On RNA. Oligo(dT) was used as RTion-primer. A control experiment was systematically performed without RT to look for the absence of DNA contamination. Quantitative PCR assay was achieved with 2.5 of RT reaction or 125 ng of cellular DNA sample and SYBR Green kit (Roche) using the RotorGene (Labgene) systems. The products were GNF-7 amplified by 35 cycles: 95uC for 15s; 60uC for 15s and 72uC for 20s. The following oligonucleotides pairs (0.5 mM) were used: for gRNA and PolcDNA, sMLV3350: 59-TATCGGGCCTCGGCAAGAAAG sense and aMLV3600: 59-AAACAGAGTCCCCGTTTTGGTG antisense; for ss-cDNA, sensMLV+1: 59-GCGCCAGTCCTCCGATTGACTGAG sense and aMLV142: 59-GAAAGACCCCCGCTGACGGGTAGTC antisense; for FL cDNA, sMLV-368 59-AGAATAGAGAAGTTCAGATC sense and aMLV290 59- GCTAACTAGTACCGACGCAGGCGC; for SD’, sMLV1450: 59-CTG CTG ACG GGA GAA GAA AAA CA sense and aMLV5620 59-GCGGACCCACACTGTGTC antisense; for GAPDH, sGAPDH721 59-GCTCACTGGCATGGCCTTCCGTGT sense and aGAPD931 59-TGGAGGAGTGGGTGTCGCTGTTGA antisense; for plasmid transfected detection spRR88-784: 59CACAGAACTAGTCAGAGACAGCAT sense and aMLV-431:Results Role of the NC basic sequence and zinc finger in virus assemblyThe conserved basic residues and the unique ZF of MuLV NC are important functional determinants in virus replication (Fig 1). To study their role in MuLV assembly, we used full-length molecular clones with mutation or deletion in the NC domain of Gag (called NC here) (Fig 1). The N-terminal basic residues of NC (Fig 1) were deleted generating the NC D16?3 mutant clone lacking residues R16 through R23. The ZF motif was either mutated by changing Cys-39 to a Ser (C39S), or completely deleted (DZF). In parallel, we used a molecular clone (PR-) where the protease was inactivated by a deletion (nt 2421?546). Mutating the protease enzyme prevented processing of the two Gag and GagPol precursors. Importantly, inactivating the HIV-1 protease has an impact on intravirion DNA level [29]. Each MuLV molecular clone was transfected into 293T cells and levels of Gag proteins in pelletable viral particles were SPI 1005 site monitored by immunoblotting, as previously reported [37]. To detect Gag and mature capsid (CA) in cellular and viral samples, immunoblotting was conducted with an anti-CA primary antibody (Fig 2). When virus release was efficient (wt and PR-), Gag did 1317923 not accumulate in cells. As shown in Fig 2B, the levels of Gag processing variedRoles of the NC in HIV-1 and MuLV Replicationssomewhat, as illustrated by the ratios of CA to Gag proteins. To determine the level of virus produced, signals were quantified with ImageQuant software, normalized to wt level and average values from three independent experiments are given in Fig 2B. Results indicate that the ZF mutants, C39S and DZF, produced wt level of viral particles in the culture medium, but these mutant particles contained incompletely processed Gag. This partial Gag processing might explain, at least in part, the loss of MuLV infectivity when mutating the NC cysteines in the zinc finger [8,38]. As expected, the PR- mutant produced immature virions at wt level. In contrast, deleting the N-ter basic residues (D16?3) induced a severe decrease (86 ) of MuLV production (Fig 2). The deletion of the basic residues caused a dramatic release defect, while ZF mutation or deletion induced only a default in Gag processing.Quantitative analysis of the impact of NC mutations on genomic RNA packaging into virionsNC is thought to drive the interaction of Gag with nucleic acids and as such.On RNA. Oligo(dT) was used as RTion-primer. A control experiment was systematically performed without RT to look for the absence of DNA contamination. Quantitative PCR assay was achieved with 2.5 of RT reaction or 125 ng of cellular DNA sample and SYBR Green kit (Roche) using the RotorGene (Labgene) systems. The products were amplified by 35 cycles: 95uC for 15s; 60uC for 15s and 72uC for 20s. The following oligonucleotides pairs (0.5 mM) were used: for gRNA and PolcDNA, sMLV3350: 59-TATCGGGCCTCGGCAAGAAAG sense and aMLV3600: 59-AAACAGAGTCCCCGTTTTGGTG antisense; for ss-cDNA, sensMLV+1: 59-GCGCCAGTCCTCCGATTGACTGAG sense and aMLV142: 59-GAAAGACCCCCGCTGACGGGTAGTC antisense; for FL cDNA, sMLV-368 59-AGAATAGAGAAGTTCAGATC sense and aMLV290 59- GCTAACTAGTACCGACGCAGGCGC; for SD’, sMLV1450: 59-CTG CTG ACG GGA GAA GAA AAA CA sense and aMLV5620 59-GCGGACCCACACTGTGTC antisense; for GAPDH, sGAPDH721 59-GCTCACTGGCATGGCCTTCCGTGT sense and aGAPD931 59-TGGAGGAGTGGGTGTCGCTGTTGA antisense; for plasmid transfected detection spRR88-784: 59CACAGAACTAGTCAGAGACAGCAT sense and aMLV-431:Results Role of the NC basic sequence and zinc finger in virus assemblyThe conserved basic residues and the unique ZF of MuLV NC are important functional determinants in virus replication (Fig 1). To study their role in MuLV assembly, we used full-length molecular clones with mutation or deletion in the NC domain of Gag (called NC here) (Fig 1). The N-terminal basic residues of NC (Fig 1) were deleted generating the NC D16?3 mutant clone lacking residues R16 through R23. The ZF motif was either mutated by changing Cys-39 to a Ser (C39S), or completely deleted (DZF). In parallel, we used a molecular clone (PR-) where the protease was inactivated by a deletion (nt 2421?546). Mutating the protease enzyme prevented processing of the two Gag and GagPol precursors. Importantly, inactivating the HIV-1 protease has an impact on intravirion DNA level [29]. Each MuLV molecular clone was transfected into 293T cells and levels of Gag proteins in pelletable viral particles were monitored by immunoblotting, as previously reported [37]. To detect Gag and mature capsid (CA) in cellular and viral samples, immunoblotting was conducted with an anti-CA primary antibody (Fig 2). When virus release was efficient (wt and PR-), Gag did 1317923 not accumulate in cells. As shown in Fig 2B, the levels of Gag processing variedRoles of the NC in HIV-1 and MuLV Replicationssomewhat, as illustrated by the ratios of CA to Gag proteins. To determine the level of virus produced, signals were quantified with ImageQuant software, normalized to wt level and average values from three independent experiments are given in Fig 2B. Results indicate that the ZF mutants, C39S and DZF, produced wt level of viral particles in the culture medium, but these mutant particles contained incompletely processed Gag. This partial Gag processing might explain, at least in part, the loss of MuLV infectivity when mutating the NC cysteines in the zinc finger [8,38]. As expected, the PR- mutant produced immature virions at wt level. In contrast, deleting the N-ter basic residues (D16?3) induced a severe decrease (86 ) of MuLV production (Fig 2). The deletion of the basic residues caused a dramatic release defect, while ZF mutation or deletion induced only a default in Gag processing.Quantitative analysis of the impact of NC mutations on genomic RNA packaging into virionsNC is thought to drive the interaction of Gag with nucleic acids and as such.
