Journal: Nature microbiology
Article Title: Deep sequencing of HIV-1 reverse transcripts reveals the multifaceted anti-viral functions of APOBEC3G
Figure Lengend Snippet: Effects of A3G on profiles of nascent HIV-1 cDNA products in infected T cells. a) Early steps of the HIV-1 life cycle illustrating three proposed anti-retroviral mechanisms for A3G that are deaminase-dependent (pathways 1 and 2) or -independent (pathway 3). b) Diagram of HIV-1 reverse transcription. The first full intermediate, (-)sss cDNA, is completed in step 3. PBS: primer binding site, PPT: polypurine tract. c) Basic steps of sequencing library preparation. During infection, HIV-1 produces nascent viral cDNAs of increasing length (see step 2 in b). Sequencing reads reveal precise 3’-termini at the points of adaptor-viral DNA ligation (red box). d) Immunoblot analysis of HIV-1 virion lysates from one of six independent virus preparations. ‘Low’ or ‘High’ A3G refers to producer cell transfection ratios of 1:10 or 1:4, respectively (A3G expression plasmid to NL4.3/ΔVif). e) Single-cycle virion infectivity measured by β-galactosidase activity in challenged TZM-bl reporter cells. f) Quantitative PCR measuring cDNA abundance in CEM-SS cells at 4 h post-infection. For e) and f) the individual data points with their mean and standard deviation of eight independent infections from six virus preparations are shown. *** indicates p-value of
Article Snippet: Unless otherwise noted, the reactions were initiated by addition of 10 nM HIV-1 RT (Worthington), and incubated for the indicated amount of time at 37°C.
Techniques: Infection, Binding Assay, Sequencing, DNA Ligation, Transfection, Expressing, Plasmid Preparation, Activity Assay, Real-time Polymerase Chain Reaction, Standard Deviation