Variations Of Env Gene And The Role Of LTR During The Attenuation Of Chinese Equine Infectious Anemia Virus Donkey Leukocyte Attenuated Strain

Equine infectious anemia virus (EIAV) is a lentivirus within the family Retroviridae and the causative agent of equine infectious anemia (ElA). The Chinese donkey leukocyte attenuated strain of EIAV (EIAV-DLA) was the only successful lentivirus vaccine up to now, which has been applied extensively in China against EIA. EIAV-DLA can serve as a model for the study of lentivirus. The EIAV proviral genomic long-terminal repeat (LTR) is a eukaryotic promotor, which plays an important regulatory role on transcription and replication of the virus. The envelope protein of the lentivirus initiates great interests for its frequent variations. Comparisons of the nucleotide sequence of EIAV-DLA with that of EIAV-L indicate that both LTR and env gene (especially in gp90 coding region) are relatively highly varied.To elucidate the role of EIAV env gene in the attenuation process of Chinese EIAV-DLA, the env genes containing whole S2 and gp90 of EIAV-L and EIAV-DLA were amplified from different sera of EIAV-L challenged ponies and EIAV-DLA infected donkey leukocytes by RT-PCR, respectively. Thirty-env genes of EIAV-L and EIAV-DLA were obtained. We compared the nucleotide acids and amino acids of the 30 env gp90 genes and found that there existed a series of regular variations among the gp90 genes of EIAV-L and EIAV-DLA. These variations included mutations, insertions and deletions of amino acids. The polarity change and deletions or insertions of amino acids affect the TV-glycosylation sites between EIAV-L and EIAV-DLA. Other research on lentivirus such as HIV and SIV indicated that iV-glycosylation played important roles on virus cell tropism, virulence and escape neutralization antibodies. Further compareation of env genes revealed that the V3 to V5 regions appeared significant differences between EIAV-L and EIAV-DLA whereas appeared few differences on EIAV-L or EIAV-DLA, respectively. We compared the nucleotide acids and amino acids of the 27 S2 genes and found that there existed 3 regular mutations among the S2 genes of EIAV-L and EIAV-DLA and without deletions or insertions of amino acid.On the basis of variation research on env gene of EIAV-L and EIAV-DLA in China, the full gp90 and partial gp90 (V3-V5 region) were cloned into retroviral vector pBABE-puro and obtained recombinant plasmids pBABE-puro-L-gp90, pBABE-puro-DLA-gp90, pBABE-puro-L-V3-V5, and pBABE-puro- DLA-V3-V5. The recombinant plasmids were transfected into retrovirus package cell line PA317 and stable cell lines were generated by puromycin selection. The gp90 and V3-V5 were then cloned into mammalian expression vector pcDNA3.1(+), resulting in recombinant plasmids pcDNA-L-gp90, pcDNA-DLA-gp90, pcDNA L-V3-V5, and pcDNA-DLA-V3-V5. The recombinant plasmids were transfected into 293T ceil line and stable cell lines were obtained by G418 selection and subsequent subclonings. The stable cell lines expressing the env genes of EIAV-L and EIAV-DLA provide a good platform for further developing the configuration and function of env genes. However, the attenuation and immuno-protection mechanisms of EIAV-DLA are still not known. Researches on HIV indicated that N-glycosylation played important roles on virus cell tropism, virulence and escape...