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[Expression of baculovirus AcMNPV p48 gene in insect cell]
Meijin Yuan1, Zhenqiu Huang, Zhaoyang Hu
1State Key Laboratory of Biocontrol, Sun Yat-sen University, Guangzhou 510275, China. lssymj@mail.sysu.edu.cn
Objective:
p48 (ac103) gene is highly conserved in baculovirus, implying that p48 might play a fundamental role in the life cycle of baculovirus. We studied the expression of p48 gene of Autographa californica multiple nucleopolyhedrovirus (AcMNPV)--the type baculovirus species.
Methods:
With Bac-to-Bac system, we constructed two p48 repair viruses, in which hemagglutinin (HA)-tag was fused to C-terminus and N-terminus of P48, respectively. To examine the effect on occlusion body morphogenesis and to facilitate examination of virus infection, the green fluorescence protein (gfp) gene and polyhedrin (polh) gene were also inserted into the recombinant viruses. Sf9 cells were transfected with each bacmid constructed, and the supernatants containing the budded viruses (BVs) were used to infect Sf9 cells. At the indicated time points, cells were harvested and used for SDS-PAGE and Western blot analysis. The expression of fusion protein was detected with the monoclonal antibody to the HA-epitope.
Results:
Western blot analysis indicated that a specific 43 kDa protein was detected at 12 hours postinfection (hpi) and remained detectable up to 96 hpi in cells infected with p48 repair virus with HA-tag fused in C-terminus. Meanwhile, another protein of 26 kDa was also detected from 48 hpi to 96 hpi. However, no signals were detected in cells infected with p48 repair virus with HA-tag fused in N-terminus even at 96 hpi.
Conclusion:
p48 is a late gene and expressed in late infection. P48 might be cleaved in N-terminal when it is expressed in insect cells.
Insights
The baculovirus p48 gene is expressed late in infection, with the protein potentially cleaved at the N-terminus. This study investigated p48 gene expression in Autographa californica multiple nucleopolyhedrovirus (AcMNPV).
Area of Science:
- Molecular Biology
- Virology
- Insect Pathology
Background:
- The p48 (ac103) gene is conserved across baculoviruses, suggesting a crucial role in their life cycle.
- Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a model organism for studying baculovirus infection.
Purpose of the Study:
- To investigate the expression pattern of the p48 gene in AcMNPV-infected insect cells.
- To determine the functional significance of p48 during baculovirus replication.
Main Methods:
- Construction of recombinant AcMNPV with HA-tagged p48 genes at C-terminus and N-terminus using the Bac-to-Bac system.
- Infection of Sf9 cells with recombinant viruses and analysis of protein expression via SDS-PAGE and Western blot.
- Detection of fusion protein expression using an anti-HA monoclonal antibody.
Main Results:
- A 43 kDa protein, corresponding to C-terminally tagged p48, was detected from 12 to 96 hours post-infection (hpi).
- An additional 26 kDa protein was observed from 48 to 96 hpi in cells infected with the C-terminally tagged virus.
- No detectable protein signal was observed for the N-terminally tagged p48 construct even at 96 hpi.
Conclusions:
- The p48 gene is a late gene, with its protein product expressed during the late stages of baculovirus infection.
- Evidence suggests that the p48 protein may undergo N-terminal cleavage within infected insect cells.
