Molecular cloning and characterization of the RNA packaging-defective retrovirus SE21Q1b

Abstract

The nonconditional RNA packaging mutant SE21Q1b contains cis-and transacting defects which cause cellular mRNA, rather than viral genomic RNA, to be nonspecifically packaged into SE21Q1b viral particles. Using genomic libraries of the c-SE21Qlb quail cell line, we have been able to construct a molecular clone of the SE21Q1b provirus. Upon transfection into primary quail embryo fibroblasts, the SE21Q1b molecular clone is able to recapitulate the nonspecific RNA packaging phenotype of the c-SE21Q1b cell line. The RNA packaging phenotypes displayed by several SE21Q1b/avian sarcoma-leukemia virus hybrid provirus constructs have further indicated that sequences responsible for the altered RNA packaging phenotype of SE21Q1b are localized in the left third of the SE21Qlb proviral genome. DNA sequence analysis of this region has revealed that the 5' SE21Q1b deletion has removed 179 bp from the SE21Qlb left long terminal repeat and leader regions. Several differences were detected at the carboxyl terminus of the deduced SE21Q1b nucleocapsid protein sequence in comparison with that of Rous sarcoma virus PR-C. Results of site-directed oligonucleotide mutagenesis experiments indicate, however, that the presence of these residues in the nucleocapsid protein alone is not responsible for the decreased RNA packaging specificity of SE21Qlb.

Efficiency and selectivity of RNA packaging by Rous sarcoma virus Gag deletion mutants

“…A cluster of differences in the C terminus of the NC domain, when tested in a wild-type background, did not reconstitute the mutant phenotype (2). The results reported here are consistent with those of Anderson et al (2), who suggest that the behavior of SE21Q1b Gag is not due to a single defect.…”

Retroviral nucleocapsid domains mediate the specific recognition of genomic viral RNAs by chimeric Gag polyproteins during RNA packaging in vivo

“…We also observed that the HIV-1 mutant containing the M-MuLV NC domain was able to encapsidate a nonviral RNA 20-fold more efficiently than was wild-type HIV-1, indicative of an increased nonspecific packaging activity in the mutant. This phenotype is reminiscent of the avian SE21Q1b mutant, which packages cellular RNAs because of ill-defined mutations in the Gag polyprotein (2,34). However, the HIV-1 mutant containing the M-MuLV NC domain appears not to package RNAs strictly on a nonspecific basis, as the mutant also displayed an ability to specifically recognize the M-MuLV ⌿ element.…”

Usage of retrovirus vectors for gene transfer into birds

“…Для вырезания (сплайсинга) части транскрипта, соот ветствующей генам gag и рої, в провирусе имеются две сигнальные последовательности -sa, sd. Ге номная РНК упаковывается вирусными белками при участии цис-активных упаковочных последова тельностей (40, находящихся между уникальным Ш-участком и началом gag [2][3][4][5][6][7][8][9].…”

“…-сигналы упаковки; У -ген неомицинфосфотрансферазы; 2 -ген гигромицинфосфотрансферазы; 3 -тимидинкиназный ген HSV; Promoter -внутренний промотор; IRES -внутренний рибосомный повторяющийся сайт [2,10]. Делегирование ^-упаковочных последова тельностей и точечные мутации в gag-гене наруша ют процесс узнавания Ga^-белком специфической вирусной РНК.…”

“…Делегирование ^-упаковочных последова тельностей и точечные мутации в gag-гене наруша ют процесс узнавания Ga^-белком специфической вирусной РНК. В этом случае происходит сборка вириона нормальной структуры, в который упако вывается клеточная РНК или РНК другого вируса, имеющего сигналы упаковки и находящегося в клетке наряду с дефектным по упаковочным после довательностям вирусом [2,10].…”