ◽  
Sharon Reed ◽  
Michael Goodin ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Protein Interactions ◽  
Matrix Protein ◽  
Fluorescent Protein ◽  
Streak Virus ◽  
In Planta ◽  
Nuclear Localization Signals ◽  
N Protein ◽  
P Protein ◽  
Maize Fine Streak Virus

ABSTRACT The genome of the nucleorhabdovirus maize fine streak virus (MFSV) consists of 13,782 nucleotides of nonsegmented, negative-sense, single-stranded RNA. The antigenomic strand consisted of seven open reading frames (ORFs), and transcripts of all ORFs were detected in infected plants. ORF1, ORF6, and ORF7 had significant similarities to the nucleocapsid protein (N), glycoprotein (G), and polymerase (L) genes of other rhabdoviruses, respectively, whereas the ORF2, ORF3, ORF4, and ORF5 proteins had no significant similarities. The N (ORF1), ORF4, and ORF5 proteins localized to nuclei, consistent with the presence of nuclear localization signals (NLSs) in these proteins. ORF5 likely encodes the matrix protein (M), based on its size, the position of its NLS, and the localization of fluorescent protein fusions to the nucleus. ORF2 probably encodes the phosphoprotein (P) because, like the P protein of Sonchus yellow net virus (SYNV), it was spread throughout the cell when expressed alone but was relocalized to a subnuclear locus when coexpressed with the MFSV N protein. Unexpectedly, coexpression of the MFSV N and P proteins, but not the orthologous proteins of SYNV, resulted in accumulations of both proteins in the nucleolus. The N and P protein relocalization was specific to cognate proteins of each virus. The subcellular localizations of the MFSV ORF3 and ORF4 proteins were distinct from that of the SYNV sc4 protein, suggesting different functions. To our knowledge, this is the first comparative study of the cellular localizations of plant rhabdoviral proteins. This study indicated that plant rhabdoviruses are diverse in genome sequence and viral protein interactions.

scholarly journals Lettuce necrotic yellows cytorhabdovirus protein localization and interaction map, and comparison with nucleorhabdoviruses

2012 ◽  
Vol 93 (4) ◽  
pp. 906-914 ◽  
Author(s):  
Kathleen M. Martin ◽  
Ralf G. Dietzgen ◽  
Renyuan Wang ◽  
Michael M. Goodin
Keyword(s):  
Protein Interactions ◽  
Matrix Protein ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Bimolecular Fluorescence Complementation ◽  
Cell Periphery ◽  
In Planta ◽  
Interaction Map ◽  
Yellow Dwarf Virus ◽  
P Proteins

Lettuce necrotic yellows virus (LNYV), Sonchus yellow net virus (SYNV) and Potato yellow dwarf virus (PYDV) are members of the family Rhabdoviridae that infect plants. LNYV is a cytorhabdovirus that replicates in the cytoplasm, while SYNV and PYDV are nucleorhabdoviruses that replicate in the nuclei of infected cells. LNYV and SYNV share a similar genome organization with a gene order of nucleoprotein (N), phosphoprotein (P), putative movement protein (Mv), matrix protein (M), glycoprotein (G) and polymerase (L). PYDV contains an additional predicted gene of unknown function located between N and P. In order to gain insight into the associations of viral structural and non-structural proteins and the mechanisms by which they may function, we constructed protein localization and interaction maps. Subcellular localization was determined by transiently expressing the viral proteins fused to green or red fluorescent protein in leaf epidermal cells of Nicotiana benthamiana. Protein interactions were tested in planta by using bimolecular fluorescence complementation. All three viruses showed Mv to be localized at the cell periphery and the G protein to be membrane associated. Comparing the interaction maps revealed that only the N–P and M–M interactions are common to all three viruses. Associations unique to only one virus include P–M for LNYV, G–Mv for SYNV and M–Mv, M–G and N–M for PYDV. The cognate N–P proteins of all three viruses interacted and exhibited characteristic changes in localization when co-expressed.

scholarly journals Novel Phosphoprotein-Interacting Region in Nipah Virus Nucleocapsid Protein and Its Involvement in Viral Replication

2010 ◽  
Vol 84 (19) ◽  
pp. 9793-9799 ◽  
Author(s):  
Mio Omi-Furutani ◽  
Misako Yoneda ◽  
Kentaro Fujita ◽  
Fusako Ikeda ◽  
Chieko Kai
Keyword(s):  
Amino Acid ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Nipah Virus ◽  
Live Cells ◽  
N Protein ◽  
P Protein ◽  
P Proteins ◽  
Fluorescent Protein Tags ◽  
Protein Tags

ABSTRACT The interaction of Nipah virus (NiV) nucleocapsid (N) protein with phosphoprotein (P) during nucleocapsid assembly is the essential process in the viral life cycle, since only the encapsidated RNA genome can be used for replication. To identify the region responsible for N-P interaction, we utilized fluorescent protein tags to visualize NiV N and P proteins in live cells and analyzed their cellular localization. N protein fused to monomeric enhanced cyan fluorescence protein (N-ECFP) exhibited a dotted pattern in transfected cells, while P protein fused to monomeric red fluorescent protein (P-mRFP) showed diffuse distribution. When the two proteins were coexpressed, P-mRFP colocalized with N-ECFP dots. N-ECFP mutants with serial amino acid deletions were generated to search for the region(s) responsible for this N-P colocalization. We found that, in addition to the 467- to 496-amino-acid (aa) region reported previously, aa 135 to 146 were responsible for the N-P colocalization. The residues crucial for N-P interaction were further investigated by introducing alanine substitutions into the untagged N protein. Alanine scanning in the region of aa 135 to 146 has revealed that there are distinct regions essential for the interaction of N-P and the function of N. This is the first study to visualize Nipah viral proteins in live cells and to assess the essential domain of N protein for the interaction with P protein.

scholarly journals Role of the Sonchus Yellow Net Virus N Protein in Formation of Nuclear Viroplasms

2007 ◽  
Vol 81 (10) ◽  
pp. 5362-5374 ◽  
Author(s):  
Min Deng ◽  
Jennifer N. Bragg ◽  
Steven Ruzin ◽  
Denise Schichnes ◽  
David King ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Protein Import ◽  
Protein Targeting ◽  
Affinity Purification ◽  
N Protein ◽  
Helix Loop Helix ◽  
P Protein ◽  
Importin Α ◽  
P Proteins

ABSTRACT Sonchus yellow net virus is a plant nucleorhabdovirus whose nucleocapsid (N), phosphoprotein (P), and polymerase (L) proteins form large viroplasms in the nuclei of infected plants (C. R. F. Martins, J. A. Johnson, D. M. Lawrence, T. J. Choi, A. Pisi, S. L. Tobin, D. Lapidus, J. D. O. Wagner, S. Ruzin, K. McDonald, and A. O. Jackson, J. Virol. 72:5669-5679, 1998). When expressed alone, the N protein localizes to the nuclei of plant and yeast (Saccharomyces cerevisiae) cells and the P protein is distributed throughout the cells, but coexpression of N and P results in formation of subnuclear viroplasm-like foci (M. M. Goodin, J. Austin, R. Tobias, M. Fujita, C. Morales, and A. O. Jackson, J. Virol. 75:9393-9406, 2001; M. M. Goodin, R. G. Dietzgen, D. Schichnes, S. Ruzin, and A. O. Jackson, Plant J. 31:375-383, 2002). We now show that the N protein and various fluorescent derivatives form similar subnuclear foci in plant cells and that homologous interactions mediated by a helix-loop-helix region near the amino terminus are required for formation of the foci. Mutations within the helix-loop-helix region also interfere with N- and P-protein interactions that are required for N and P colocalization in the subnuclear foci. Affinity purification of N proteins harboring single mutations within the motif revealed that Tyr40 is critical for N-N and N-P interactions. Additional in vitro binding assays also indicated that the N protein binds to yeast and plant importin α homologues, whereas mutations in the carboxy-terminal nuclear localization signal abrogate importin α binding. The P protein did not bind to the importin α homologues, suggesting that the N and P proteins use different pathways for nuclear entry. Our results in toto support a model suggesting that during infection, the N and P proteins enter the nucleus independently, that viroplasm formation requires homologous N-protein interactions, and that P protein targeting to the viroplasm requires N-P protein interactions that occur after N and P protein import into the nucleus.

scholarly journals Identification of the domains of the influenza A virus M1 matrix protein required for NP binding, oligomerization and incorporation into virions

2007 ◽  
Vol 88 (8) ◽  
pp. 2280-2290 ◽  
Author(s):  
Sarah L. Noton ◽  
Elizabeth Medcalf ◽  
Dawn Fisher ◽  
Anne E. Mullin ◽  
Debra Elton ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Protein Interactions ◽  
Influenza A ◽  
Matrix Protein ◽  
Fluorescent Protein ◽  
Viral Envelope ◽  
Major Protein ◽  
Middle Domain ◽  
Self Association

The matrix (M1) protein of influenza A virus is a multifunctional protein that plays essential structural and functional roles in the virus life cycle. It drives virus budding and is the major protein component of the virion, where it forms an intermediate layer between the viral envelope and integral membrane proteins and the genomic ribonucleoproteins (RNPs). It also helps to control the intracellular trafficking of RNPs. These roles are mediated primarily via protein–protein interactions with viral and possibly cellular proteins. Here, the regions of M1 involved in binding the viral RNPs and in mediating homo-oligomerization are identified. In vitro, by using recombinant proteins, it was found that the middle domain of M1 was responsible for binding NP and that this interaction did not require RNA. Similarly, only M1 polypeptides containing the middle domain were able to bind to RNP–M1 complexes isolated from purified virus. When M1 self-association was examined, all three domains of the protein participated in homo-oligomerization although, again, the middle domain was dominant and self-associated efficiently in the absence of the N- and C-terminal domains. However, when the individual fragments of M1 were tagged with green fluorescent protein and expressed in virus-infected cells, microscopy of filamentous particles showed that only full-length M1 was incorporated into budding virions. It is concluded that the middle domain of M1 is primarily responsible for binding NP and self-association, but that additional interactions are required for efficient incorporation of M1 into virus particles.

scholarly journals Biosensors Used for Epifluorescence and Confocal Laser Scanning Microscopies to Study Dickeya and Pectobacterium Virulence and Biocontrol

2021 ◽  
Vol 9 (2) ◽  
pp. 295
Author(s):  
Yvann Bourigault ◽  
Andrea Chane ◽  
Corinne Barbey ◽  
Sylwia Jafra ◽  
Robert Czajkowski ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Fluorescent Protein ◽  
Infected Plant ◽  
Soft Rot ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Cell Physiology ◽  
In Planta ◽  
Reporter System ◽  
Cell Wall Lysis

Promoter-probe vectors carrying fluorescent protein-reporter genes are powerful tools used to study microbial ecology, epidemiology, and etiology. In addition, they provide direct visual evidence of molecular interactions related to cell physiology and metabolism. Knowledge and advances carried out thanks to the construction of soft-rot Pectobacteriaceae biosensors, often inoculated in potato Solanum tuberosum, are discussed in this review. Under epifluorescence and confocal laser scanning microscopies, Dickeya and Pectobacterium-tagged strains managed to monitor in situ bacterial viability, microcolony and biofilm formation, and colonization of infected plant organs, as well as disease symptoms, such as cell-wall lysis and their suppression by biocontrol antagonists. The use of dual-colored reporters encoding the first fluorophore expressed from a constitutive promoter as a cell tag, while a second was used as a regulator-based reporter system, was also used to simultaneously visualize bacterial spread and activity. This revealed the chronology of events leading to tuber maceration and quorum-sensing communication, in addition to the disruption of the latter by biocontrol agents. The promising potential of these fluorescent biosensors should make it possible to apprehend other activities, such as subcellular localization of key proteins involved in bacterial virulence in planta, in the near future.

scholarly journals Targeted disruption of PKC from AKAP Signaling Complexes

2021 ◽  
Author(s):  
Ameya J. Limaye ◽  
George N. Bendzunas ◽  
Eileen Kennedy
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Signaling Pathways ◽  
Protein Interactions ◽  
Protein Protein Interactions ◽  
Targeted Disruption ◽  
Physiological Processes ◽  
Kinase C ◽  
P Protein

Protein Kinase C (PKC) is a member of the AGC subfamily of kinases and regulates a wide array of signaling pathways and physiological processes. Protein-protein interactions involving PKC and its...

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