R than 75 ,Table 1 Specific infectivity (infectious units per ng p24) of
R than 75 ,Table 1 Specific infectivity (infectious units per ng p24) of EU-labeled virusesVirus No DNA No EU EU for 1st 16 h EU for 2nd 24 h EU for 48 h D443N p24 (ng/ml) ?SD <0.008 191 ?3 28 ?4 165 ?4 20 ?1 107 ?10 Specific infectivity 0 1510 2230 500 820ResultsHIV-1 RNA staining in virions requires CA core openingTo label and track HIV-1 RNA intracellularly, we incorporated a modified nucleoside, 5-ethynyl uridine (EU), into vRNA during virus production. Detection of alkynemodified nucleosides in vRNA was performed by selective ligation of an azide-containing fluorescent dye to the modified nucleoside, in PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28878015 a so-called a “click” reaction [17], and subsequent fluorescent confocal microscopy of infected target cells. Because HIV-1 particles can incorporate cellular mRNA from the producer cell [18-20], theSD, standard deviation of duplicate measurements.Xu et al. Retrovirology 2013, 10:70 http://www.retrovirology.com/content/10/1/Page 3 ofwhereas particles without MS2-binding sites purchase GW9662 showed a background of 8 co-localization of EU staining and GFP (Additional file 1: Table S1 and Figure S2). Particles were fixed to slides and stained for EU (Figure 1A). Few particles stained for vRNA when treated with buffer alone, which permeabilizes the viral membrane. However, normal cell lysate increased staining of the vRNA, suggesting that cellular factors may promote dye entry into the core. vRNA staining was also observed in particles subjected to cell lysate containing rhTRIM5, whichleads to disruption of the capsid. Without an intact CA core to protect it, vRNA should be sensitive to RNase A treatment, leading to its degradation. With RNase A added to the particles exposed to rhTRIM5, significantly fewer vRNA puncta were detected by staining in vitro compared to virions without RNase A treatment (Figure 1B). However, virions treated with normal 293T cell lysate and RNase A showed no decrease in vRNA staining, suggesting that the small dye (approximately 268 Da), but not the larger enzyme (12.7 kDa), couldA5bufferMS2-GFPEUmerge 5293T lysateMS2-GFPEUmerge 5293TrhTRIM5a lysateMS2-GFPEUmergeBMS2-GFP + EU/MS2-GFP1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1p < 0.05 NSbuffer 293T 293T-rhTRIM5amediumRNase AFigure 1 EU staining of HIV-1 RNA in vitro. (A) EU-labeled virus encoding MS2-binding sites and produced in the presence of MS2-GFP (green) was treated with cell extraction buffer, 293T cell lysate, or 293T cell lysate containing rhTRIM5 for 15 minutes, fixed onto slides, and then stained for EU (red). Particles were visualized by fluorescent confocal microscopy and shown for each individual color and with both colors (merge). (B) MS2-GFP+ and EU+ virions were counted per field after treatment with buffer, 293T cell extract, or 293T cell extract expressing rhTRIM5 in the presence of 10 g/ml RNase A. Data represent the ratio of double positive particles of the total counted MS2-GFP+ particles ?SEM of at least 4 fields. Results are representative of 2 independent experiments. A student's t test was performed on medium vs. RNase A treatment of virions incubated with either 293T or 293T-rhTRIM5 lysate. NS denotes a non-significant p value (>0.05).Xu et al. Retrovirology 2013, 10:70 http://www.retrovirology.com/content/10/1/Page 4 ofaccess the RNA. Particles made without MS2-GFP also showed significant loss of PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26795252 vRNA staining in the presence of 10 and 100 g/ml RNase A (Additional file 1: Figure S3).Labeled HIV-1 RNA is detected in infected cellsTo visualize EU-labele.