Base-pair alterations in the epsilon-lower stem due to a novel double substitution in the precore gene of HBV-e negative variant were recovered by secondary mutations

The HBe negative phenotype, a natural precore mutant (G1896A/G1897A) of HBV with aborted HBeAg expression is known to cause chronic hepatitis. The des tabilized C : G base-pairing in the lower stem of epsilon-hairpin due to G1 896A substitution is reportedly compensated by a second C1858T mutation and suggested to play an important role in enhanced selection of the HBe negat ive variant. We undertook to investigate presence of such compensatory muta tions at other positions by analyzing epsilon-sequences (nts. 1847-1907) as well as to look for their effect(s), if any, on the consensus sequence of the overlapping core-initiator of HBe negative HBV variants in CLD patients . Three of the 5 HBe negative patients had classical G1896A mutation having a second compensatory mutation at nt. 1858. One patient showed an addition al G1897A substitution, presenting as a novel precore stop codon mutation ( UGG-->UAA), followed by a compensatory mutation at position 1857. In the th ird patient, a G1899A substitution was seen which compensated the impaired U at position 1855. Other substitution and deletion mutations were also obs erved in the remaining epsilon-hairpin, which however, did not produce any compensatory mutation. Further, all the precore variants showed a conserved G at position 1904, important for the optimal context of their core-initia tor which however, remained impaired with A (nt. 1850). Our results suggest that the nts. 1851-1859 and nts. 1895-1904 in the lower stem, and restorat ion of authentic base-pairings therein, maintain the structural integrity a nd stability of the epsilon-hairpin. This may have a role in the enhanced s election of the HBe negative variants and persistence of HBV infection in c hronic liver disease patients.