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Baculovirus LEF-11 Hijack Host ATPase ATAD3A to Promote Virus Multiplication in Bombyx mori cells

View Article: PubMed Central - PubMed

ABSTRACT

Research on molecular mechanisms that viruses use to regulate the host apparatus is important in virus infection control and antiviral therapy exploration. Our previous research showed that the Bombyx mori nucleopolyhedrovirus (BmNPV) LEF-11 localized to dense regions of the cell nucleus and is required for viral DNA replication. Herein, we examined the mechanism of LEF-11 on BmNPV multiplication and demonstrated that baculovirus LEF-11 interacts with Bombyx mori ATAD3A and HSPD1 (HSP60) protein. Furthermore, we showed that LEF-11 has the ability to induce and up-regulate the expression of ATAD3A and HSPD1, phenomena that were both reversed upon knockdown of lef-11. Our findings showed that ATAD3A and HSPD1 were necessary and contributed to BmNPV multiplication in Bombyx mori cells. Moreover, ATAD3A was found to directly interact with HSPD1. Interestingly, ATAD3A was required for the expression of HSPD1, while the knockdown of HSPD1 had no obvious effect on the expression level of ATAD3A. Taken together, the data presented in the current study demonstrated that baculovirus LEF-11 hijacks the host ATPase family members, ATAD3A and HSPD1, efficiently promote the multiplication of the virus. This study furthers our understanding of how baculovirus modulates energy metabolism of the host and provides a new insight into the molecular mechanisms of antiviral research.

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Identification of LEF-11-associated proteins by Co-IP and mass spectrometry.(A) Co-IP assays of LEF-11-associated protein analyzed by SDS-PAGE. Marker, protein molecular weight marker; Input, input cell lysates; IgG, IP with control mouse IgG; α-cMYC, IP with anti-cMYC antibody. The specific bands represented by the arrows. IP No.1 and IP No.2 is representative of two repeated experiments. (B) Co-immunoprecipitation of LEF-11 examined by Western blotting. BmN-SWU1 cells were co-transfected with LEF-11 and candidate protein. At 48 hours after transfection, cells were lysed and immunoprecipitation performed with α-FLAG/HA, and the bound of target protein using α-HA/FLAG to detected. The label on the top of each panel shows the antibodies used for immunoprecipitation. The labels on the right of each panel show the antibodies used for analysis of Western blotting. The apparent molecular size of each band is shown on the left of each panel.
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f1: Identification of LEF-11-associated proteins by Co-IP and mass spectrometry.(A) Co-IP assays of LEF-11-associated protein analyzed by SDS-PAGE. Marker, protein molecular weight marker; Input, input cell lysates; IgG, IP with control mouse IgG; α-cMYC, IP with anti-cMYC antibody. The specific bands represented by the arrows. IP No.1 and IP No.2 is representative of two repeated experiments. (B) Co-immunoprecipitation of LEF-11 examined by Western blotting. BmN-SWU1 cells were co-transfected with LEF-11 and candidate protein. At 48 hours after transfection, cells were lysed and immunoprecipitation performed with α-FLAG/HA, and the bound of target protein using α-HA/FLAG to detected. The label on the top of each panel shows the antibodies used for immunoprecipitation. The labels on the right of each panel show the antibodies used for analysis of Western blotting. The apparent molecular size of each band is shown on the left of each panel.

Mentions: To analyze the regulatory mechanism of LEF-11 on viral multiplication, immunoprecipitation assays were performed to identify the binding partners of LEF-11. BmN-SWU1 cells were infected with vBmlef11cMYC and IP was performed using α-cMYC or mouse IgG antibody. The results showed that protein samples immunoprecipitated with α-cMYC had obvious differences in bands compared with IgG control. These proteins of 3 differential bands were located at 100 kDa, 60–70 kDa and 45–50 kDa, respectively (Fig. 1A). Protein bands were excised and subjected to digestion, and then analysis followed by tandem mass spectrometry (MS/MS). A total of 8 related proteins were screened by protein peptides and molecular mass analysis. These results showed that 5 candidate proteins with the corresponding sizes were identified in Bombyx mori and only 3 candidate proteins were identified from BmNPV by bioinformatics analysis. The candidate proteins include Bombyx mori ATAD3A, HSPD1, PP2A, Actin, PP5 and BmNPV LEF-8, LEF-3, and Chitinase protein (see Table 1 for specific information on all candidate proteins).


Baculovirus LEF-11 Hijack Host ATPase ATAD3A to Promote Virus Multiplication in Bombyx mori cells
Identification of LEF-11-associated proteins by Co-IP and mass spectrometry.(A) Co-IP assays of LEF-11-associated protein analyzed by SDS-PAGE. Marker, protein molecular weight marker; Input, input cell lysates; IgG, IP with control mouse IgG; α-cMYC, IP with anti-cMYC antibody. The specific bands represented by the arrows. IP No.1 and IP No.2 is representative of two repeated experiments. (B) Co-immunoprecipitation of LEF-11 examined by Western blotting. BmN-SWU1 cells were co-transfected with LEF-11 and candidate protein. At 48 hours after transfection, cells were lysed and immunoprecipitation performed with α-FLAG/HA, and the bound of target protein using α-HA/FLAG to detected. The label on the top of each panel shows the antibodies used for immunoprecipitation. The labels on the right of each panel show the antibodies used for analysis of Western blotting. The apparent molecular size of each band is shown on the left of each panel.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5385504&req=5

f1: Identification of LEF-11-associated proteins by Co-IP and mass spectrometry.(A) Co-IP assays of LEF-11-associated protein analyzed by SDS-PAGE. Marker, protein molecular weight marker; Input, input cell lysates; IgG, IP with control mouse IgG; α-cMYC, IP with anti-cMYC antibody. The specific bands represented by the arrows. IP No.1 and IP No.2 is representative of two repeated experiments. (B) Co-immunoprecipitation of LEF-11 examined by Western blotting. BmN-SWU1 cells were co-transfected with LEF-11 and candidate protein. At 48 hours after transfection, cells were lysed and immunoprecipitation performed with α-FLAG/HA, and the bound of target protein using α-HA/FLAG to detected. The label on the top of each panel shows the antibodies used for immunoprecipitation. The labels on the right of each panel show the antibodies used for analysis of Western blotting. The apparent molecular size of each band is shown on the left of each panel.
Mentions: To analyze the regulatory mechanism of LEF-11 on viral multiplication, immunoprecipitation assays were performed to identify the binding partners of LEF-11. BmN-SWU1 cells were infected with vBmlef11cMYC and IP was performed using α-cMYC or mouse IgG antibody. The results showed that protein samples immunoprecipitated with α-cMYC had obvious differences in bands compared with IgG control. These proteins of 3 differential bands were located at 100 kDa, 60–70 kDa and 45–50 kDa, respectively (Fig. 1A). Protein bands were excised and subjected to digestion, and then analysis followed by tandem mass spectrometry (MS/MS). A total of 8 related proteins were screened by protein peptides and molecular mass analysis. These results showed that 5 candidate proteins with the corresponding sizes were identified in Bombyx mori and only 3 candidate proteins were identified from BmNPV by bioinformatics analysis. The candidate proteins include Bombyx mori ATAD3A, HSPD1, PP2A, Actin, PP5 and BmNPV LEF-8, LEF-3, and Chitinase protein (see Table 1 for specific information on all candidate proteins).

View Article: PubMed Central - PubMed

ABSTRACT

Research on molecular mechanisms that viruses use to regulate the host apparatus is important in virus infection control and antiviral therapy exploration. Our previous research showed that the Bombyx mori nucleopolyhedrovirus (BmNPV) LEF-11 localized to dense regions of the cell nucleus and is required for viral DNA replication. Herein, we examined the mechanism of LEF-11 on BmNPV multiplication and demonstrated that baculovirus LEF-11 interacts with Bombyx mori ATAD3A and HSPD1 (HSP60) protein. Furthermore, we showed that LEF-11 has the ability to induce and up-regulate the expression of ATAD3A and HSPD1, phenomena that were both reversed upon knockdown of lef-11. Our findings showed that ATAD3A and HSPD1 were necessary and contributed to BmNPV multiplication in Bombyx mori cells. Moreover, ATAD3A was found to directly interact with HSPD1. Interestingly, ATAD3A was required for the expression of HSPD1, while the knockdown of HSPD1 had no obvious effect on the expression level of ATAD3A. Taken together, the data presented in the current study demonstrated that baculovirus LEF-11 hijacks the host ATPase family members, ATAD3A and HSPD1, efficiently promote the multiplication of the virus. This study furthers our understanding of how baculovirus modulates energy metabolism of the host and provides a new insight into the molecular mechanisms of antiviral research.

No MeSH data available.


Related in: MedlinePlus