RESUMO
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.
Assuntos
Nucleopoliedrovírus/química , Nucleopoliedrovírus/fisiologia , Spodoptera/virologia , Proteínas do Envelope Viral/química , Motivos de Aminoácidos , Animais , Sistemas CRISPR-Cas , Teste de Complementação Genética , Larva/virologia , Mariposas/virologia , Nucleopoliedrovírus/genética , Filogenia , Domínios Proteicos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Células Sf9 , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
Baculoviruses infect insects, producing two distinct phenotypes during the viral life cycle: the budded virus (BV) and the occlusion-derived virus (ODV) for intra- and inter-host spread, respectively. Since the 1980s, several countries have been using Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) as a biological control agent against the velvet bean caterpillar, A. gemmatalis. The genome of AgMNPV isolate 2D (AgMNPV-2D) carries at least 152 potential genes, with 24 that possibly code for structural proteins. Proteomic studies have been carried out on a few baculoviruses, with six ODV and two BV proteomes completed so far. Moreover, there are limited data on virion proteins carried by AgMNPV-2D. Therefore, structural proteins of AgMNPV-2D were analysed by MALDI- quadrupole-TOF and liquid chromatography MS/MS. A total of 44 proteins were associated with the ODV and 33 with the BV of AgMNPV-2D. Although 38 structural proteins were already known, we found six new proteins in the ODV and seven new proteins carried by the AgMNPV-2D BV. Eleven cellular proteins that were found on several other enveloped viruses were also identified, which are possibly carried with the virion. These findings may provide novel insights into baculovirus biology and their host interaction. Moreover, our data may be helpful in subsequent applied studies aiming to improve AgMNPV use as a biopesticide and a biotechnology tool for gene expression or delivery.
Assuntos
Nucleopoliedrovírus/química , Proteoma/análise , Proteínas Estruturais Virais/análise , Animais , Linhagem Celular , Cromatografia Líquida , Corpos de Inclusão Viral , Lepidópteros , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Liberação de VírusRESUMO
The aim of this study was to encapsulate the occlusion bodies (OBs) of Spodoptera frugiperda nucleopolyhedrovirus (SfNPV) in Eudragit S100 microparticles (MPs), considering this technique as a possible alternative to protect them from deleterious environmental conditions. The MPs were prepared by oil-in-oil emulsion (O/O) solvent evaporation method. Experimental conditions were established according to a previous multi-level experimental design involving the core/polymer ratio as independent variable. The effects of these parameters on particle size and process yield were investigated observing that polymer concentration had a significant effect on particle size. After adequate conditions for MPs formation were determined, virus was encapsulated. The virus microparticles presented a particle size between 50-300 microm and concentration was 2.62 x 10(9) OBs g(-1). Microencapsulation efficiency was 53.43% and virus release adjusted to Higuchi model suggesting diffusion as the release mechanism. Evaluated microencapsulation process protected viral particles of UV-inactivation, suggesting its potential for a biopesticide development.
Assuntos
Resinas Acrílicas/química , Sistemas de Liberação de Medicamentos/métodos , Luz , Nucleopoliedrovírus/química , Spodoptera , Proteínas Virais/química , Liberação de Vírus , Animais , Físico-Química , Composição de Medicamentos/métodos , Estabilidade de Medicamentos , Microscopia Eletrônica de Varredura , Liberação de Vírus/efeitos da radiaçãoRESUMO
ORF 31 is a unique baculovirus gene in the genome of Anticarsia gemmatalis multiple nucleopolyhedrovirus isolate 2D (AgMNPV-2D). It encodes a putative polypeptide of 369 aa homologous to poly (ADP-ribose) polymerase (PARP) found in the genomes of several organisms. Moreover, we found a phylogenetic association with Group I PARP proteins and a 3D homology model of its conserved PARP C-terminal catalytic domain indicating that had almost an exact spatial superimposition of <1 A with other PARP available structures. The 5' end of ORF 31 mRNA was located at the first nucleotide of a CATT motif at position -27. Using real-time PCR we detected transcripts at 3 h post-infection (p.i.) increasing until 24 h p.i., which coincides with the onset of DNA replication, suggestive of a possible role in DNA metabolism.