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Published on: January 8, 2015
Characterization of a new Autographa californica multiple nucleopolyhedrovirus (AcMNPV) polyhedra mutant
Bergmann Morais Ribeiro1, Ana Paula Montenegro Generino, Cláudia Natércia Lima Acacio
1Laboratório de Microscopia Eletrônica, Departamento de Biologia Celular, Universidade de Brasília, Brasília, DF, CEP 70910-900, Brazil. bergmann@unb.br
Abstract:
In the very late phase of baculovirus infection, virions are occluded in a crystalline matrix called polyhedra, which is mainly composed of polyhedrin. This protein is highly conserved among baculoviruses and changes in its amino acid sequence may lead to mutant polyhedra. During the purification of an AcMNPV recombinant virus, a mutant virus was isolated. Structural and ultrastrutural analysis by light and transmission electron microscopy (TEM) of insect cells infected with this mutant virus did not show polyhedra formation and differed from the wild-type infection by the presence of a proteinaceous mass dispersed in the cytoplasm and nucleus of the infected cells, which was confirmed by immunogold labelling to be polyhedrin. The polyhedrin gene was amplified by PCR and sequenced. The only change observed was the substitution of a G to a T at the nucleotide +352, which resulted in a Val to Phe change. A recombinant virus was constructed by transferring the mutant gene into a polyhedrin negative virus. The phenotype of this recombinant virus was the same as the mutant one, confirming that this single mutation alone was responsible for the mutant phenotype.
Insights
A single gene mutation in Autographa californica multiple nucleopolyhedrovirus (AcMNPV) prevents polyhedra formation. This study identifies the specific nucleotide change responsible for the altered polyhedrin protein structure and its impact on viral occlusion.
Area of Science:
- Molecular Biology
- Virology
- Insect Pathology
Background:
- Baculovirus infection involves virion occlusion within polyhedra, a crystalline matrix primarily composed of polyhedrin.
- Polyhedrin is a highly conserved protein among baculoviruses, and alterations in its sequence can lead to mutant polyhedra.
- Understanding polyhedrin's role is crucial for baculovirus applications in biotechnology and pest control.
Purpose of the Study:
- To investigate the cause of aberrant polyhedra formation in a mutant Autographa californica multiple nucleopolyhedrovirus (AcMNPV).
- To identify the specific genetic mutation responsible for the observed mutant phenotype.
- To confirm the role of the identified mutation in polyhedra assembly.
Main Methods:
- Structural and ultrastructural analysis using light and transmission electron microscopy (TEM).
- Immunogold labeling to confirm the identity of proteinaceous masses.
- Polymerase chain reaction (PCR) amplification and sequencing of the polyhedrin gene.
- Construction of a recombinant virus with the mutant polyhedrin gene.
Main Results:
- Infected cells with the mutant virus failed to form polyhedra, exhibiting dispersed polyhedrin in the cytoplasm and nucleus.
- Sequencing revealed a single nucleotide substitution (G to T at +352) in the polyhedrin gene, resulting in an amino acid change (Val to Phe).
- A recombinant virus carrying the mutant gene replicated the observed mutant phenotype, confirming the mutation's causative role.
Conclusions:
- A single nucleotide mutation in the AcMNPV polyhedrin gene is sufficient to abolish polyhedra formation.
- The Valine to Phenylalanine substitution at amino acid position 352 disrupts the structural integrity required for polyhedra assembly.
- This finding provides critical insights into the structure-function relationship of polyhedrin and baculovirus occlusion.
