Reconstitution of retroviral fusion and uncoating in a cell-free system

The molecular events underlying the immediate steps of retroviral uncoating, occurring after membrane fusion and leading to the formation of an active reverse transcription complex, are not known. To better understand these processes, we have developed a cell-free system that recapitulates these early steps of retroviral replication by using avian sarcoma and leukosis virus as a model retrovirus. The substrates used in this system are viral particles that are trapped before completing membrane fusion. These virions are induced to fuse out of endosomes and the viral cores are released into solution where they are amenable to biochemical manipulation. This system revealed that membrane fusion is not sufficient to stimulate the formation of a reverse transcription complex. Instead, ATP hydrolysis and cellular factors >5 kDa in size are required. Furthermore, later steps of avian sarcoma and leukosis virus reverse transcription were stimulated by nuclear factors. The cell-free system should now allow for the definition of retroviral uncoating mechanisms and facilitate the identification and characterization of the cellular factors involved.