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.hepatitis B virus ͉ viral maturation ͉ posttranslational modification ͉ tandem mass spectrometry ͉ vibrational cooling MALDI-Fourier transform MS H epatitis B viruses (HBVs), or hepadnaviruses, are pararetroviruses that replicate their DNA genome via an RNA intermediate (pregenomic RNA, pgRNA) by reverse transcription (1, 2). Similar to classical retroviruses, hepadnaviruses begin assembly with the formation of intracellular nucleocapsid (NC) particles that specifically package viral pgRNA. However, they undergo reverse transcription before membrane envelopment of NCs and secretion as virions, rather than subsequent to entry into new target cells, like classical retroviruses (2, 3). This replication strategy is manifested in the hepadnaviral maturation phenomenon: secreted hepadnaviral virions contain only the mature, double-stranded relaxed circular (or the mature, doublestranded linear) DNA species, whereas intracellular pools of NCs contain a mix of nucleic acid species, including the pgRNA and DNA species from all of the intermediate stages of reverse transcription (2).The maturation phenomenon implies that mature DNAcontaining NCs may be selectively enveloped and secreted. It was proposed that DNA synthesis and NC envelopment may be coupled through a maturation signal (2). Strong support for this maturation signal model has been obtained (4-6), particularly through the use of a synchronized duck HBV (DHBV) replication system (7,8)...

Hepatitis B virus replication

“…In fact, capsids from core protein variants lacking part of this region still package pgRNA but appear unable to produce full-length RC-DNA; instead they preferentially reverse transcribe the fraction of spliced genomic RNAs [46,47] . Phosphorylation/dephosphorylation events at the S and T residues in the nucleic acid binding domain clearly accompany genome maturation [48] , and mutations affecting the core phosphorylation status [46,49] can influence DNA synthesis in various ways. Outside capsids, or in the absence of core protein, apparently no full-length DNA can be formed.…”

Avian hepatitis B viruses: Molecular and cellular biology, phylogenesis, and host tropism

“…However, the S245A mutant was deficient in genome maturation [48] . It has been shown that the CTD contains several phosphosites, which are heterogeneously phosphorylated intracellularly and hypophosphorylated or non-phosphorylated in the secreted virion [40,43] . This dephosphorylation, which occurs as nucleocapsids mature (meaning that the pgRNA is reverse transcribed into the rcDNA genome), is thought to be a maturation signal that results in secretion of only fully matured virions containing the DNA genome [43] .…”

“…It has been shown that the CTD contains several phosphosites, which are heterogeneously phosphorylated intracellularly and hypophosphorylated or non-phosphorylated in the secreted virion [40,43] . This dephosphorylation, which occurs as nucleocapsids mature (meaning that the pgRNA is reverse transcribed into the rcDNA genome), is thought to be a maturation signal that results in secretion of only fully matured virions containing the DNA genome [43] . In addition, it has been shown that binding of hepadnaviral capsids to the nuclear pore complex depends on the phosphorylation status of the core protein [35] .…”

“…The N-terminal region of the core protein (up to about 144 aa) is acetylated [43] and sufficient for assembly of the capsid shell [44,45] . The carboxyterminal end of the protein, the so called C-terminal domain (CTD), is extremely basic and binds nucleic acids.…”

Molecular Virology of Hepatitis B Virus for Clinicians

“…When the polymerase reaches the 5' end of pgRNA, an RNA oligomer containing DR1 and the 6 to 7 nucleotides at the 5' end of DR1 sequence is left uncleaved. It is this RNA oligomer that is subsequently translocated and annealed to the direct repeat 2 (DR2) and primes plus strand DNA synthesis to yield relaxed circular DNA (rcDNA) [31].When rc DNA are formed, the nucleocapsids are matured and can be enveloped and secreted out of cells [49,50].…”