HEPATITIS-B VIRUS NUCLEOCAPSID ENVELOPMENT DOES NOT OCCUR WITHOUT GENOMIC DNA-SYNTHESIS

Assembly of the enveloped hepatitis B virus (HBV) is initiated by pack aging of the RNA pregenome and the viral reverse transcriptase-DNA pol ymerase into a nucleocapsid, The pregenome is then reverse transcribed into single-stranded minus-polarity DNA, which is subsequently replic ated to double-stranded DNA. All replicative intermediates are observa ble in capsids within infected liver, but only relatively mature nucle ocapsids containing partially double stranded DNA are found in secrete d virions. This observation suggests that maturation of the genome wit hin the capsid is required for envelopment and secretion, We show that the differential distribution of replicative intermediates between in tracellular nucleocapsids and secreted virions is also observable in h uman hepatoma cells transfected with wild-type HBV genomes. However, n ucleocapsids were not enveloped or secreted when they were produced by an HBV genome carrying a missense mutation in the DNA polymerase that eliminates all DNA synthesis, An HBV missense mutant defective in the RNase H activity of the polymerase which allowed minus-strand DNA syn thesis but not formation of double-stranded DNA was able to form virio n-like particles. These experiments demonstrate that immature nucleoca psids containing pregenomic RNA are incompetent for envelopment and th at minus-strand DNA synthesis in the interior lumen of the capsid is c oupled to the appearance of a signal on the exterior of the nucleocaps id that is essential for its envelopment.