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Characterization of a baculovirus lacking the DBP (DNA-binding protein) gene
Adam L Vanarsdall1, Victor S Mikhailov, George F Rohrmann
1Department of Microbiology, Nash Hall Room 220, Oregon State University, Corvallis, OR 97331-3804, USA.
Abstract:
Autographa californica multiple nucleopolyhedrovirus (AcMNPV) encodes two proteins that possess properties typical of single-stranded DNA-binding proteins (SSBs), late expression factor-3 (LEF-3), and a protein referred to as DNA-binding protein (DBP). Whereas LEF-3 is a multi-functional protein essential for viral DNA replication, transporting helicase into the nucleus, and forms a stable complex with the baculovirus alkaline nuclease, the role for DBP in baculovirus replication remains unclear. Therefore, to better understand the functional role of DBP in viral replication, a DBP knockout virus was generated from an AcMNPV bacmid and analyzed. The results of a growth curve analysis indicated that the dbp knockout construct was unable to produce budded virus indicating that dbp is essential. The lack of DBP does not cause a general shutdown of the expression of viral genes, as was revealed by accumulation of early (LEF-3), late (VP39), and very late (P10) proteins in cells transfected with the dbp knockout construct. To investigate the role of DBP in DNA replication, a real-time PCR-based assay was employed and showed that, although viral DNA synthesis occurred in cells transfected with the dbp knockout, the levels were less than that of the control virus suggesting that DBP is required for normal levels of DNA synthesis or for stability of nascent viral DNA. In addition, analysis of the viral DNA replicated by the dbp knockout by using field inversion gel electrophoresis failed to detect the presence of genome-length DNA. Furthermore, analysis of DBP from infected cells indicated that similar to LEF-3, DBP was tightly bound to viral chromatin. Assessment of the cellular localization of DBP relative to replicated viral DNA by immunoelectron microscopy indicated that, at 24 h post-infection, DBP co-localized with nascent DNA at distinct electron-dense regions within the nucleus. Finally, immunoelectron microscopic analysis of cells transfected with the dbp knockout revealed that DBP is required for the production of normal-appearing nucleocapsids and for the generation of the virogenic stroma.
Insights
Autographa californica multiple nucleopolyhedrovirus DNA-binding protein (DBP) is essential for viral replication, nucleocapsid formation, and production of infectious budded virus. Knockout viruses lacking DBP show impaired DNA synthesis and abnormal virion assembly.
Area of Science:
- Virology
- Molecular Biology
- Insect Pathology
Background:
- Autographa californica multiple nucleopolyhedrovirus (AcMNPV) has two single-stranded DNA-binding proteins: LEF-3 and DNA-binding protein (DBP).
- LEF-3 is crucial for viral DNA replication and nuclear transport.
- The specific role of DBP in baculovirus replication is not well understood.
Purpose of the Study:
- To elucidate the functional role of DBP in AcMNPV replication.
- To generate and analyze a DBP knockout virus to understand its essentiality.
Main Methods:
- Generation of a DBP knockout AcMNPV bacmid.
- Growth curve analysis to assess viral production.
- Western blot to analyze viral protein expression.
- Real-time PCR to quantify viral DNA synthesis.
- Field inversion gel electrophoresis to analyze replicated DNA.
- Immunoelectron microscopy to determine DBP localization and effect on virion assembly.
Main Results:
- DBP is essential for AcMNPV replication, as the knockout virus failed to produce budded virus.
- Viral gene expression (early, late, and very late) was not globally affected by DBP absence.
- DBP knockout resulted in reduced viral DNA synthesis and failure to produce genome-length DNA.
- DBP binds tightly to viral chromatin and co-localizes with nascent viral DNA.
- DBP is required for the formation of normal nucleocapsids and the virogenic stroma.
Conclusions:
- DBP plays a critical, essential role in AcMNPV replication, particularly in DNA synthesis and virion morphogenesis.
- DBP is required for efficient viral DNA replication and the assembly of infectious progeny virions.
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