Heat shock protein 90-independent activation of truncated hepadnavirus reverse transcriptase.
The reverse transcriptase (RT) encoded by hepadnaviruses (hepatitis B viruses) is a multifunctional protein critical for several aspects of viral assembly and replication. Reverse transcription is triggered by the specific interaction between the RT and an RNA signal located on the viral pregenomic RNA, termed epsilon, and is initiated through a novel protein priming mechanism whereby the RT itself serves as a protein primer and epsilon serves as the obligatory template. Using the RT from duck hepatitis B virus as a model, we previously demonstrated that RT-epsilon interaction and protein priming require the assistance of a host cell chaperone complex, heat shock protein 90 (Hsp90) and its co-chaperones, which associates with the RT and facilitates the folding of the RT into an active conformation. We now report that extensive truncation removing the entire C-terminal RNase H domain and part of the central RT domain could relieve this dependence on Hsp90 for RT folding such that the truncated RT variants could function in epsilon interaction and protein priming independently of Hsp90. The presence of certain nonionic or zwitterionic detergent was sufficient to establish and maintain the truncated RT proteins in an active, albeit labile, state. Furthermore, we were able to refold an RT truncation variant de novo after complete denaturation. In contrast, the full-length RT and also RT variants with less-extensive C-terminal truncations required Hsp90 for activation. Surprisingly, the presence of detergent plus some yet-to-be-identified cytoplasmic factor(s) led to a dramatic suppression of the RT activities. These results have important implications for RT folding and conformational maturation, Hsp90 chaperone function, and potential inhibition of RT functions by host cell factors.
Wang
Xingtai
X
Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
Qian
Xiaofeng
X
Guo
Hwai-Chen
HC
Hu
Jianming
J
D000818:Animals; D002460:Cell Line; D003902:Detergents / Q000494:pharmacology; D015967:Gene Expression Regulation, Viral; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D015611:Hepatitis B Virus, Duck / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D009154:Mutation; D017510:Protein Folding; D012367:RNA, Viral / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012156:Reticulocytes; D014158:Transcription, Genetic
D003902:Detergents; D018841:HSP90 Heat-Shock Proteins; D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase