Identification of DNA and RNA from retroviruses using ribonuclease A

Retroviruses, such as human immunodeficiency virus (HIV), can be disrupted with chemical agents and made to disgorge their encapsidated nucleic acid. The products can be visualized by atomic force microscopy (AFM). Retroviruses may contain both viral genomic RNA and reverse transcribed DNA produced prior to integration into the host cell genome. It is necessary to know which molecules are RNA and which are DNA in order to interpret the events that transpire during infection. DNA, when imaged by AFM, is generally between one and two nanometers in thickness, more regular in its contours, and it is relatively uniform in height over its entire length; RNA, on the other hand, is less than a nanometer in thickness within single stranded regions, but varies dramatically in height over its length due to the presence of secondary structural domains. These observations, however, are often not definitive. Nonetheless, we have been able to tell one from the other using AFM, by exposing the molecules, in buffer, to moderate concentrations of RNase A. Upon exposure to the enzyme, the DNA, which cannot be cleaved, becomes coated with the protein, and the nucleic acid-protein complex exhibits a height of about three times that of the native molecule, appearing as thick cords. RNA, however, is degraded by the single strand specific RNase A into short, stable, presumably double-stranded segments, reflecting its pattern of secondary structure. Using this approach, we obtained evidence that reverse transcription of RNA into DNA may occur within the retroviral capsid.