A novel aptamer-based dNTP assay reveals that intact HIV virions are highly stable and do not contain enough dNTPs to...

HIV virions contain high concentrations of IP6 (inositol hexakisphosphate), which stabilize capsids by binding to pores that allow dNTPs to import into capsids during infection. The concentration of dNTPs in virions, however, is not known. The low cellular concentrations (~5 µM each) predict only ~1 copy of each dNTP in a virion, unless a mechanism for concentrating exists, as it does for IP6. Reports indicating significant DNA synthesis occurring in virions would require larger amounts of packaged dNTPs or import of dNTPs. We developed a novel aptamer-based assay capable of detecting 1 femtomole of dNTP, enabling dNTP measurements in virions. Virions from HEK 293T cells contained dNTPs with dTTP >dCTP ≈ dATP >dGTP. This order was also observed in uninfected cells. Concentrations of dNTPs in virions were 2-fold to 4-fold lower than in cells, although it was unclear if this resulted from uncertainties associated with estimating virion and cell volumes or real differences. Virions were highly stable and did not "leak" dNTPs as associated dNTPs were not released, even after incubation at 60°C for 90 min or several freeze-thaw cycles, a result that implies diffusion-mediated import of dNTPs into virions is also unlikely. Overall, results show that dNTPs in intact virions are at or below concentrations in cells, and even the most abundant dNTP (dTTP) is present at ~1 copy per virion. This precludes significant DNA synthesis in intact virions, although it is important to note that our assays may not represent all virion states. The results support a mechanism where dNTPs are passively captured during virion budding.

HIV virions presumably package small molecules (e.g., amino acids, cations, and nucleotides) passively during budding. Therefore, they would be present in virions at cellular levels, unless there is a mechanism for concentration or exclusion of the molecule (e.g., inositol hexakisphosphate (IP6), which stabilizes the viral capsid, is present at several-fold greater concentration in the virion compared with cells). Reports of DNA synthesis occurring in HIV virions suggest that HIV may also package dNTPs at levels much higher than predicted by their cellular concentrations, or virions are permeable to dNTPs. However, we show here that virion dNTP levels are at or below cellular levels with 1 or fewer molecules of each dNTP in an average virion. Furthermore, virions did not leak dNTPs when exposed to elevated temperatures or freeze-thawing, suggesting that they were not permeable. The results suggest that virions demonstrating significant DNA synthesis may be structurally altered relative to those measured here.