Figure 6: Effect of zinc coordinating activity of NC on the kinetics of 591R primer extension. Reactions were performed with RT in the presence or absence of WT NC, SSHS NC (32), or zinc-less NC (69). The data were plotted as % FL DNA versus Time (min). Symbols: minus NC, squares; WT NC, triangles; SSHS NC, open circles, dashed line; zinc-less NC, diamonds. Note that the minus NC and WT NC curves are the same as those shown in Figure 5 for reactions with WT RT, but the scale on the y-axis is expanded here.

Mentions: Examination of the data presented in Figure 6 shows that both SSHS NC and zinc-less NC behaved in an identical manner. Throughout the 3 h incubation period, primer extension was ∼1.7–2-fold lower than extension in the absence of NC, but by 3 h, there was no difference between the three curves. This result indicates that SSHS NC and zinc-less NC inhibited mispriming to only a very small extent. In support of this conclusion, we also observed that extent of mispriming was reduced ∼5-fold more efficiently with WT NC than with the NCs that do not coordinate zinc. Thus, the data of Figure 6 demonstrate that zinc coordination is essential for maximal NC function in our system. The results also imply that NC nucleic acid chaperone activity is responsible for destabilization of the non-PPT duplexes.

Post K, Kankia B, Gopalakrishnan S, Yang V, Cramer E, Saladores P, Gorelick RJ, Guo J, Musier-Forsyth K, Levin JG - Nucleic Acids Res. (2009)

Bottom Line: NC reduced priming by these RNAs to essentially base-line level, whereas PPT priming was unaffected.Binding studies in reactions with both NC and RT ruled out a competition binding model to explain NC's observed effects on mispriming efficiency.Taken together, these results demonstrate that NC chaperone activity has a major role in ensuring the fidelity of plus-strand priming.

Affiliation: Laboratory of Molecular Genetics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.

Abstract: During minus-strand DNA synthesis, RNase H degrades viral RNA sequences, generating potential plus-strand DNA primers. However, selection of the 3' polypurine tract (PPT) as the exclusive primer is required for formation of viral DNA with the correct 5'-end and for subsequent integration. Here we show a new function for the nucleic acid chaperone activity of HIV-1 nucleocapsid protein (NC) in reverse transcription: blocking mispriming by non-PPT RNAs. Three representative 20-nt RNAs from the PPT region were tested for primer extension. Each primer had activity in the absence of NC, but less than the PPT. NC reduced priming by these RNAs to essentially base-line level, whereas PPT priming was unaffected. RNase H cleavage and zinc coordination by NC were required for maximal inhibition of mispriming. Biophysical properties, including thermal stability, helical structure and reverse transcriptase (RT) binding affinity, showed significant differences between PPT and non-PPT duplexes and the trends were generally correlated with the biochemical data. Binding studies in reactions with both NC and RT ruled out a competition binding model to explain NC's observed effects on mispriming efficiency. Taken together, these results demonstrate that NC chaperone activity has a major role in ensuring the fidelity of plus-strand priming.