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Effect of ac68 knockout and lef3 leading sequence disruption on viral propagation

Guohui Li1, Huiqing Chen, Qi Tang

  • 1Institute of Life Sciences, Jiangsu University, 301# Xuefu Road, Zhenjiang, 212013, China.

Current Microbiology
|June 23, 2010
PubMed

Insights

Autographa californica multiple nucleopolyhedrovirus Orf68 (ac68) is an early gene. Deleting ac68 and its associated lef3 sequences did not impact virus propagation in Sf-9 cells, indicating ac68 is non-essential.

Area of Science:

  • Molecular virology
  • Insect molecular biology
  • Genetics of baculoviruses

Background:

  • Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a significant insect pathogen.
  • Orf68 (ac68) is classified as an early gene, but its precise transcription start site and functional necessity were undetermined.
  • The ac68 gene overlaps with the lef3 gene, raising questions about potential regulatory interactions.

Purpose of the Study:

  • To determine the transcription start site of the AcMNPV ac68 gene.
  • To investigate the impact of ac68 deletion on viral propagation.
  • To assess the effect of disrupting the lef3 gene's leading sequence on AcMNPV replication.

Main Methods:

  • 5' RACE (Rapid Amplification of Complementary Ends) analysis was employed to identify the transcription start site of ac68.
  • A bacmid with a targeted ac68 knockout was generated, which also involved deleting a portion of the lef3 leading sequence.
  • Viral propagation was assessed in Sf-9 cells infected with the mutant bacmid, monitoring budded virus production, nucleocapsid formation, and occlusion body development.

Main Results:

  • The transcription start site of ac68 was mapped to 18 nucleotides upstream of its start codon.
  • Virus propagation, including infectious budded virus production and the formation of nucleocapsids and occlusion bodies, proceeded normally in Sf-9 cells infected with the ac68 knockout mutant.
  • Complete deletion of ac68 and disruption of the lef3 leading sequence did not impede viral propagation, confirming ac68 as a non-essential gene.

Conclusions:

  • Ac68 is a non-essential gene for AcMNPV propagation in Sf-9 cells.
  • The overlapping sequence and potential regulatory elements between ac68 and lef3 do not appear critical for viral replication.
  • Ac68 represents the second identified auxiliary gene among the core genes of AcMNPV, alongside Ac133 (alk-exo).

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