Coexistence of Two Distinct Secretion Mutations (P5T and I97L) in Hepatitis B Virus Core Produces a Wild-Type Pattern of Secretion

Abstract

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transcription (33) or any instability of core proteins and particles (31, 33; M. Newman, F. M. Suk, and C. Shih, unpublished results).Although an immature secretion-like phenotype has also been found to occur in vivo in woodchuck and snow goose hepadnaviruses (4, 26), it is puzzling that it has not been found so far in natural infection in humans (F. M. Suk, M. H. Lin, and C. Shih, unpublished results).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 ...

Reduced Secretion of Virions and Hepatitis B Virus (HBV) Surface Antigen of a Naturally Occurring HBV Variant Correlates with the Accumulation of the Small S Envelope Protein in the Endoplasmic Reticulum and Golgi Apparatus

“…The immature secreted virions may have a selective disadvantage in vivo (e.g., less infectious), leading to the emergence of compensatory mutations, such as core mutants P5T and P130T (11,49). The possible existence of unknown compensatory mutations could explain the lack of abundant immature genomes in serum samples 9, 17, and 18, despite the presence of 97L muta- FIG.…”

“…Even though the immature secretion phenotype has been observed with woodchuck and snowgoose hepadnaviruses (7,42), it has not been reported with human patients. This may be due to the presence of naturally occurring compensatory mutations for 97L in the core protein at positions 5 (11) or 130 (49), both changing a highly conserved proline to threonine.…”

“…1, sequence analysis of the core gene from serum samples 9, 17, and 18 revealed the presence of a 97L mutation, but not compensatory P5T or P130T mutations (references 11 and 49 and data not shown). Based on all the published HBV core 97L mutant sequences, compensatory core mutations P5T or P130T are associated with approximately 76% of the 97L mutants (11). Previously, we demonstrated by a genetic approach that the core-envelope interaction is important for virion secretion (27).…”

Stability and Morphology Comparisons of Self-AssembledVirus-Like Particles from Wild-Type and Mutant Human Hepatitis B VirusCapsidProteins

“…To date, the structural basis for either the immature secretion or replication advantage phenotypes has remained unclear. Furthermore, it is also unclear what could be the structural basis for compensatory mutations that can offset the I97L immature secretion (4,16,35). Since isoleucine and leucine are isomers with identical molecular weights, it is puzzling that such a subtle structural difference in side chains can cause a significant functional consequence on viral replication and virion secretion.…”

The Leeuwenhoek lecture 2006. Microscopy goes cold: frozen viruses reveal their structural secrets

“…A frequent natural mutation, observed in chronically infected patients, of isoleucine 97 to leucine (I97L) leads to the secretion of immature virus particles containing single-stranded DNA (Yuan et al 1999). Two other natural mutations, proline 5 to threonine (P5T) and leucine 60 to valine (L60V ), give very low secretion of virus (Le Pogam et al 2000), and, strikingly, the P5T mutation corrects for the immature secretion of the I97L mutant when present as the double mutant I97L, P5T (Chua et al 2003). Two other residues close by (leucine 95 and lysine 96) when changed to alanine result in cores in the cytoplasm that do not become enveloped or secreted (Ponsel & Bruss 2003).…”