一例HIV-1急性感染者奠基病毒的gp160基因序列特征及其假病毒构建

Sequence characteristics and pseudovirus construction of a full-length gp160 of HIV-1 subtype B from an acutely infected patient

目的观察急性感染期艾滋病病毒I型（HIV-1）gp160的全长基因序列及感染特征。方法从一例处于Feibig I期HIV-1感染者血浆中提取RNA,扩增gp160全长基因并测序,分析其生物学信息;将gp160全长基因与pcDNA3.1His/V5真核表达载体连接,构建Env-pcDNA3.1真核表达质粒,与骨架质粒pNL4-3.Luc.R-E-共转染293细胞,包装出假病毒。用包装的假病毒感染ghost细胞,测定感染细胞的荧光素酶活性（RLU）,鉴定假病毒的感染活性。结果成功扩增出gp160全长基因,嗜性预测为CCR5,N-糖基化位点数与标准株HXB2相同,但gp120糖基化程度更高,氨基酸变异主要集中在V1-V5区。假病毒感染试验显示,RLU值达到7log。结论获得了处于急性感染期的HIV-1gp160基因序列和高感染活性的假病毒。

Objective To observe characteristics of a full-length gpl60 sequence of HIV-1 subtype B from an a- cutely infected patient. Methods HIV-1 RNA was extracted from the plasma of a acutely infected patients in Feibig stage I, and then the full-length of gpl60 was amplified and sequenced directly. The Env containing PCR amplicons was ligated into the pcDNA3.1 His/V5 Topl0 expression vector directly to construct the Env-pcDNA3.1 expression plasmid. The Env-pseudovirus was made in 293 cells which cotransfected with Env-pcDNA3. 1 and the backbone plasmid pNL4--3. Luc. R-E-. Then the ghost cells with CCR5/CXCR4 coreceptor were infected by Env-pseudotyped viruses to calculate the relative luciferase unit （RLU） and infectivity of the pseudovirus. Results A HIV-1 subtype B full-length gpl60 gene was amplified successfully from the plasma of an acutely HIV-1 infected patient in Feibig stage I. Phylogenetic analysis of full-length gpl60 nucleotide sequences confirmed that the functional clone was grouped in clade B. The total number of Nlinked glycosylation sites were the same to the standard strain HXB2. However, the degree of gpl20 glycosylation was higher, and amino acid variation was found mainly in V1 ~ V5 variable regions compared with the other reference strains of subtype B. HIV pesudovirus containing luciferase gene was capable of infecting ghost cells with the RLU reached to 7 Log. Conclusion The full length gpl60 sequence was amplified from an acutely infected patient, and Env-pseudotyped HIV-1 was successfully produced and had the ability of infecting ghost cells.