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Figure 4 | Retrovirology

Figure 4

From: The dUTPase-related gene of bovine immunodeficiency virus is critical for viral replication, despite the lack of dUTPase activity of the encoded protein

Figure 4

The dUTPase activity in virions and in cell extracts. The PPiLight pyrophosphate detection assay was performed to measure the dUTPase activity in viral extracts of WT BIV and EIAV, in Cf2Th cells that were chronically-infected by WT BIV (generating ~107 viral particles/ml), or in uninfected Cf2Th cells. A dUTPase activity was also tested in a complementation assay of recombinant BIV dUTPase and the extract of uninfected Cf2Th cells. The assay conditions are described in Methods and in the text. All viruses and cells were lysed and then incubated at 37°C for 30 min with dUTP-containing reaction buffer. Viral extracts were prepared from ~8.8 × 107 viral particles of either EIAV or WT BIV. Viruses were lysed using 0.5% Triton X-100 in the DMEM-FCS medium. Cell extracts were prepared from the pellets of ~5 × 104 infected or uninfected Cf2Th cells by disruption in 100 μl of 0.5% Triton X-100 in PBS for 30 minutes on ice (followed by removing the insoluble material at 15000 rpm at 4°C). The activities in cells were then tested with equal amounts of the appropriate cell extracts. The complementation assay was performed by incubating 25 ng (in 5 μl) of the purified recombinant BIV dUTPase (the 74-residues version) with 10 μl of uninfected Cf2Th cells extract (prepared from ~5 × 104 cells). All reactions were initiated by adding the 40 μl of dUTP-containing assay buffer for 30 min at 37°C, followed by heat inactivation, and then assayed as described in Methods. The shown values were obtained after subtracting the non-specific background produced with lysis buffer.

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