Mutational analysis of the hepatitis B virus P gene product: domain structure and RNase H activity

Abstract

To correlate the hepatitis B virus P gene with the enzymatic activities predicted to participate in hepadnavirus reverse transcription, a series of P gene mutants containing missense mutations, in-phase insertions, and in-phase deletions was constructed by site-directed mutagenesis. These mutants were tested in the context of otherwise intact hepatitis B virus genomes for the ability to produce core particles containing the virus-associated polymerase activity. The results obtained suggest that the P protein consists of three functional domains and a nonessential spacer arranged in the following order: terminal protein, spacer, reverse transcriptase/DNA polymerase, and RNase H. The first two domains are separated by a spacer region which could be deleted to a large extent without significant loss of endogenous polymerase activity. In cotransfection experiments, all P gene mutants could be complemented in trans by constructs expressing the wild-type gene product but not by a second P gene mutant. This indicates that the multifunctional P gene is expressed as a single translational unit and independent of the core gene and furthermore that the gene product is freely diffusible and not processed before core assembly.

Hepadnaviral assembly is initiated by polymerase binding to the encapsidation signal in the viral RNA genome.

“…by its absence in mutant P11, which carried a stop codon at the very beginning of the P gene (lanes 1 and 2) and (ii) by a strong reduction of its immunoprecipitation by competition with the homologous peptides P6 and P8 (lane 4). To prove further that the 32P-labelled 90 kDa protein detected was a P gene product, mutant PA1-P12, which was derived from construct P12 by introducing a 90 amino acid deletion into the spacer region (Radziwill et al, 1990; Table I) was analysed in parallel. As shown in lane 5, a protein with an expected size of -80 kDa was detected and again immunoprecipitation could be competed with the homologous peptides (lane 6).…”

“…The basic constructs, pCH3097, pMTpol and pCH3142, are described in Bartenschlager et al (1992). Radziwill et al (1990) and Junker-Niepmann et al (1990). Construction of the HBV-lacZ hybrid plasmids ( Figure 6) is described in Bartenschlager et al (1990).…”

“…Construction of the HBV-lacZ hybrid plasmids ( Figure 6) is described in Bartenschlager et al (1990). P gene mutants, P1, P2, P1 I and PA 1. are described in Radziwill et al (1990) and Bartenschlager et al (1990). Mutant P19 was generated by site-directed mutagenesis (Zoller and Smith, 1984) using the following oligonucleotide (mismatch to the wild type sequence underlined): CTGGGCTTTTTTCTTCTA.…”

“…This raises the question of how a freely diffusible hepadnaviral reverse transcriptase/DNA polymerase is directed specifically onto the viral RNA template and how promiscuous reverse transcription of cellular RNAs is prevented. Substantial progress towards answering these questions has been made by mutation and complementation analysis of P gene functions, which revealed that the hepadnaviral P gene product is a multidomain protein that differs from the retroviral pol gene product by lacking the protease and integrase domains, and instead containing amino-terminally the DNA terminal protein (TP) involved in the HBV-specific initiation of reverse transcription (Gerlich and Robinson, 1980;Bartenschlager and Schaller, 1988;Radziwill et al, 1990). Moreover, and again in contrast to the retroviral polymerase, the hepadnaviral P protein was found to participate as an essential structural component in the packaging of the RNA pregenome Hirsch et al, 1990).…”

Functional incorporation of green fluorescent protein into hepatitis B virus envelope particles

“…For replication of HBV in the HuH-7 liver cell line, plasmid pHBV was used that carries a 1.1 mer of the HBV DNA genome (Radziwill et al, 1990). This plasmid was cotransfected with pEGFP.S by the calcium phosphate precipitation technique.…”