Journal: Journal of Virology
Article Title: Requirement for Integrase during Reverse Transcription of Human Immunodeficiency Virus Type 1 and the Effect of Cysteine Mutations of Integrase on Its Interactions with Reverse Transcriptase
Figure Lengend Snippet: cDNA synthesis and RT activities of wild-type and mutant viruses. (A) Quantitative analysis of viral cDNA synthesis by real-time PCR. The amount of early reverse-transcribed viral DNA present in CEM cells was measured 8 h after infection with the wild-type (WT) or mutant viruses (shaded bars). All viral stocks were treated with RNase-free DNase I to remove potential contamination by plasmid DNA. The culture medium alone was used in the mock infection (hatched bar), and equivalent amounts of the respective heat-inactivated viruses (solid bars) were used as negative controls for infection. For normalization of the DNA input of each sample, the human β-globin gene was amplified under identical PCR conditions. The results are expressed as the number of copies of HIV-1 cDNA detected per microgram of total DNA extracted from infected cells. The values shown represent the averages of two independent experiments. In each experiment, the standards and test samples were run in duplicate. (B) Particle-associated RT activities of wild-type and C130S-containing mutant viruses. After transient transfection of 293T cells, WT (shaded bar) and mutant (open bars) viruses were harvested, concentrated, lysed, and assayed for particle-associated RT activity as described in Materials and Methods. The result for each viral clone is shown as the mean ± standard error of three experiments using independently derived virus stocks.
Article Snippet: Fifty nanograms of p24 equivalent of viral stock was treated with 5 U of RNase-free DNase I (Amersham Pharmacia) at room temperature for 1 h. An equal amount of viral stock was heat inactivated by incubation at 65°C for 2 h after DNase I treatment.
Techniques: Mutagenesis, Real-time Polymerase Chain Reaction, Infection, Plasmid Preparation, Amplification, Polymerase Chain Reaction, Transfection, Activity Assay, Derivative Assay