The formation of 2-LTR circles requires the action of the cellular non-homologous DNA end-joining pathway. Specifically the cellular Ku, XRCC4 and ligase IV proteins are needed. Evidence for this is provided by the observation that cells mutant in these functions do not support detectable formation of 2-LTR circles, though integration and formation of 1-LTR circles are mostly normal. The reaction takes place in the nucleus, and formation of 2-LTR circles has been used as a surrogate assay for nuclear transport. It has also been suggested that the NHEJ system affects the toxicity of retroviral infection.