HBV Minus Strand Nucleotide Sequence Region Redundancy Composition On Replication

Objective: To observe the effect of base composition of terminalredundancies(r) alterations on the minus-strand on the replication capacityand the third template switch in hepatitis B virus (HBV).Methods: HBV1.1×genome mutants with a point mutation on either3’ror5’r or both r were constructed by restriction digestion andligation-independent cloning (RILC). These mutans were transfected intoHEK293cells. HBV DNA replication intermediates were extracted fromHEK293cells and then determined by southern blotting. HBV RNAsisolated from HEK293cells co-transfected with HBV1.1×and the plasmidsPEGFP-N1were assayed by Northern blot.Results: Plasmids with various mutations in the two redundanceregions were constructed. Compared with the wild type, the amount of thetotal DNA replication intermediates and rcDNA produced by mutants withthe single point mutations on the3’r and5’r did not change significantly.T1821G mutations on both of the two “r” led the mutant replicate at asignificantly lower level, featured by an apparant decrease in the amount ofvarious forms HBV DNA, while the replication capacity of the mutants withnt1820or nt1822or nt1823mutations on both side “r” were similar to that of the wild type. The amount of the pgRNA transcribed by the T1821Gdouble-mutant was only43.9%of that of the wild-type, while the3’r and the5’r T1821G single-mutants produced pgRNA at a similar level as the wildtype did.Conclusions: The whole3’r and at least part of5’r can tolerate singlebase change without affecting the replication capability of HBV and the thirdtemplate switch. T1821G double-mutant reduces significantly andspecifically the level of HBV DNA replication, and the phenotype of thismutant can be partially at least explained by the decreased transcription levelof pgRNA. Further studies are warrantted to elucidate the mechanismresponsible for the reduction of pgRNA transcription. Objective: To probe the influence of the N-terminal sequence of Mprotein on envelopment function of its S domain.Methods: HBV1.1×genome mutant without M gene and S geneexpression was constructed by restriction digestion and ligation-independentcloning (RILC), and the recombinant plasmids expressing M protein, or Sprotein, or different lengths of N-terminally truncated M proteins wereconstructed by the restriction digestion and ligation. The mutant plasmid andexpression plasmid were co-transfected into HepG2cells. Culturesupernatant was harvested at the5th day post-transfection to determine ifthere were viral replication intermediates by southern blotting.Results: The HBV1.1×genome mutant without M gene and S geneexpression, the plasmids expressing M protein, or S protein, or differentlengths of N-terminally truncated M proteins were successfully constructed.Viral replication intermediates were detectable in the culture supernatant ofHepG2cells transfected with HBV DNA with deletion of M protein and Sprotein expression, whether or not the S protein was provided bytrans-complementation.Conclusions: HBV nucleocapsids can be secreted out of cells even without being enveloped by envelope proteins from HBV-replicating cells. Amethod of immunoprecipitation (IP) to specifically separate virions isneeded to determine whether the culture supernatant contains completelyenveloped virions.