Defective herpes simplex virus type 1 vectors harboring gag, pol, and env genes can be used to rescue defective retrovirus vectors
Defective herpes simplex virus type 1 vectors harboring gag, pol, and env genes can be used to rescue defective retrovirus vectors
复制标题
含有 gag、pol 和 env 基因的有缺陷的单纯疱疹病毒 1 型载体可用于拯救有缺陷的逆转录病毒载体
作者：
N. Savard;F. Cosset;A. Epstein
构建了一个逆转录病毒包装转录单元，在该转录单元中，Moloney小鼠白血病病毒(MoMLV)的Gag-Poll和env基因在疱疹病毒调控序列的控制下表达。将该转录单位克隆到单纯疱疹病毒1型(HSV-1)扩增载体中，构建成Gag-Polenv[GPE]载体，并以缺失的HSV-1载体为辅助病毒构建扩增载体。用GPE载体感染携带LacZ前病毒的人TE671细胞(ATCC CRL 8805)，在感染后不同时间收集感染细胞的上清液并进行筛选。逆转录-聚合酶链式反应和对小鼠NIH3T3细胞转导β-半乳糖苷酶活性的能力以及对人TE671细胞转导β-半乳糖苷酶活性的能力均表明，这些上清液含有具有感染性的生态型LacZ逆转录病毒颗粒。GPE载体感染的TE-lac2细胞释放的逆转录病毒载体滴度随感染扩增颗粒剂量的增加而增加。辅助病毒的重叠感染抑制了逆转录病毒载体的产生，说明辅助病毒的混合感染对逆转录病毒的产生有负面的干扰作用。GPE载体对逆转录病毒载体的诱导可被抗HSV-1抗血清中和，但不能被抗MoMLV抗血清中和，而GPE载体感染的TE-lac2细胞上清液对NIH3T3细胞的β-半乳糖苷酶活性的转导可被抗MoMLV抗血清中和。这些结果表明，HSV-1 GPE扩增载体可以挽救缺陷型LacZ逆转录病毒载体，并提示它们可以作为一种发射斜坡，从几乎任何含有缺陷型逆转录病毒载体的体外或体内细胞内激发缺陷型逆转录病毒载体。
A retroviral packaging transcription unit was constructed in which the Moloney murine leukemia virus (MoMLV) gag-pol and env genes are expressed under the control of herpesvirus regulatory sequences. This transcription unit, lacking long terminal repeats, primer binding sites, and most of the retrovirus packaging signal but retaining both retroviral donor and acceptor splice sites, was cloned into a herpes simplex virus type 1 (HSV-1) amplicon plasmid, and amplicon vectors (the gag-pol-env [GPE] vectors) were generated by using a defective HSV-1 vector as helper virus. The GPE vector population was used to infect human TE671 cells (ATCC CRL 8805), harboring a lacZ provirus (TE-lac2 cells), and supernatants of infected cells were collected and filtered at different times after infection. These supernatants were found to contain infectious ecotropic lacZ retroviral particles, as shown both by reverse transcription-PCR and by their ability to transduce a beta-galactosidase activity to murine NIH 3T3 cells but not to human TE671 cells. The titer of retroviral vectors released by GPE vector-infected TE-lac2 cells increased with the dose of infectious amplicon particles. Retrovirus vector production was inhibited by superinfection with helper virus, indicating that helper virus coinfection negatively interfered with retrovirus production. Induction of retrovirus vectors by GPE vectors was neutralized by anti-HSV-1 but not by anti-MoMLV antiserum, while transduction of beta-galactosidase activity to NIH 3T3 cells by supernatants of GPE vector-infected TE-lac2 cells was neutralized by anti-MoMLV antiserum. These results demonstrate that HSV-1 GPE amplicon vectors can rescue defective lacZ retrovirus vectors and suggest that they could be used as a sort of launching ramp to fire defective retrovirus vectors from within virtually any in vitro or in vivo cell type containing defective retroviral vectors.
登录
查看更多内容
使用牛痘/T7聚合酶互补系统分析逆转录病毒组装。
影响因子：
3.7
作者：
Dong,J;Hunter,E
通讯作者：
Hunter,E
产生具有非常广泛宿主范围的高滴度假型逆转录病毒载体。
影响因子：
--
作者：
Yee,JK;Friedmann,T;Burns,JC
通讯作者：
Burns,JC
DOI：
10.1073/pnas.90.17.8033
发表时间：
1993-09-01
影响因子：
11.1
作者：
BURNS, JC;FRIEDMANN, T;YEE, JK
通讯作者：
YEE, JK
DOI：
10.1073/pnas.84.7.1926
发表时间：
1987-04-01
影响因子：
11.1
作者：
KWONG, AD;FRENKEL, N
通讯作者：
FRENKEL, N
DOI：
10.1073/pnas.89.3.991
发表时间：
1992-02-01
影响因子：
11.1
作者：
ISFORT, R;JONES, D;KUNG, HJ
通讯作者：
KUNG, HJ