| During attempts to develop aptamers that bind to the nucleocapsid protein of HIV-1 (i.e. NCp7) with high affinity, a heretofore unreported property of the protein---the ability to mediate degradation of nucleic acids, was discovered. Using lambda DNA as a model nucleic acid system, it was shown that NCp7-mediated degradation of nucleic acids is non-specific, depends on incubation time, the concentration of NCp7, and the presence of divalent and monovalent cations. It was further demonstrated that degradation can be abrogated if NCp7 is pre-incubated with NCp7-inhibitors. Lyophilization of NCp7 can induce irreversible changes in its secondary structure that result in loss of its nucleic acid degrading ability. HIV-1 Gag and SIV Gag also showed nucleic acid degrading ability, implying that this property of NCp7 extends to the Gag polyproteins of which it forms a part. Zinc finger mutants of NCp7 showed differential degradation towards DNA, with a proximal zinc finger mutant (NCp7SSHS/CCHC) showing enhanced nucleic acid degrading ability, compared to WT and a distal zinc finger mutant (NCp7CCHC/SSHS ). It was further shown that, relative to WT NCp7, the NCp7SSHS/CCHC mutant had lost the ability to discriminate between virus-derived RNA and non-viral RNA, so that it degraded both. This observation provides an explanation for the observed reduction or absence of viral RNA packaged in budding virions in whole cell assays performed with that mutant. The results have implications for the poorly understood viral packaging mechanism, in that the enrichment of viral nucleic acids over host cell derived nucleic acids could occur through destruction of the latter by the protein. This is referred to as the "needle in a haystack" hypothesis.;These findings not only raise questions about the potential utility of nucleic acid-based antiretrovirals, but also provide a rationale for why those reported so far are of limited effectiveness. However it also determined that NCp7-binding aptamers that are susceptible to NCp7-mediated degradation can be protected by appending them to HIV-1 Psi packaging signal stem loops. |
