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

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.

Keywords: AcMNPV; CRISPR/Cas9; HearSNPV; P74; Rachiplusia nu; SeMNPV; Spodoptera exigua; baculovirus.

Figure 1

Phylogenetic analysis of the P74 proteins. Evolutionary inference of the P74 protein considering species of the four baculovirus genera and representative species of other invertebrate viruses (nudivirus, hytrosavirus, and nimavirus). Bootstrapping values greater than 40 are shown. Alphabaculoviruses, betabaculoviruses, gammabaculoviruses, and deltabaculoviruses are shaded in green, yellow, blue, and gray, respectively. The three species of alphabaculoviruses considered in the experimental studies are in red letters.

Figure 2

Domain architecture of P74 proteins. Bioinformatics studies for P74 protein consensus based on sequences from species of four baculovirus genera. (A) P74 similarity plot. The dashed line indicates the average value. Sequence logos highlight the six cysteines perfectly conserved in all P74 (orange boxes). Dashed red lines show the connectivity predicted with Disulfind server. The putative NES is also indicated (yellow box), and the point of separation between the Nt and Ct regions is marked with a red asterisk. The WDP triad contains a conserved BamHI site at the nucleotide sequence level (or the possibility of introducing it through a same-sense point mutation) further used on the construction of hybrid proteins. (B) P74 hydropathy profile (black line). Variability was considered as the standard deviation (in blue). The three putative transmembrane regions (TMs) are indicated, with one of them (TM-1) containing a putative NES sequence. (C) Sequence logos of each of the TM detected. (D) TM predictions. These regions are highlighted in gray in all analyses.

Figure 3

AcMNPV knockout in p74 gene. Sequence result of the p74-knockout AcMNPV-bacmid. The cRNA recognition site is highlighted in yellow, and the protospacer-adjacent motif (PAM) sequence is indicated in red. The dotted line shaded in light blue corresponds to the nucleotide deletion observed. The red box marks the early stop codon generated by the mutation, and the amino acids in blue are substitutions also resulting from the 4 bp deletion. Both initial and stop codons are in bold. The orange triangle shows where the DSB is expected to be generated. Mutant 3 was selected (highlighted with a yellow star).

Figure 4

Production of viral OBs with P74 variants. AcMNPV per os infective virions with P74 variants were generated in Sf21 cells by the coinfection of BVs expressing those proteins and GFP, with BVs expressing polyhedrin and mCherry. Both BVs are based on the p74-knockout bacmid. Representative microscopy photos (400×) are shown. (A) Green fluorescent cells showing that the infection induced GFP expression. (B) Red fluorescent cells showing that the infection induced mCherry expression. (C) Bright field (the presence of OBs can be seen). (D) Merged image obtained through combination of the previous images (A,B).

Figure 5

Recombinant P74 protein detection in OBs. Immunodetection of P74 variants in OBs by Western blot using antibodies anti-6×His-tag. The source of the OBs evaluated is indicated on each lane.

Figure 6

Infectivity of complemented AcMNPV p74 knockout. S. exigua larvae exposed to virions derived from AcMNPV knocked out in p74 and supplemented with the ORF variant of the same virus, AcMNPV-p74AcAc. (A) Representative larva after BV injection showing GFP and (B) mCherry expression. (C) Representative larva after per os infection with OBs showing GFP and (D) mCherry expression. All images (0.8×) were taken 120 h after starting treatment.

Figure 7

Virulence of the recombinant AcMNPVs in S. exigua. Mortality percentages of third-instar larvae of S. exigua orally infected by the droplet feeding method with the different variants of OBs (1 × 10⁷ OBs/mL). Bioassay results (48 larvae in experimental units of 16 individuals for each treatment) are shown, and standard deviations are included. Exposed insects were maintained at 26 °C, and mortality was recorded every 12 h.

Figure 8

Virulence of the recombinant AcMNPVs in R. nu. Mortality percentages of third-instar larvae of R. nu orally infected by the droplet feeding method with the different variants of OBs (1 × 10⁷ OBs/mL). Bioassay results (48 larvae in experimental units of 16 individuals for each treatment) are shown, and standard deviations are included. Exposed insects were maintained at 26 °C, and mortality was recorded every 24 h.