Into 293T cells and levels of Gag proteins in pelletable viral

Into 293T cells and levels of Gag proteins in pelletable viral 125-65-5 web 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 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 SPDP site 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 drives the specific incorporation of the gRNA into assembling viral particles [12] by binding to the 59 UTR of the gRNA with high affinity (for review [16,39]). Subsequently, GaggRNA complexes reach the plasma membrane where formation of viral particles is completed (for review [18]). As for other retroviral NC’s [40,41] the NC packaging function primarily relies on its ability to interact with nucleic acid sequences, notably the 59 UTR of the gRNA in a very tight mode, which drives gRNA selection. At the same time NC binding to the gRNA causes genome dimerization chaperoned by the NC annealing activity [42]. Recently, we reported that mutating the NC ZF of HIV-1 resulted in virions where the newly made viral DNA replaced the gRNA, due to the RTion of the gRNA before virus release. This study also showed a correlation between intravirion levels of viralDNA and gRNA among the HIV-1 NC-mutant particles [43]. To determine whether this property was conserved in gammaretroviruses such as MuLV, we first examined the impact of NC mutations on the level of gRNA packaging in a quantitative manner by RT-qPCR. For the first time, the ability of MuLV NC to package the gRNA was monitored by RT-qPCR. Identical volumes of MuLV containing medium were collected and MuLV particles pelleted by centrifugation through a sucrose cushion. Next, MuLV samples were treated by RNAse-free DNase before particle lysis to remove any transfected plasmid DNA, which could interfere with the qPCR assays. As an internal control, we used aliquots of NCmutant HIV-1 virions that contain 18204824 a high level of viral DNA. This allowed us to monitor the level of the MuLV particle recovery after ultracentrifugation and DNase treatment. Nucleic acids were purified by two successive phenol-chloroform treatments. The recovered RNAs were reverse transcribed using an oligodT primer and quantitative analyses were carried out using PCR primer pairs that specifically target the intronic region of the v.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 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 drives the specific incorporation of the gRNA into assembling viral particles [12] by binding to the 59 UTR of the gRNA with high affinity (for review [16,39]). Subsequently, GaggRNA complexes reach the plasma membrane where formation of viral particles is completed (for review [18]). As for other retroviral NC’s [40,41] the NC packaging function primarily relies on its ability to interact with nucleic acid sequences, notably the 59 UTR of the gRNA in a very tight mode, which drives gRNA selection. At the same time NC binding to the gRNA causes genome dimerization chaperoned by the NC annealing activity [42]. Recently, we reported that mutating the NC ZF of HIV-1 resulted in virions where the newly made viral DNA replaced the gRNA, due to the RTion of the gRNA before virus release. This study also showed a correlation between intravirion levels of viralDNA and gRNA among the HIV-1 NC-mutant particles [43]. To determine whether this property was conserved in gammaretroviruses such as MuLV, we first examined the impact of NC mutations on the level of gRNA packaging in a quantitative manner by RT-qPCR. For the first time, the ability of MuLV NC to package the gRNA was monitored by RT-qPCR. Identical volumes of MuLV containing medium were collected and MuLV particles pelleted by centrifugation through a sucrose cushion. Next, MuLV samples were treated by RNAse-free DNase before particle lysis to remove any transfected plasmid DNA, which could interfere with the qPCR assays. As an internal control, we used aliquots of NCmutant HIV-1 virions that contain 18204824 a high level of viral DNA. This allowed us to monitor the level of the MuLV particle recovery after ultracentrifugation and DNase treatment. Nucleic acids were purified by two successive phenol-chloroform treatments. The recovered RNAs were reverse transcribed using an oligodT primer and quantitative analyses were carried out using PCR primer pairs that specifically target the intronic region of the v.