RESUMO
Chicken anemia virus gene encoding apoptin, a selective killer of cancer cells was synthesized and inserted into vaccinia virus (strain L-IVP) genome. The insertion has replaced major part of the viral C11R gene encoding viral growth factor (VGF), which is important for the virulence. The recombinant virus VVdGF-ApoS24/2 was obtained through the transient dominant selection technique with the use of puromycin resistance gene as the selective marker. The expression apoptin gene from a synthetic early-late promoter of vaccinia virus effectively provides accumulation of the protein in the cells infected with the VVdGF-ApoS24/2 virus. Despite the presence of virus growth factor signal peptide at apoptin N-terminal secretion of the recombinant protein into culture medium did not occur. The recombinant virus VVdGF-ApoS24/2 was found to have a significantly greater selective lyticactivity on human cancer cell lines (A549, A431, U87MG, RD and MCF7) as compared with the parent strain L-IVP and its variant VVdGF2/6 with the deletion of the C11R gene. The results suggest that the use of apoptin represents a promising approach for improving the natural anticancer activities of vaccinia virus.
Assuntos
Vacinas Anticâncer/genética , Proteínas do Capsídeo/genética , Neoplasias/genética , Vírus Oncolíticos/genética , Vaccinia virus/genética , Animais , Proteínas do Capsídeo/uso terapêutico , Vírus da Anemia da Galinha/genética , Galinhas/genética , Galinhas/virologia , Vetores Genéticos , Genoma Viral , Humanos , Células MCF-7 , Neoplasias/terapia , Neoplasias/virologia , Terapia Viral Oncolítica/métodos , Replicação Viral/genéticaRESUMO
Mousepox (ectromelia) virus genome contains four genes encoding for kelch-like proteins EVM018, EVM027, EVM150 and EVM167. A complete set of insertion plasmids was constructed to allow the production of recombinant ectromelia viruses with targeted deletions of one to four genes of kelch family both individually (single mutants) and in different combinations (double, triple and quadruple mutants). It was shown that deletion of any of the three genes EVMO18, EVM027 or EVM167 resulted in reduction of 50% lethal dose (LD50) by five and more orders in outbred white mice infected intraperitoneally. Deletion of mousepox kelch-gene EVM150 did not influence the virus virulence. Two or more kelch-genes deletion also resulted in high level of attenuation, which could evidently be due to the lack of three genes EVM167, EVM018 and/or EVM027 identified as virulence factors. The local inflammatory process on the model of intradermal injection of mouse ear pinnae (vasodilatation level, hyperemia, cutaneous edema, arterial thrombosis) was significantly more intensive for wild type virus and virulent mutant deltaEVM150 in comparison with avirulent mutant AEVM167.
Assuntos
Vírus da Ectromelia/genética , Vírus da Ectromelia/patogenicidade , Ectromelia Infecciosa/genética , Deleção de Genes , Genes Virais/genética , Proteínas Virais/genética , Animais , Linhagem Celular , Chlorocebus aethiops , Vírus da Ectromelia/metabolismo , Ectromelia Infecciosa/metabolismo , CamundongosRESUMO
The biological properties of cowpox virus (CPXV) mutants with target deletion of 4 of the 6 BTB/kelch genes (D11L, C18L, G3L, and A56R) were examined in CV-1 cell cultures. There were changes in mutant temperature sensitivity and a reduction in a viral cytopathic effect. The mutant-infected culture yielded a smaller number of cells with actin-related long cellular protrusions (63 of 300 cells) as compared with wild CPXV (127 of 300). The length of the protrusions was 20-60 and 40-120 microm, respectively). Confocal microscopy revealed the formation of large globed structures containing both actin and CPXV antigens in the cells infected with quadruple mutants. These globed structures were recognized as incomplete protrusions. The findings show that the formation of long protrusions in the cells infected with wild type CPXV represents a type of specific viral potency related to the activity of BTB/kelch genes whose deletion results in cellular insufficiency to form full-fledged protrusions.