Generation of a helper cell line for packaging avian leukosis virus-based vectors

Abstract

We constructed an avian leukosis virus-based packaging cell line, pHF-g, containing Rous-associated virus DNA with several alterations to abolish RNA packaging. One of them is a 52-base-pair deletion encompassing the putative encapsidation signal in the leader region. The 3' long terminal repeat was also removed and replaced by the polyadenylation sequence from the herpes simplex virus thymidine kinase gene. When pHF-g cells were transfected by an avian leukosis virus-based vector, they produced replication-defective virus at high titer but they did not release any replication-competent particles. Proviral DNA was shown to be correctly integrated as well as correctly expressed. Viral RNAs were shown to be correctly translated into gag-related polypeptides.

Usage of retrovirus vectors for gene transfer into birds

“…Ранние упаковочные клеточные линии пред ставляли собой провирус вируса-помощника, в ко тором делетировали упаковочный сигнал. Однако рекомбинации между гомологичными последова тельностями вектора и упаковочного генома как в процессе обратной транскрипции, так и при совме стной упаковке последовательностей вектора и упаковочного мутанта в вирион [12] [42,[46][47][48] в геноме RAV-1 произвели следующие изменения: ^-упако вочный сигнал делетировали, З'-LTR заменили на сигналы полиаделирования; gag-pol-и env-гены экспрессируются в различных плазмидах. Таким образом, для восстановления репликативной ком петентности вируса-помощника необходим не один раунд рекомбинации с последовательностями ре тровирусного вектора, как в случае Q4dh упако вочной клеточной линии, а три раунда рекомбина ции, что маловероятно.…”

Homologous and nonhomologous retroviral recombinations are both involved in the transfer by infectious particles of defective avian leukosis virus-derived transcomplementing genomes

“…The NLB vector possesses a wild-type leader sequence. The removal of the ⌿ packaging signal in the leader region of the transcomplementing PhE genome created a SacI restriction site (30). Thus, results of the digestion of the U33-Ph2 PCR products with the SacI enzyme would allow us to determine whether the ⌿ packaging signal is present or absent in the leader region of the Phleo r proviruses.…”

Packaging cells for avian leukosis virus-based vectors with various host ranges

“…A (8, 33,34). The availability of other ALV-based packaging cells that provide different subgroups of envelopes is of particular interest, since it would extend the host ranges of the recombinant vectors.…”

“…ND, not determined. These NLB viral supernatants were also checked for the absence of replication-competent viruses, as previously described (33). The QT6 cell line, kindly provided by C. Moscovici, was derived from a fibro-sarcoma chemically induced in a Japanese quail (27).…”

“…The genetically controlled sensitivity to ALV infections of chickens implies that not all chicken strains can be infected by subgroup A viruses obtained from previously described helper cell lines (8,33,34). The availability of helper cells producing viruses with B, C, and E tropisms abolishes this limitation.…”