MUTATIONS IN THE PRECORE REGION OF HEPATITIS-B VIRUS SERVE TO ENHANCETHE STABILITY OF THE SECONDARY STRUCTURE OF THE PRE-GENOME ENCAPSIDATION SIGNAL

We conducted a large-scale survey to determine the frequency and clini cal significance of mutations in the pre-core region of hepatitis B vi rus (HBV). Sera from 263 patients with chronic HBV infection were anal yzed by direct sequencing of PCR-amplified HBV DNA. Four major missens e/nonsense mutations (M) were found: (M1) C --> T at nucleotide positi on 1856, Pro --> Ser at codon 15; (M2) G --> A at position 1896, Trp - -> stop at codon 28; (M3) G --> A at position 1898, Gly --> Ser at cod on 29; and (M4) G --> A at position 1899, Gly --> Asp at codon 29. The commonest conserved mutation was M0: T --> C at position 1858, Pro -- > Pro at codon 15. We found that M1 and M2 were mutually exclusive, M3 was only found in association with M1, and M4 was predominantly found in association with M2. All patients with M1 but none of those with M 2 had M0. The invariable coexistence of certain mutations in codon 15 and codons 28 and 29 and the mutual exclusion of other mutations in th ese two noncontiguous regions is related to the stem-loop structure of the pre-genome encapsidation sequence located in the precore/core reg ion. M2 and M4 enhance the stability of the stem by providing two addi tional paired sites. M1 destroys an existing base pair. However, M1 on ly occurred in the presence of M0, which provides an extra paired site , and 50% of patients with M1 had M3, a compensatory mutation that res tores base pairing at this site. Our data support the proposed seconda ry structure of the pre-genome encapsidation sequence. The primary fun ction of the mutations in the pre-core region is to enhance stability of this secondary structure to ensure perpetuation of viral replicatio n.