title: TP-RT domain interactions of duck hepatitis B virus reverse transcriptase in cis and in trans during protein-primed initiation of DNA synthesis in vitro.
abstract: The hepadnavirus reverse transcriptase (RT) has the unique ability to initiate viral DNA synthesis using RT itself as a protein primer. Protein priming requires complex interactions between the N-terminal TP (terminal protein) domain, where the primer (a specific Y residue) resides, and the central RT domain, which harbors the polymerase active site. While it normally utilizes the cis-linked TP to prime DNA synthesis (cis-priming), we found that the duck hepatitis B virus (DHBV) RT domain, in the context of the full-length RT protein or a mini-RT construct containing only truncated TP and RT domains, could additionally use a separate TP or RT domain in trans as a primer (trans-priming). trans interaction could also be demonstrated by the inhibitory effect (trans-inhibition) on cis-priming by TP and RT domain sequences provided in trans. Protein priming was further shown to induce RT conformational changes that resulted in TP-RT domain dissociation, altered priming site selection, and a gain of sensitivity to a pyrophosphate analog inhibitor. trans-priming, trans-inhibition, and trans-complementation, which requires separate TP and RT domains to reconstitute a functional RT protein, were employed to define the sequences in the TP and RT domains that could mediate physical or functional inter- and intradomain interactions. These results provide new insights into TP-RT domain interactions and conformational dynamics during protein priming and suggest novel means to inhibit protein priming by targeting these interactions and the associated conformational transitions.
authors.affiliation: Department of Microbiology and Immunology, The Penn State University College of Medicine, Hershey, Pennsylvania, USA.
authors.forename: Rajeev K
authors.initials: RK
authors.lastname: Boregowda
authors.forename: Christina
authors.initials: C
authors.lastname: Adams
authors.forename: Jianming
authors.initials: J
authors.lastname: Hu
mesh_terms: D017931:DNA Primers / Q000235:genetics* / Q000378:metabolism; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics* / Q000378:metabolism; D015611:Hepatitis B Virus, Duck / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D004279:DNA, Viral; D014764:Viral Proteins; D012194:RNA-Directed DNA Polymerase