[Construction of recombinant lentivirus vaccine with single round replication].
OBJECTIVE: To develop a safe and effective lentivirus vaccine model and provide insights into the development of other lentivirus vaccines. METHODS: In this study, a construct of pGPT was made by deleting env gene in the infectious Equine infectious anemia virus (EIAV) molecular clone of WU57. Since the overlaping of EIAV Rev gene with env gene, there was no Rev gene in the construct of pGPT. For compensation of Rev function, the construct of pGPTC was made by inserting 4 copies of constitutive RNA transport elements (CTEs) from Mason-Pfizer monkey virus into the construct of pGPT. In addition, a construct designated pTEB expressing EIAV Env protein was made while env gene-minus viruses were made by co-transfection of pGPT/pTEB or pGPTC/pTEB into 293 cells. Western blot was used to identify the development of recombinant virus particles. Then immunofluorescence assay was used to evaluate the infectivity of recombinant virus particles in vitro. RESULTS: EIAV proteins expression was detected in the supernatant of transfected 293 cells by Western blot within pGPTC/pTEB transfected cells. However, no evidence of EIAV proteins expression was observed within pGPT/pTEB transfected cells. EIAV proteins expression was detected in the first round but not in the second round infected EK cells with EIAV(GPTC) by immunofluorescence assay. CONCLUSION: Rev/RRE was necessary for expression of viral structural proteins; CTEs from Mason-Pfizer monkey virus was functionally interchangeable with EIAV Rev/RRE to help RNAs transportation out of nucleus to express structural proteins and EIAV particles were produced in the transfected 293 cells. A live EIAV recombinant virus with single round infection had been developed.