REVERSE TRANSCRIPTION-ASSOCIATED DEPHOSPHORYLATION OF HEPADNAVIRUS NUCLEOCAPSID

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Hepatitis B Virus (HBV) is a major global health problem, with currently more than 300 million chronically infected worldwide. Its extremely high degree of infectivity has been in part linked to the nature of its assembly and morphogenesis. Dynamic protein phosphorylation is paramount to the regulation of viral, as well as normal, cellular processes, although, it presents numerous challenges for MS study due to the phosphate modification?s transience and labile nature. Phosphorylation of the HBV capsid protein may regulate its numerous functional roles in the HBV lifecycle, including, during morphogenesis, transmitting the viral maturation signal that triggers envelopment and secretion of mature infectious virions. To identify biophysical and biochemical correlates of viral maturation, we have characterized the gross structure of purified capsids from three stages of viral maturation by atomic force microscopy (AFM), and the phosphorylation of the capsid protein at these stages by MALDI-TOF MS ESI-Q-oTOF MS/MS, and vibrational cooling (VC) MALDI-FT MS, which minimizes phosphate loss. Duck HBV (DHBV) nucleocapsids from three extremes of viral maturation (immature, RNA-containing; mature, DNA-containing; and virion-derived nucleocapsids) were isolated from virus-expressing cell lines and purified to homogeneity by gradient ultracentrifugation. Intact nucleocapsids were spotted directly onto AFM targets and visualized with a Veeco Multimode? AFM attached to a Nanoscope? IIIa controller. Denatured capsids were subjected to SDS-PAGE and digested with Lys-C or trypsin in-gel. Peptides were purified by ZipTip microchromatography. Comparative analysis of phosphopeptides from different stages of viral maturation was conducted with a Bruker Reflex IV MALDI-TOF MS. Phosphopeptides were subjected to MSn with a VC MALDI-FTICR MS, constructed in-house, or were subjected to tandem MS sequencing with an MDS Sciex/ABI QStar ESI Q-oTOF MS. Using VC MALDI-FTICR MS, we were able to detect a novel DHBV capsid phosphopeptide, then obtain phosphosite localization (to S230) by conducting successive SORI-CAD experiments (MS2, MS3) upon it. Furthermore, we obtained complete sequencing and phosphosite localization for a novel DHBV capsid proline-rich pentaphosphorylated peptide using ESI-Q-oTOF MS/MS, confirming four known sites of capsid phosphorylation (T239, S245, S257, and S259) and identifying a second novel phosphorylation site (S232). AFM analysis of the intact purified capsids from the three different stages of viral maturation demonstrated a correlation between viral maturation and a progressively increased capsid size. Comparative MALDI-TOF MS analysis of peptides derived from these same three capsid populations revealed that the capsid maturation process is correlated to a dramatic dephosphorylation of the capsid protein at all six phosphosites. These results, together with the known requirement of capsid phosphorylation at earlier stages in the viral lifecycle, suggest that the hepadnaviral capsid undergoes a dynamic change in phosphorylation in order to fulfill its multiple roles at different stages in the viral lifecycle. Dephosphorylation of the maturing capsids may provide the signal that triggers their envelopment and secretion, perhaps facilitated by the capsid structural changes that we detected by AFM. These maturation-associated differences in the capsids' capacity for expansion, as well as the dramatic dephosphorylation of the capsid protein, reflect previously unappreciated capsid dynamics, which we speculate may be important for capsid stability, envelopment, and/or disassembly during infection.