Quantitative DNA Binding, Looping, and Compaction Properties of the HIV-1 Viral Protein R

Abstract

Human immunodeficiency virus type 1 (HIV-1) Viral protein R (Vpr) is packaged into virions (∼200 molecules) and is essential for viral replication. While several in vivo functions have been attributed to Vpr, one primary function is believed to be transport of the HIV-1 pre-integration complex into the nucleus. Because the nuclear pore diameter is approximately 25 nm, the DNA must be highly compact in order to enter the nucleus. To understand the mechanism by which Vpr may facilitate this nuclear transport, we combine single molecule stretching and atomic force microscopy (AFM). We measure the DNA binding affinity of Vpr for the first time. We then investigate the ability of Vpr to both bind and compact DNA. To do this, we hold DNA at low force for fixed times, allowing it to form loops and other compact structures. We find that the timescale required for the formation of large loops due to protein-DNA bridging is several minutes. In contrast, by holding the DNA molecule at a constant force of 15 pN and measuring its length change, Vpr can also actively compact DNA on the timescale of tens of seconds (15 ± 2 s), representing a different compaction process involving DNA shortening likely due to Vpr binding alone. The persistence length determined from AFM at a low concentration is much longer than that of bare DNA, suggesting that Vpr forms shorter but more rigid structures upon binding DNA. AFM images also demonstrate DNA bridging between strands, consistent with the looping observed in optical tweezers experiments. These results support a model in which Vpr translocates the viral DNA into the nucleus by forming compact structures mediated by protein-DNA and protein-protein interactions.