The biosynthesis of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) requires the removal of the covalently linked viral polymerase from the 5= end of the minus strand [()strand] of viral relaxed circular DNA (rcDNA), which generates a deproteinated rcDNA (DP-rcDNA) intermediate. In the present study, we systematically characterized the four termini of cytoplasmic HBV DP-rcDNA by 5=/3= rapid amplification of cDNA ends (RACE), 5= radiolabeling, and exonuclease digestion, which revealed the following observations: (i) DP-rcDNA and rcDNA possess an identical 3= end of ()strand DNA; (ii) compared to rcDNA, DP-rcDNA has an extended but variable 3= end of plus strand [()strand] DNA, most of which is in close proximity to direct repeat 2 (DR2); (iii) DP-rcDNA exhibits an RNA primer-free 5= terminus of ()strand DNA with either a phosphate or hydroxyl group; and (iv) the 5= end of the DP-rcDNA ()strand is unblocked at nucleotide G1828, bearing a phosphate moiety, indicating the complete removal of polymerase from rcDNA via unlinking the tyrosyl-DNA phosphodiester bond during rcDNA deproteination. However, knockout of cellular 5= tyrosyl-DNA phosphodiesterase 2 (TDP2) did not markedly affect rcDNA deproteination or cccDNA formation. Thus, our work sheds new light on the molecular mechanisms of rcDNA deproteination and cccDNA biogenesis. [ABSTRACT FROM AUTHOR]