Four conserved cysteine residues of the hepatitis B virus polymerase are critical for RNA pregenome encapsidation.
Hepadnaviruses replicate via reverse transcription of an RNA template, the pregenomic RNA (pgRNA). Although hepadnaviral polymerase (Pol) and retroviral reverse transcriptase are distantly related, some of their features are distinct. In particular, Pol contains two additional N-terminal subdomains, the terminal protein and spacer subdomains. Since much of the spacer subdomain can be deleted without detrimental effects to hepatitis B virus (HBV) replication, this subdomain was previously thought to serve only as a spacer that links the terminal protein and reverse transcriptase subdomains. Unexpectedly, we found that the C terminus of the spacer subdomain is indispensable for the encapsidation of pgRNA. Alanine-scanning mutagenesis revealed that four conserved cysteine residues, three at the C terminus of the spacer subdomain and one at the N terminus of the reverse transcriptase subdomain, are critical for encapsidation. The inability of the mutant Pol proteins to incorporate into nucleocapsid particles, together with other evidence, argued that the four conserved cysteine residues are critical for RNA binding. One implication is that these four cysteine residues might form a putative zinc finger motif. Based on these findings, we speculate that the RNA binding activity of HBV Pol may be mediated by this newly identified putative zinc finger motif.
Kim
Seahee
S
Department of Biochemistry, Yonsei University, Seoul, Korea.
Lee
Jehan
J
Ryu
Wang-Shick
WS
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002460:Cell Line; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016679:Genome, Viral; D006515:Hepatitis B virus / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D019251:Nucleocapsid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D012315:RNA Caps / Q000235:genetics* / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D012315:RNA Caps; D012367:RNA, Viral; D014764:Viral Proteins; D004259:DNA-Directed DNA Polymerase; D003545:Cysteine