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 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 An integrated protein localization and interaction map for Potato yellow dwarf virus, type species of the genus Nucleorhabdovirus

Virology ◽  
2010 ◽  
Vol 402 (1) ◽  
pp. 61-71 ◽  
Author(s):  
Anindya Bandyopadhyay ◽  
Kristin Kopperud ◽  
Gavin Anderson ◽  
Kathleen Martin ◽  
Michael Goodin
Keyword(s):  
Virus Type ◽  
Type Species ◽  
Protein Localization ◽  
Yellow Dwarf ◽  
Dwarf Virus ◽  
Interaction Map ◽  
Yellow Dwarf Virus

Live-cell visualization of transmembrane protein oligomerization and membrane fusion using two-fragment haptoEGFP methodology

2012 ◽  
Vol 32 (3) ◽  
pp. 333-343 ◽  
Author(s):  
Derek J. Quinn ◽  
Neil V. McFerran ◽  
John Nelson ◽  
W. Paul Duprex
Keyword(s):  
Protein Interactions ◽  
Mammalian Cells ◽  
Measles Virus ◽  
Fluorescent Protein ◽  
Transmembrane Protein ◽  
Bimolecular Fluorescence Complementation ◽  
Type I ◽  
Protein Oligomerization ◽  
Fluorescent Signal ◽  
Enveloped Virus

Protein interactions play key roles throughout all subcellular compartments. In the present paper, we report the visualization of protein interactions throughout living mammalian cells using two oligomerizing MV (measles virus) transmembrane glycoproteins, the H (haemagglutinin) and the F (fusion) glycoproteins, which mediate MV entry into permissive cells. BiFC (bimolecular fluorescence complementation) has been used to examine the dimerization of these viral glycoproteins. The H glycoprotein is a type II membrane-receptor-binding homodimeric glycoprotein and the F glycoprotein is a type I disulfide-linked membrane glycoprotein which homotrimerizes. Together they co-operate to allow the enveloped virus to enter a cell by fusing the viral and cellular membranes. We generated a pair of chimaeric H glycoproteins linked to complementary fragments of EGFP (enhanced green fluorescent protein) – haptoEGFPs – which, on association, generate fluorescence. Homodimerization of H glycoproteins specifically drives this association, leading to the generation of a fluorescent signal in the ER (endoplasmic reticulum), the Golgi and at the plasma membrane. Similarly, the generation of a pair of corresponding F glycoprotein–haptoEGFP chimaeras also produced a comparable fluorescent signal. Co-expression of H and F glycoprotein chimaeras linked to complementary haptoEGFPs led to the formation of fluorescent fusion complexes at the cell surface which retained their biological activity as evidenced by cell-to-cell fusion.

scholarly journals Tula hantavirus L protein is a 250 kDa perinuclear membrane-associated protein

2004 ◽  
Vol 85 (5) ◽  
pp. 1181-1189 ◽  
Author(s):  
Sami K. J. Kukkonen ◽  
Antti Vaheri ◽  
Alexander Plyusnin
Keyword(s):  
Matrix Protein ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Expression System ◽  
Peak Level ◽  
Cell Fractionation ◽  
L Protein ◽  
System L ◽  
Infected Cells ◽  
In Cells

The complete open reading frame of Tula hantavirus (TULV) L RNA was cloned in three parts. The middle third (nt 2191–4344) could be expressed in E. coli and was used to immunize rabbits. The resultant antiserum was then used to immunoblot concentrated TULV and infected Vero E6 cells. The L protein of a hantavirus was detected, for the first time, in infected cells and was found to be expressed as a single protein with an apparent molecular mass of 250 kDa in both virions and infected cells. Using the antiserum, the expression level of the L protein was followed and image analysis of immunoblots indicated that there were 104 copies per cell at the peak level of expression. The antiserum was also used to detect the L protein in cell fractionation studies. In cells infected with TULV and cells expressing recombinant L, the protein pelleted with the microsomal membrane fraction. The membrane association was confirmed with membrane flotation assays. To visualize L protein localization in cells, a fusion protein of L and enhanced green fluorescent protein, L–EGFP, was expressed in Vero E6 cells with a plasmid-driven T7 expression system. L–EGFP localized in the perinuclear region where it had partial co-localization with the Golgi matrix protein GM130 and the TULV nucleocapsid protein.

scholarly journals pFPL Vectors for High-Throughput Protein Localization in Fungi: Detecting Cytoplasmic Accumulation of Putative Effector Proteins

2015 ◽  
Vol 28 (2) ◽  
pp. 107-121 ◽  
Author(s):  
Xiaoyan Gong ◽  
Oscar Hurtado ◽  
Baohua Wang ◽  
Congqing Wu ◽  
Mihwa Yi ◽  
...  
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Protein Localization ◽  
Yellow Fluorescent Protein ◽  
Effector Proteins ◽  
Fungal Transformation ◽  
In Planta ◽  
Green Fluorescent

As part of a large-scale project whose goal was to identify candidate effector proteins in Magnaporthe oryzae, we developed a suite of vectors that facilitate high-throughput protein localization experiments in fungi. These vectors utilize Gateway recombinational cloning to place a gene's promoter and coding sequences upstream and in frame with enhanced cyan fluorescent protein, green fluorescent protein (GFP), monomeric red fluorescence protein (mRFP), and yellow fluorescent protein or a nucleus-targeted mCHERRY variant. The respective Gateway cassettes were incorporated into Agrobacterium-based plasmids to allow efficient fungal transformation using hygromycin or geneticin resistance selection. mRFP proved to be more sensitive than the GFP spectral variants for monitoring proteins secreted in planta; and extensive testing showed that Gateway-derived fusion proteins produced localization patterns identical to their “directly fused” counterparts. Use of plasmid for fungal protein localization (pFPL) vectors with two different selectable markers provided a convenient way to label fungal cells with different fluorescent proteins. We demonstrate the utility of the pFPL vectors for identifying candidate effector proteins and we highlight a number of important factors that must be taken into consideration when screening for proteins that are translocated across the host plasma membrane.

scholarly journals Strategies to improve the fluorescent signal of the tripartite sfGFP system

2020 ◽  
Vol 52 (9) ◽  
pp. 998-1006
Author(s):  
Jing Shen ◽  
Wenlu Zhang ◽  
Chunyang Gan ◽  
Xiafei Wei ◽  
Jie Li ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Bimolecular Fluorescence Complementation ◽  
Fluorescence Signal ◽  
Recovery Efficiency ◽  
Good Recovery ◽  
Protein Protein Interactions ◽  
Bifc Assay ◽  
Green Fluorescent

Abstract Bimolecular fluorescence complementation (BiFC) is a popular method used to detect protein–protein interactions. For a BiFC assay, a fluorescent protein is usually split into two parts, and the fluorescence is recovered upon the interaction between the fused proteins of interest. As an elegant extension of BiFC, a tripartite superfold green fluorescent protein (sfGFP) system that has the advantages of low background fluorescence and small fusion tag size has been developed. However, the tripartite system exhibits a low fluorescence signal in some cases. To address this problem, we proposed to increase the affinity between the two parts, G1–9 and G11, of the tripartite system by adding affinity pairs. Among the three affinity pairs tested, LgBiT-HiBiT improved both the signal and signal-to-noise (S/N) ratio to the greatest extent. More strikingly, the direct covalent fusion of G11 to G1–9, which converted the tripartite system into a new bipartite system, enhanced the S/N ratio from 20 to 146, which is superior to the bipartite sfGFP system split at 157/158 or 173/174. Our results implied that the 10th β-strand of sfGFP has a low affinity and a good recovery efficiency to construct a robust BiFC system, and this concept might be applied to other fluorescent proteins with similar structure to construct new BiFC systems.

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