Related Experiment Videos
Mapping functional domains in AcMNPV pp31
D R Broussard1, L A Guarino, D L Jarvis
1Departments of Entomology and Biology, Texas A&M University, College Station 77843, USA.
Abstract:
Autographa californica nuclear polyhedrosis virus (AcMNPV) replicates in the nucleus and produces a viral-modified form of the nuclear matrix called the virogenic stroma. The virogenic stroma is the site of viral DNA packaging and nucleocapsid assembly and is thought to be the site of viral DNA replication and RNA transcription. AcMNPV encodes a phosphoprotein, pp31, which localizes to the nucleus of uninfected insect cells and to the virogenic stroma of infected insect cells. pp31 has DNA binding activity and has been identified as a late expression factor. Thus, the intracellular location of pp31, its DNA binding activity, and its identification as a late transcription factor suggest that it participates in replicative events that occur in the virogenic stroma during AcMNPV infection. The purpose of this study was to map the pp31 domains needed for nuclear localization, virogenic stroma localization, and DNA binding. We focused on four basic amino acid regions (BRs 1-4) and used site-directed mutagenesis and gene fusion techniques to probe their functions. The amino-terminal basic region (BR1) was most important for nuclear localization of pp31 in uninfected cells. Three of the four BRs were needed to efficiently localize pp31 to the nucleus and virogenic stroma in infected cells. BR3 was identified as the DNA binding domain of pp31. These data indicated that BR1, BR3, and BR4 are important functional or multifunctional domains within the AcMNPV pp31 protein.
Insights
Autographa californica nuclear polyhedrosis virus (AcMNPV) phosphoprotein pp31
Area of Science:
- Molecular Virology
- Insect Cell Biology
- Viral Replication Mechanisms
Background:
- Autographa californica nuclear polyhedrosis virus (AcMNPV) replicates within the insect cell nucleus.
- A viral-modified nuclear matrix, the virogenic stroma, serves as the site for viral DNA packaging, assembly, replication, and transcription.
- The AcMNPV phosphoprotein pp31 is found in the nucleus and virogenic stroma, possesses DNA binding activity, and functions as a late expression factor.
Purpose of the Study:
- To map the specific domains of the AcMNPV pp31 protein responsible for its nuclear localization, virogenic stroma targeting, and DNA binding.
- To elucidate the functional roles of basic regions (BRs) within the pp31 protein during viral infection.
Main Methods:
- Site-directed mutagenesis was employed to alter specific basic regions (BRs 1-4) within the pp31 gene.
- Gene fusion techniques were utilized to track the localization of modified pp31 proteins within insect cells.
- Analysis of pp31 localization in both uninfected and AcMNPV-infected insect cells was performed.
Main Results:
- The amino-terminal basic region (BR1) is crucial for pp31 nuclear localization in uninfected cells.
- Efficient localization to the nucleus and virogenic stroma in infected cells requires three of the four basic regions.
- Basic region 3 (BR3) was identified as the specific DNA binding domain of pp31.
Conclusions:
- The study identifies key functional domains within AcMNPV pp31.
- BR1, BR3, and BR4 are essential for pp31's localization and DNA binding activities.
- These findings contribute to understanding pp31's role in AcMNPV replication within the virogenic stroma.