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Development of reverse genetics for Ibaraki virus to produce viable VP6-tagged IBAV.

Matsuo E, Saeki K, Roy P, Kawano J - FEBS Open Bio (2015)

Bottom Line: Moreover, several tags were inserted into the truncated region to produce VP6-tagged IBAV.Further, tagged-VP6 proteins were first assembled into puncta in cells infected with VP6-tagged IBAV.Our data suggests that, in order to initiate primary replication, IBAV VP6 is likely to accumulate in some parts of infected cells to assemble efficiently into the primary replication complex (subcore).

View Article: PubMed Central - PubMed

Affiliation: Microbiology & Immunology, Division of Animal Science, Department of Bioresource Science, Graduate School of Agricultural Science, Kobe University, 1-1, Rokkodai, Nada-ku, Kobe-city 657-8501, Japan.

ABSTRACT
Ibaraki virus (IBAV) is a member of the epizootic hemorrhagic disease virus (EHDV) serogroup, which belongs to the Orbivirus genus of the Reoviridae family. Although EHDV, including IBAV, represents an ongoing threat to livestock in the world, molecular mechanisms of EHDV replication and pathogenesis have been unclear. The reverse genetics (RG) system is one of the strong tools to understand molecular mechanisms of virus replication. Here, we developed a RG system for IBAV to identify the nonessential region of a minor structural protein, VP6, by generating VP6-truncated IBAV. Moreover, several tags were inserted into the truncated region to produce VP6-tagged IBAV. We demonstrated that all VP6-tagged IBAV could replicate in BHK cells in the absence of any helper VP6 protein. Further, tagged-VP6 proteins were first assembled into puncta in cells infected with VP6-tagged IBAV. Our data suggests that, in order to initiate primary replication, IBAV VP6 is likely to accumulate in some parts of infected cells to assemble efficiently into the primary replication complex (subcore).

No MeSH data available.


Related in: MedlinePlus

Translocation of tagged VP6 in the infected cells. (A) Flag-tagged VP6 was detected using anti-rabbit DDDDK antibody at 2, 7, 24, and 36 h post-infection with IBAV d1Flag at MOI of 0.5. Nuclei were stained with DAPI. Arrowheads indicate puncta formation of VP6. (B) Magnified images of BHK21A11 cells infected with either IBAVd1Flag (left) or IBAVd1HA (right) at 7 h post-infection. Nuclei were stained with DAPI. Arrowheads indicate puncta formation of VP6. Fluorescent images were merged with bright field images.
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f0035: Translocation of tagged VP6 in the infected cells. (A) Flag-tagged VP6 was detected using anti-rabbit DDDDK antibody at 2, 7, 24, and 36 h post-infection with IBAV d1Flag at MOI of 0.5. Nuclei were stained with DAPI. Arrowheads indicate puncta formation of VP6. (B) Magnified images of BHK21A11 cells infected with either IBAVd1Flag (left) or IBAVd1HA (right) at 7 h post-infection. Nuclei were stained with DAPI. Arrowheads indicate puncta formation of VP6. Fluorescent images were merged with bright field images.

Mentions: Interestingly, TC-tagged IBAV formed puncta in infected cells at 18 h post-infection (Fig. 6B). To further confirm if VP6 forms puncta in early infection, BHK21A11 cells were infected with VP6-tagged IBAV and at 2, 7, 24 and 36 h post-infection (Fig. 7). At 7 h post-infection, Flag-tagged VP6 already assembled into puncta, while the proteins were detectable throughout the cytosol at 24 h and 36 h post-infection (Fig. 7A). HA-tagged VP6 also assembled into puncta at 7 h post-infection in BHK21A11 cells (Fig. 7B right panel). The same results were obtained using BSR cells (data not shown). As orbivirus VP6 is likely to be important for assembly of the primary replicase complex [37,40], these data suggest that VP6 may be necessary to be concentrated in cytosol of the infected cells in order to initiate primary replication efficiently.


Development of reverse genetics for Ibaraki virus to produce viable VP6-tagged IBAV.

Matsuo E, Saeki K, Roy P, Kawano J - FEBS Open Bio (2015)

Translocation of tagged VP6 in the infected cells. (A) Flag-tagged VP6 was detected using anti-rabbit DDDDK antibody at 2, 7, 24, and 36 h post-infection with IBAV d1Flag at MOI of 0.5. Nuclei were stained with DAPI. Arrowheads indicate puncta formation of VP6. (B) Magnified images of BHK21A11 cells infected with either IBAVd1Flag (left) or IBAVd1HA (right) at 7 h post-infection. Nuclei were stained with DAPI. Arrowheads indicate puncta formation of VP6. Fluorescent images were merged with bright field images.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4472822&req=5

f0035: Translocation of tagged VP6 in the infected cells. (A) Flag-tagged VP6 was detected using anti-rabbit DDDDK antibody at 2, 7, 24, and 36 h post-infection with IBAV d1Flag at MOI of 0.5. Nuclei were stained with DAPI. Arrowheads indicate puncta formation of VP6. (B) Magnified images of BHK21A11 cells infected with either IBAVd1Flag (left) or IBAVd1HA (right) at 7 h post-infection. Nuclei were stained with DAPI. Arrowheads indicate puncta formation of VP6. Fluorescent images were merged with bright field images.
Mentions: Interestingly, TC-tagged IBAV formed puncta in infected cells at 18 h post-infection (Fig. 6B). To further confirm if VP6 forms puncta in early infection, BHK21A11 cells were infected with VP6-tagged IBAV and at 2, 7, 24 and 36 h post-infection (Fig. 7). At 7 h post-infection, Flag-tagged VP6 already assembled into puncta, while the proteins were detectable throughout the cytosol at 24 h and 36 h post-infection (Fig. 7A). HA-tagged VP6 also assembled into puncta at 7 h post-infection in BHK21A11 cells (Fig. 7B right panel). The same results were obtained using BSR cells (data not shown). As orbivirus VP6 is likely to be important for assembly of the primary replicase complex [37,40], these data suggest that VP6 may be necessary to be concentrated in cytosol of the infected cells in order to initiate primary replication efficiently.

Bottom Line: Moreover, several tags were inserted into the truncated region to produce VP6-tagged IBAV.Further, tagged-VP6 proteins were first assembled into puncta in cells infected with VP6-tagged IBAV.Our data suggests that, in order to initiate primary replication, IBAV VP6 is likely to accumulate in some parts of infected cells to assemble efficiently into the primary replication complex (subcore).

View Article: PubMed Central - PubMed

Affiliation: Microbiology & Immunology, Division of Animal Science, Department of Bioresource Science, Graduate School of Agricultural Science, Kobe University, 1-1, Rokkodai, Nada-ku, Kobe-city 657-8501, Japan.

ABSTRACT
Ibaraki virus (IBAV) is a member of the epizootic hemorrhagic disease virus (EHDV) serogroup, which belongs to the Orbivirus genus of the Reoviridae family. Although EHDV, including IBAV, represents an ongoing threat to livestock in the world, molecular mechanisms of EHDV replication and pathogenesis have been unclear. The reverse genetics (RG) system is one of the strong tools to understand molecular mechanisms of virus replication. Here, we developed a RG system for IBAV to identify the nonessential region of a minor structural protein, VP6, by generating VP6-truncated IBAV. Moreover, several tags were inserted into the truncated region to produce VP6-tagged IBAV. We demonstrated that all VP6-tagged IBAV could replicate in BHK cells in the absence of any helper VP6 protein. Further, tagged-VP6 proteins were first assembled into puncta in cells infected with VP6-tagged IBAV. Our data suggests that, in order to initiate primary replication, IBAV VP6 is likely to accumulate in some parts of infected cells to assemble efficiently into the primary replication complex (subcore).

No MeSH data available.


Related in: MedlinePlus