Post-embedding in situ hybridization for localization of viral nucleic acid in ultra-thin sections.

Lin, Na-Sheng; Chen, Chin-Chieh; Hsu, Yau‐Heiu

doi:10.1177/41.10.8245409

Cited by 31 publications

(20 citation statements)

References 12 publications

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“…The ORF2 protein inclusions might be sites where processing reactions required for virus transport take place. This hypothesis is supported by the fact that BaMV RNA was detected within EDCBs by in situ hybridization using a BaMV genomic RNA 3h-end-specific probe (Lin et al, 1993). RNA-binding activity Triple-gene-block of BaMV Triple-gene-block of BaMV has been observed for the insoluble aggregates of cauliflower mosaic caulimovirus P1 protein (Thomas & Maule, 1995) and the 66 kDa cytoplasmic inclusion of tamarillo mosaic potyvirus, which also possesses NTPase and RNA-helicase activities (Eagles et al, 1994).…”

supporting

confidence: 52%

Subcellular localization of the 28 kDa protein of the triple-gene-block of bamboo mosaic potexvirus.

Chang

Lin

Liou

et al. 1997

Journal of General Virology

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Open reading frame 2 of the bamboo mosaic potexvirus (BaMV) genome encodes a 28 kDa protein, the first of the ' triple-gene-block ' of BaMV which is believed to play a role in cell-to-cell movement of the virus in host plants. The 28 kDa protein was expressed in Escherichia coli and polyclonal antiserum was raised in a rabbit. Western blot analyses showed that the 28 kDa protein was associated mainly with components in the cell wall and 30 000 g pellet fractions of a BaMV-infected leaf homogenate. Immunogold electron microscopy of infected leaf tissues revealed that the 28 kDa protein was associated with electron-dense crystalline bodies (EDCBs) in the cytoplasm and nuclei. Nuclear EDCBs were found closely associated with nucleoli. Gold-labelled EDCB-like structures were also detected in the cytoplasm, but not within nuclei, in protoplasts up to 48 h post-inoculation. No specific labelling of the 28 kDa protein was found within any cytoplasmic structures or within cell walls.

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supporting

confidence: 52%

Subcellular localization of the 28 kDa protein of the triple-gene-block of bamboo mosaic potexvirus.

Chang

Lin

Liou

et al. 1997

Journal of General Virology

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“…Plasmid pJP1 containing the cDNA of ORF2 (Chang et al, 1997) served as template for construction of the ORF2 derivatives by PCR. Plasmids pBaHB (Lin et al, 1993), pJP1 (Chang et al, 1997), pCT14 and pWC1 (B. Y. Chang, unpublished data), which contain cDNA fragments of the whole genome, ORF2, ORF3 and ORF4 of BaMV, respectively, were linearized and used as templates for in vitro transcription.…”

Section: Methodsmentioning

confidence: 99%

Identification of the RNA-binding sites of the triple gene block protein 1 of bamboo mosaic potexvirus.

Wung

Hsu

Liou

et al. 1999

Journal of General Virology

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The triple gene block protein 1 (TGBp1) encoded by open reading frame 2 of bamboo mosaic potexvirus (BaMV) was overexpressed in Escherichia coli and purified in order to test its RNAbinding activity. UV crosslinking assays revealed that the RNA-binding activity was present mainly in the soluble fraction of the refolded TGBp1. The binding activity was nonspecific and salt concentration-dependent : activity was present at 0-50 mM NaCl but was almost abolished at 200 mM. The RNA-binding domain was located by deletion mutagenesis to the N-terminal 3-24 amino acids of TGBp1. Sequence alignment analysis of the N-terminal 25 amino acids of the TGBp1 homologues of potexviruses identified three arginine residues. Arg-to-Ala substitution at any one of the three arginines eliminated most of the RNA-binding activity, indicating that they were all critical to the RNA-binding activity of the TGBp1 of BaMV.

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“…Interestingly, the TGB1 proteins of other potexviruses also form aggregates (Rouleau et al, 1994;Chang et al, 1997) that associate with viral RNA (Lin et al, 1993), and many potexviruses produce protein aggregates surrounded by accumulating virions (Zettler et al, 1968;Diaz-Ruiz and Feldman, 1990;Lin et al, 1993;Rouleau et al, 1994;Chang et al, 1997), indicating a possible conserved regulatory mechanism.…”

Section: The X-body Is Not Required For Virus Accumulation and Encapsmentioning

confidence: 99%

The TGB1 Movement Protein ofPotato virus XReorganizes Actin and Endomembranes into the X-Body, a Viral Replication Factory

et al. 2012

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Potato virus X (PVX) requires three virally encoded proteins, the triple gene block (TGB), for movement between cells. TGB1 is a multifunctional protein that suppresses host gene silencing and moves from cell to cell through plasmodesmata, while TGB2 and TGB3 are membrane-spanning proteins associated with endoplasmic reticulum-derived granular vesicles. Here, we show that TGB1 organizes the PVX “X-body,” a virally induced inclusion structure, by remodeling host actin and endomembranes (endoplasmic reticulum and Golgi). Within the X-body, TGB1 forms helically arranged aggregates surrounded by a reservoir of the recruited host endomembranes. The TGB2/3 proteins reside in granular vesicles within this reservoir, in the same region as nonencapsidated viral RNA, while encapsidated virions accumulate at the outer (cytoplasmic) face of the X-body, which comprises a highly organized virus “factory.” TGB1 is both necessary and sufficient to remodel host actin and endomembranes and to recruit TGB2/3 to the X-body, thus emerging as the central orchestrator of the X-body. Our results indicate that the actin/endomembrane-reorganizing properties of TGB1 function to compartmentalize the viral gene products of PVX infection.

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Post-embedding in situ hybridization for localization of viral nucleic acid in ultra-thin sections.

Cited by 31 publications

References 12 publications

Subcellular localization of the 28 kDa protein of the triple-gene-block of bamboo mosaic potexvirus.

Subcellular localization of the 28 kDa protein of the triple-gene-block of bamboo mosaic potexvirus.

Identification of the RNA-binding sites of the triple gene block protein 1 of bamboo mosaic potexvirus.

The TGB1 Movement Protein ofPotato virus XReorganizes Actin and Endomembranes into the X-Body, a Viral Replication Factory

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