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Autographa californica multiple nucleopolyhedrovirus me53 (ac140) is a nonessential gene required for efficient
Jondavid de Jong1, Basil M Arif, David A Theilmann
1Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Abstract:
me53 is a highly conserved baculovirus gene found in all lepidopteran baculoviruses that have been fully sequenced to date. The putative ME53 protein contains a zinc finger domain and has been previously described as a major early transcript. We generated an me53-null bacmid (AcDeltame53GFP), as well as a repair virus (AcRepME53:HA-GFP) carrying me53 with a C-terminal hemagglutinin (HA) tag, under the control of its native early and late promoter elements. Sf9 and BTI-Tn-5b1 cells transfected with AcDeltame53GFP resulted in a 3-log reduction in budded-virus (BV) production compared to both the parental Autographa californica multiple nucleopolyhedrosis virus and the repair bacmids, demonstrating that although me53 is not essential for replication, replication is compromised in its absence. Our data also suggest that me53 does not affect DNA replication. Cell fractionation showed that ME53 is found in both the nucleus and the cytoplasm as early as 6 h postinfection. Deletion of the early transcriptional start site resulted in a 10- to 360-fold reduction of BV yield; however, deletion of the late promoter (ATAAG) resulted in a 160- to 1,000-fold reduction, suggesting that, in the context of BV production, ME53 is required both early and late in the infection cycle. Additional Western blot analysis of purified virions from the repair virus revealed that ME53:HA is associated with both BV and occlusion-derived virions. Together, these results indicate that me53, although not essential for viral replication, is required for efficient BV production.
Insights
The baculovirus me53 gene is crucial for efficient budded virus (BV) production, impacting both early and late infection stages. While not essential for viral replication, its absence significantly reduces BV yield, highlighting its role in virus assembly and release.
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- The me53 gene is a conserved, highly expressed early transcript in lepidopteran baculoviruses.
- The ME53 protein contains a zinc finger domain, suggesting a potential role in nucleic acid binding or regulation.
- Previous studies identified ME53 as a major early transcript, but its specific function remained unclear.
Purpose of the Study:
- To investigate the function of the baculovirus me53 gene in viral replication and propagation.
- To determine the impact of me53 deletion on budded virus (BV) production and viral DNA replication.
- To elucidate the temporal requirement and localization of the ME53 protein during baculovirus infection.
Main Methods:
- Generation of an me53-null bacmid (AcDeltame53GFP) and a repair virus with a tagged ME53 protein (AcRepME53:HA-GFP).
- Transfection of insect cells (Sf9 and BTI-Tn-5b1) and measurement of budded virus (BV) production.
- Cell fractionation to determine ME53 protein localization and Western blot analysis of purified virions.
Main Results:
- Deletion of me53 resulted in a 3-log reduction in BV production, indicating compromised but not abolished replication.
- ME53 deletion did not significantly affect viral DNA replication.
- ME53 protein was detected in both the nucleus and cytoplasm as early as 6 hours post-infection.
- Deletion of the early and late transcriptional start sites of me53 significantly reduced BV yield, suggesting a role throughout the infection cycle.
- ME53 protein was found to be associated with both budded virus (BV) and occlusion-derived virions.
Conclusions:
- The me53 gene is essential for efficient budded virus (BV) production in baculoviruses.
- ME53 plays a critical role in both early and late stages of the viral infection cycle concerning BV propagation.
- While not indispensable for viral replication itself, me53 is vital for maximizing viral progeny release and infectivity.
