Reverse transcription-associated dephosphorylation of hepadnavirus nucleocapsids

Abstract

Hepatitis B viruses are pararetroviruses that contain a partially dsDNA genome and replicate this DNA through an RNA intermediate (the pregenomic RNA, pgRNA) by reverse transcription. Viral assembly begins with the packaging of the pgRNA into nucleocapsids (NCs), with subsequent reverse transcription within NCs converting the pgRNA into the characteristic dsDNA genome. Only NCs containing this dsDNA (the so-called "mature" NCs) are enveloped by the viral envelope proteins and secreted as virions; "immature" NCs, i.e., those containing pgRNA or immature reverse transcription intermediates, are excluded from virion formation. This phenomenon is thought to be caused by the emergence of an intrinsic maturation signal only on the mature NCs. To define the maturation signal, we have devised a method to separate mature from immature duck hepatitis B virus NCs and have compared them to NCs derived from secreted virions. Detailed mass spectrometric analyses revealed that the core protein from immature NCs was phosphorylated on at least six sites, whereas the core protein from mature NCs and that from secreted virions was entirely dephosphorylated. These results, together with the known requirement of core phosphorylation for pgRNA packaging and DNA synthesis, suggest that the NC undergoes a dynamic change in phosphorylation state to fulfill its multiple roles at different stages of viral replication. Although phosphorylation of the NCs is required for efficient RNA packaging and DNA synthesis by the immature NCs, dephosphorylation of the mature NCs may trigger envelopment and secretion.

Fig. 1.

Purification of immature and mature DHBV NC species. (Left) Intracellular NCs were harvested from Dstet-5 cells (33) and separated into immature NCs and mature NCs by successive rounds of velocity gradient centrifugation. NC DNA was isolated from gradient fractions and analyzed by Southern blotting. Immature (Imm.), mature (Mat.), and mixed maturity (Mix) NCs are indicated, as is a DNA size marker (M). (Right) Further separation of immature and mature NC species by subsequent rounds of velocity gradient ultracentrifugation. NCs from pooled mature fractions (as marked by brackets) from the first velocity gradient were subjected to second rounds of velocity gradient ultracentrifugation. NC DNA was isolated from gradient fractions and analyzed by Southern blotting. (Inset Left) The detection of immature, pgRNA-containing NCs (RNA NC) in one of the top fractions by resolving the NCs on a native agarose gel, followed by probing with a riboprobe specific for the pgRNA (37). (Insets Right) The core protein (C) levels from selected gradient fractions, as measured by SDS/PAGE and Western blotting, are shown. The presence of immature NCs at the top of the secondary gradient is again evident by the detection of core proteins but little or no viral DNA. RC, relaxed circular DNA; SS, single-stranded DNA; IC, an internal control DHBV plasmid added to all fractions during DNA extraction to verify DNA recovery. The direction of centrifugation is indicated by the long arrows at the bottom.

Fig. 2.

Maturation-associated dephosphorylation of hepadnaviral NCs revealed by SDS/PAGE. Intracellular NCs were purified as described in Fig. 1. Virions were isolated from the culture supernatant of D2 cells (9) and lysed with detergent, and virion-derived NCs were isolated by velocity gradient centrifugation. (A) Southern blot analysis of DNA from mature NCs after four rounds of enrichment by velocity gradient ultracentrifugation (lane 1). Virion DNA served as a marker for maturity (lane 2). (B) Western blot analysis of mixed maturity NCs harvested from D2 cells (lane 1), immature NCs from D2 cells (lane 2), or Dstet-5 cells (lane 3), mature NCs from D2 (lane 4) or Dstet-5 cells (lane 5), and virion-derived NCs (lane 6).

Fig. 3.

Summary of the core peptides detected by MS analyses. Coverage of detected peptide ions is shown as underlined intervals underneath the core protein sequence (the highly basic CTD is in bold). Detected Lys-C and tryptic peptides are indicated by solid and dashed lines, respectively. Vertical bars on underlined intervals represent the termini of internal or overlapping peptides. Observed posttranslational modifications are as indicated: Ac, acetylation, s, phosphoserine, and t, phosphothreonine. Previously identified sites of phosphorylation are indicated by arrows; sites identified in this study are indicated by arrowheads.

Fig. 4.

Detection of a core phosphorylation site at S230 by using VC MALDI FT-ICR MS. NCs of mixed maturity, purified, and digested in-gel by Lys-C were analyzed by using modified IonSpec FT-ICR MS, equipped with a home-built VC MALDI source (30, 41, 46, 47). (A) Mass spectrum (m/z 800–1,600) of core Lys-C peptides, showing the peptides 32–39 and 221–231, with and without phosphorylation, using the matrixα-cyano-4-hydroxycinnamic acid. The sequence from 221 to 231 is shown. (B) SORI-CAD tandem mass spectrum of the isolated ion (m/z 1,402.76). The detected fragment ion with phosphoric acid loss is as labeled. (C) MS/MS/MS mass spectrum following a second SORI-CAD experiment using the resonance frequency of m/z 1,304.788 as the target for the calculated off-resonance frequency used. Detected fragment ions, as well as predicted side-chain losses, are labeled with standard b/y-ion nomenclature. Modification by phosphate (HPO₃) is designated by a P within a circle. Phosphoric acid (H₃PO₄) loss is designated by a P within a droplet. a, dehydroalanine.

Fig. 5.

Identification of a second phosphosite at S232 by using electrospray ionization Q-oTOF MS. Tandem mass spectrum of the pentaphosphorylated 232–262 peptide ion (m/z 773.35⁵⁺) obtained from the Lys-C digest of the core protein derived from mixed NCs is shown. Major fragment ions are labeled with their corresponding b/y-ion designation and their charge state. PQR is an internal tripeptide fragment. A summary of the fragmentation ion data, including the less abundant fragment ions detected, is indicated on the structure of the pentaphosphorylated peptide (above). The sites of phosphorylation revealed by the fragment ions are displayed as S or T residues modified by a phosphate group. Ions indicated by * represent additional core peptide ions not derived from the fragmentation of the m/z 773.355+ ion. Losses of phosphate and phosphoric acid (H₃PO₄) are designated as in Fig. 4.

Fig. 6.

NC maturation-associated core dephosphorylation revealed by comparative MALDI-TOF MS of core Lys-C peptides. (A) Immature NCs. (B) Mature NCs. Both were isolated from Dstet-5 cells. (C) Virion-derived NCs. Spectra over the range m/z 800 to 4,000 are shown. Peptide ions are labeled with their corresponding sequence intervals. Phosphopeptide ions are signified by diamonds. The series of singly, doubly, triply, quadruply, and quintuply phosphorylated 232–262 peptide was only apparent in A. L, Lys-C autolysis peptides. The matrix 2,5-dihydroxybenzoic acid was used.