Figure 1.
Primary structure of MuLV NC protein and schematic representation of the mutants used here.
Numbers indicate amino acid positions. The zinc finger is drawn with the Zn ion coordinated by the CCHC residues. The broken line represents the deleted amino acids.
Figure 2.
Viral particles produced by MuLV producer cells.
(A) MuLV expression was analysed in cells by immunoblotting with an anti-CA antibody. Actin was probed as a loading control. (B) Mature capsid (CA) and Gag were detected in viral samples. Signals were quantified with ImageQuant software. For each lane, signals corresponding to all the bands were added and normalized to wt level (right part). Error bars indicate SD from at least three independent experiments.
Figure 3.
Strategy of qPCR to monitor the MuLV nucleic acid species.
Templates and primers used for qPCR analyses were schematically represented. Only the spliced SD' RNA (SD'/SA) important for this study and the gRNA are indicated. A color code was used to illustrate the specificity of the PCR-primer pairs (arrows) that were used to quantify the pR88 plasmid (blue) which generates the MuLV gRNA transcript (orange) and the spliced SD' RNA (green). Numbers refer to the position of the elongation start. Bottom panel: Products of viral reverse transcription. The primer pairs used to detect the intermediate ss-cDNA (red), Pol cDNA (orange), SD' cDNA (green) and the final product FL DNA (purple) are shown.
Figure 4.
Quantitative analysis of the nucleic acid content of viral particles released from MuLV producer cells.
(A) Quantitation of viral gRNA incorporated in wt or mutant viruses by RT-QPCR. Mock controls were subtracted from assays. Error bars indicate SD from at least four independent experiments. (B) Viral DNA levels were determined by qPCR in the wt and mutant virions. DNA was extracted from same virion samples as those used before for gRNA quantitation. Error bars indicate SD from at least seven independent experiments. (C) There is no correlation between gRNA and viral DNA levels among the MuLV mutants. For comparative purpose, data obtained with HIV-1 virions deleted of the second ZF (ΔZF2) are given (left part) [26], [38]. To facilitate the comparison, levels of viral gRNA and ss-cDNA were normalized to those measured in wt virions.
Figure 5.
Coproduction of MuLV and HIV-1 virions.
Supernatant were collected from cells cotransfected with MuLV and wt or ΔZF2 HIV-1 molecular clones (MuLV:HIV ratio of 1∶3). Released virions were pelleted and proteins analyzed by Western blotting (A). The same blot was used to probe the MuLV and HIV-1 CA proteins. The intravirion levels of MuLV and HIV-1 DNA were determined and calculated as in Fig 4C (B).