Cis-preferential recruitment of duck hepatitis B virus core protein to the RNA/polymerase preassembly complex.
Hepadnaviral replication requires the concerted action of the polymerase and core proteins to ensure selective packaging of the RNA pregenome into nucleocapsids. Virus assembly is initiated by cis-preferential binding of polymerase to the encapsidation signal straightepsilon, present on pregenomic RNA. Using the duck hepatitis B virus (DHBV) model, we analyzed how core protein is recruited to the RNA/polymerase preassembly complex. Two sets of trans-complementation assays were performed in cotransfected hepatoma cells. First, a replication-competent DHBV construct was tested for its ability to rescue replication of genomes bearing mutations within the core region. Self-packaging of wild-type pregenomes was more efficient than cross-packaging of core-deficient pregenomes, and this bias was strongly enhanced if mutant pregenomes coded for self-assembly-competent, but packaging-deficient, core proteins. Second, the site of wild-type core protein translation, i.e., pregenomic RNA (cis) or separate messenger RNA (trans), was analyzed for its effect on the phenotype of a previously described dominant-negative (DN) DHBV core protein mutant. This mutant forms chimeric nucleocapsids with wild-type core proteins and blocks reverse transcription within most, but not all, mixed particles. Strikingly, suppression of viral DNA synthesis by the mutant increased 100-fold when wild-type core protein was provided in trans. Our results suggest that recruitment of core protein to the DHBV preassembly complex occurs in a cis-preferential manner. This mechanism may account for the leakiness of DN DHBV core protein mutants targeting reverse transcription.
von Weizsäcker
Fritz
F
Department of Medicine II, University of Freiburg, Freiburg, Germany. weiz@ukl.uni-freiburg.de
Köck
Josef
J
Wieland
Stefan
S
Beck
Jürgen
J
Nassal
Michael
M
Blum
Hubert E
HE
D001665:Binding Sites; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D004279:DNA, Viral / Q000096:biosynthesis; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D019281:Dimerization; D015611:Hepatitis B Virus, Duck / Q000737:chemistry* / Q000502:physiology; D009154:Mutation; D019251:Nucleocapsid / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism*; D014158:Transcription, Genetic; D014758:Viral Core Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
D004279:DNA, Viral; D012333:RNA, Messenger; D012367:RNA, Viral; D014758:Viral Core Proteins; D012321:DNA-Directed RNA Polymerases