The Membrane-Anchoring Region of the AcMNPV P74 Protein Is Expendable or Interchangeable with Homologs from Other Species.

María Victoria Nugnes,Carolina Susana Cerrudo,Mariano Nicolás Belaich,María Victoria Miranda,Adrià Mengual-Martí,Salvador Herrero,Alexandra Marisa Targovnik

doi:10.3390/v13122416

Abstract

Baculoviruses are insect pathogens that are characterized by assembling the viral dsDNA into two different enveloped virions during an infective cycle: occluded virions (ODVs; immersed in a protein matrix known as occlusion body) and budded virions (BVs). ODVs are responsible for the primary infection in midgut cells of susceptible larvae thanks to the per os infectivity factor (PIF) complex, composed of at least nine essential viral proteins. Among them, P74 is a crucial factor whose activity has been identified as virus-specific. In this work, the p74 gene from AcMNPV was pseudogenized using CRISPR/Cas9 technology and then complemented with wild-type alleles from SeMNPV and HearSNPV species, as well as chimeras combining the P74 amino and carboxyl domains. The results on Spodoptera exigua and Rachiplusia nu larvae showed that an amino terminal sector of P74 (lacking two potential transmembrane regions but possessing a putative nuclear export signal) is sufficient to restore the virus infectivity whether alone or fused to the P74 transmembrane regions of the other evaluated viral species. These results provide novel information about the functional role of P74 and delimit the region on which mutagenesis could be applied to enhance viral activity and, thus, produce better biopesticides.

Highlights

IntroductionThe Baculoviridae family is composed of insect-specific rod-shaped viruses with large covalently closed circular double-stranded DNA (cccdsDNA), from 81,755 bp (NeleNPV)
The baculovirus per os infectivity factor (PIF) proteins are structural polypeptides associated with the occluded virions (ODVs) envelope and functionally essential for primary infection
The application of CRISPR/Cas9 technology and nonhomologous end joining (NHEJ) in baculovirus for these purposes appears as a better option due to its lower probability of unwanted side-effects and should be the preferred tool for this type of genomic study. In this sense, continuing with the work where the use of gene editing mediated by CRISPR/Cas9 is reported for optimizing the use of baculovirus as a protein expression vector [50], the present study demonstrates its usefulness for baculovirus functional genomics research and proposes a methodological detail for its implementation

Summary

Introduction

The Baculoviridae family is composed of insect-specific rod-shaped viruses with large covalently closed circular double-stranded DNA (cccdsDNA), from 81,755 bp (NeleNPV). Baculoviruses infect susceptible hosts in the larval state and produce two genetically identical but morphologically distinct viral phenotypes: occlusion-derived viruses (ODVs) and budded viruses (BVs) [3]. The first are those that initiate the infection in the midgut defining the host range (primary infection), and they are characterized by being embedded in a crystalline matrix denominated occlusion bodies (OBs) that physically protect virions against environmental stresses, while the BVs are responsible for cell-to-cell infections inside the larval body (secondary or systemic infection) [4]. The Alphabaculovirus genus is subclassified into two clades, “group I” and “group

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