Unlike a Tokyo isolate of hepatitis B virus variants, we found a Shanghai isolate that secretes few virions with an immature genome despite its core I97L mutation. Core mutations P5T and I97L were found to be mutually compensatory in offsetting their respective distinct effects on virion secretion.Hepatitis B virus (HBV) replicates by using reverse transcriptase (11,24). Due to the low fidelity of the polymerase, error-prone replication and natural selection in vivo lead to the accumulation of multiple mutations in HBV variants predominant in chronic carriers (20,22). The most frequent natural mutation in the HBV core protein occurs at amino acid 97 (5, 6, 9, 10, 13). It remains a challenge to elucidate the functional significance of these prevalent and predominant mutations, since there is no a priori knowledge about what kind of assays should be used. Recently, a so-called "immature secretion" phenotype was demonstrated to be a global phenotype in tissue culture by introducing a 97L mutation into the core gene of a wild-type HBV genetic background of subtype adr and ayw origins (31,33). This phenotype of HBV variant 97L is interesting, because it represents an exception to the dogma of preferential export of virions containing mature genomes (highermolecular-weight viral DNA in relaxed circle form) in wild-type hepadnaviruses (24). Unlike the wild-type HBV, the 97L mutant secretes similar amounts of mature and immature genomes (lower-molecular-weight viral DNA in single-strand form). This phenotype is not caused by any deficiency in reverse transcription (33) or any instability of core proteins and particles (31, 33; M. Newman, F. M. Suk, and C. Shih, unpublished results).Although an immature secretion-like phenotype has also been found to occur in vivo in woodchuck and snow goose hepadnaviruses (4, 26), it is puzzling that it has not been found so far in natural infection in humans (F. M. Suk, M. H. Lin, and C. Shih, unpublished results). Furthermore, it remains unclear whether an immature secretion phenotype can still be observed in the genetic context of naturally occurring variants, which often contain multiple mutations throughout the genome. To address these issues, we took a reciprocal approach by reverting the natural mutation at amino acid 97 in a naturally occurring HBV variant from leucine back to isoleucine (L97I) and asked whether the predicted immature secretion phenotype can be abolished by eliminating the leucine residue at position 97 of the HBV core antigen (HBcAg).This parental HBV variant clone of adr subtype origin was isolated from a hepatocellular carcinoma patient from Shanghai, China (clone 14 in references 18 and 19). In addition to the hot spot mutation I97L, it contains multiple frequent mutations, including core mutations P5T and S87G, enhancer II mutations at nucleotides 1762 and 1764, and mutations truncating the X protein and abrogating the production of the pre-S2-containing M envelope protein (18). The 3.2-kb monomeric HBV genome in clone 14 was released from the pUC18 vector ...