Limits...
The Tomato yellow leaf curl virus V2 protein forms aggregates depending on the cytoskeleton integrity and binds viral genomic DNA.

Moshe A, Belausov E, Niehl A, Heinlein M, Czosnek H, Gorovits R - Sci Rep (2015)

Bottom Line: Similar to CP, V2 localized to cytoplasmic aggregates of increasing size and as infection progressed was also found in nuclei, where it co-localized with CP.V2 was associated with viral genomic DNA molecules, suggesting that V2 functions as a DNA shuttling protein.We propose that the cytoskeleton-dependent formation and growth of V2 aggregates play an important role during TYLCV infection, and that microtubules and actin filaments are important for the delivery of V2 to the 26S proteasome.

View Article: PubMed Central - PubMed

Affiliation: Institute of Plant Sciences and Genetics in Agriculture and the Otto Warburg Minerva Center for Agricultural Biotechnology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel.

ABSTRACT
The spread of Tomato yellow leaf curl virus (TYLCV) was accompanied by the formation of coat protein (CP) aggregates of increasing size in the cytoplasm and nucleus of infected tomato (Solanum lycopersicum) cells. In order to better understand the TYLCV-host interaction, we investigated the properties and the subcellular accumulation pattern of the non-structural viral protein V2. CP and V2 are the only sense-oriented genes on the virus circular single-stranded DNA genome. Similar to CP, V2 localized to cytoplasmic aggregates of increasing size and as infection progressed was also found in nuclei, where it co-localized with CP. V2 was associated with viral genomic DNA molecules, suggesting that V2 functions as a DNA shuttling protein. The formation and the 26S proteasome-mediated degradation of V2 aggregates were dependent on the integrity of the actin and microtubule cytoskeleton. We propose that the cytoskeleton-dependent formation and growth of V2 aggregates play an important role during TYLCV infection, and that microtubules and actin filaments are important for the delivery of V2 to the 26S proteasome.

No MeSH data available.


Related in: MedlinePlus

V2 forms complexes with TYLCV genomic DNA. Detection of TYLCV DNA-V2 complexes in vitro (a) and in planta (b). a: PCR detection of viral DNA following immuno-capture with V2 antibody of mixtures of V2 and infected plant DNA; V2/DNA: V2 incubated with untreated DNA, V2/DNA + S1: V2 incubated with DNA treated with S1 nuclease, V2/-DNA: V2 incubated without DNA. M - 100 bp DNA marker. b: Southern blot analysis of viral DNA-V2 complexes from infected tomato plants; DNA: DNA from infected plants, V2/DNA: DNA extracted from immunoprecipitated V2 complexes, V2/DNA + S1: DNA extracted from immunoprecipitated V2 complexes and treated with S1 nuclease.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4419519&req=5

f3: V2 forms complexes with TYLCV genomic DNA. Detection of TYLCV DNA-V2 complexes in vitro (a) and in planta (b). a: PCR detection of viral DNA following immuno-capture with V2 antibody of mixtures of V2 and infected plant DNA; V2/DNA: V2 incubated with untreated DNA, V2/DNA + S1: V2 incubated with DNA treated with S1 nuclease, V2/-DNA: V2 incubated without DNA. M - 100 bp DNA marker. b: Southern blot analysis of viral DNA-V2 complexes from infected tomato plants; DNA: DNA from infected plants, V2/DNA: DNA extracted from immunoprecipitated V2 complexes, V2/DNA + S1: DNA extracted from immunoprecipitated V2 complexes and treated with S1 nuclease.

Mentions: Binding of the V2 protein to TYLCV DNA was demonstrated by IC-PCR, a method previously used to detect TYLCV CP-DNA complexes29. In in vitro tests, V2 over-expressed in E. coli was incubated with DNA extracted from infected plants in PCR tubes coated with anti-V2 antibody. TYLCV DNA bound to V2 was detected by PCR. When the DNA extract was treated with S1 nuclease prior to incubation with V2 (to eliminate the TYLCV genomic ssDNA, but not the dsDNA), the PCR amplicon was not obtained (Fig. 3a). This result suggested that V2 binds TYLCV ssDNA. V2-TYLCV DNA complexes in extracts of infected tomato plants were identified by immuno-precipitation with the anti-V2 antibody followed by Southern blot hybridization (Fig. 3b). When compared with viral DNA (both ss and ds) from infected tomato, it was clear that the viral DNA that immuno-precipitated with V2, was essentially ssDNA; the hybridization signal disappeared after S1 nuclease digestion (Fig. 3b). This result suggested that V2 can make complexes with viral genomic ssDNA in planta, but not with its dsDNA replicating form.


The Tomato yellow leaf curl virus V2 protein forms aggregates depending on the cytoskeleton integrity and binds viral genomic DNA.

Moshe A, Belausov E, Niehl A, Heinlein M, Czosnek H, Gorovits R - Sci Rep (2015)

V2 forms complexes with TYLCV genomic DNA. Detection of TYLCV DNA-V2 complexes in vitro (a) and in planta (b). a: PCR detection of viral DNA following immuno-capture with V2 antibody of mixtures of V2 and infected plant DNA; V2/DNA: V2 incubated with untreated DNA, V2/DNA + S1: V2 incubated with DNA treated with S1 nuclease, V2/-DNA: V2 incubated without DNA. M - 100 bp DNA marker. b: Southern blot analysis of viral DNA-V2 complexes from infected tomato plants; DNA: DNA from infected plants, V2/DNA: DNA extracted from immunoprecipitated V2 complexes, V2/DNA + S1: DNA extracted from immunoprecipitated V2 complexes and treated with S1 nuclease.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4419519&req=5

f3: V2 forms complexes with TYLCV genomic DNA. Detection of TYLCV DNA-V2 complexes in vitro (a) and in planta (b). a: PCR detection of viral DNA following immuno-capture with V2 antibody of mixtures of V2 and infected plant DNA; V2/DNA: V2 incubated with untreated DNA, V2/DNA + S1: V2 incubated with DNA treated with S1 nuclease, V2/-DNA: V2 incubated without DNA. M - 100 bp DNA marker. b: Southern blot analysis of viral DNA-V2 complexes from infected tomato plants; DNA: DNA from infected plants, V2/DNA: DNA extracted from immunoprecipitated V2 complexes, V2/DNA + S1: DNA extracted from immunoprecipitated V2 complexes and treated with S1 nuclease.
Mentions: Binding of the V2 protein to TYLCV DNA was demonstrated by IC-PCR, a method previously used to detect TYLCV CP-DNA complexes29. In in vitro tests, V2 over-expressed in E. coli was incubated with DNA extracted from infected plants in PCR tubes coated with anti-V2 antibody. TYLCV DNA bound to V2 was detected by PCR. When the DNA extract was treated with S1 nuclease prior to incubation with V2 (to eliminate the TYLCV genomic ssDNA, but not the dsDNA), the PCR amplicon was not obtained (Fig. 3a). This result suggested that V2 binds TYLCV ssDNA. V2-TYLCV DNA complexes in extracts of infected tomato plants were identified by immuno-precipitation with the anti-V2 antibody followed by Southern blot hybridization (Fig. 3b). When compared with viral DNA (both ss and ds) from infected tomato, it was clear that the viral DNA that immuno-precipitated with V2, was essentially ssDNA; the hybridization signal disappeared after S1 nuclease digestion (Fig. 3b). This result suggested that V2 can make complexes with viral genomic ssDNA in planta, but not with its dsDNA replicating form.

Bottom Line: Similar to CP, V2 localized to cytoplasmic aggregates of increasing size and as infection progressed was also found in nuclei, where it co-localized with CP.V2 was associated with viral genomic DNA molecules, suggesting that V2 functions as a DNA shuttling protein.We propose that the cytoskeleton-dependent formation and growth of V2 aggregates play an important role during TYLCV infection, and that microtubules and actin filaments are important for the delivery of V2 to the 26S proteasome.

View Article: PubMed Central - PubMed

Affiliation: Institute of Plant Sciences and Genetics in Agriculture and the Otto Warburg Minerva Center for Agricultural Biotechnology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel.

ABSTRACT
The spread of Tomato yellow leaf curl virus (TYLCV) was accompanied by the formation of coat protein (CP) aggregates of increasing size in the cytoplasm and nucleus of infected tomato (Solanum lycopersicum) cells. In order to better understand the TYLCV-host interaction, we investigated the properties and the subcellular accumulation pattern of the non-structural viral protein V2. CP and V2 are the only sense-oriented genes on the virus circular single-stranded DNA genome. Similar to CP, V2 localized to cytoplasmic aggregates of increasing size and as infection progressed was also found in nuclei, where it co-localized with CP. V2 was associated with viral genomic DNA molecules, suggesting that V2 functions as a DNA shuttling protein. The formation and the 26S proteasome-mediated degradation of V2 aggregates were dependent on the integrity of the actin and microtubule cytoskeleton. We propose that the cytoskeleton-dependent formation and growth of V2 aggregates play an important role during TYLCV infection, and that microtubules and actin filaments are important for the delivery of V2 to the 26S proteasome.

No MeSH data available.


Related in: MedlinePlus