Secretion of Genome-Free Hepatitis B Virus – Single Strand Blocking Model for Virion Morphogenesis of Para-retrovirus

Abstract

As a para-retrovirus, hepatitis B virus (HBV) is an enveloped virus with a double-stranded (DS) DNA genome that is replicated by reverse transcription of an RNA intermediate, the pregenomic RNA or pgRNA. HBV assembly begins with the formation of an “immature” nucleocapsid (NC) incorporating pgRNA, which is converted via reverse transcription within the maturing NC to the DS DNA genome. Only the mature, DS DNA-containing NCs are enveloped and secreted as virions whereas immature NCs containing RNA or single-stranded (SS) DNA are not enveloped. The current model for selective virion morphogenesis postulates that accumulation of DS DNA within the NC induces a “maturation signal” that, in turn, triggers its envelopment and secretion. However, we have found, by careful quantification of viral DNA and NCs in HBV virions secreted in vitro and in vivo, that the vast majority of HBV virions (over 90%) contained no DNA at all, indicating that NCs with no genome were enveloped and secreted as empty virions (i.e., enveloped NCs with no DNA). Furthermore, viral mutants bearing mutations precluding any DNA synthesis secreted exclusively empty virions. Thus, viral DNA synthesis is not required for HBV virion morphogenesis. On the other hand, NCs containing RNA or SS DNA were excluded from virion formation. The secretion of DS DNA-containing as well as empty virions on one hand, and the lack of secretion of virions containing single-stranded (SS) DNA or RNA on the other, prompted us to propose an alternative, “Single Strand Blocking” model to explain selective HBV morphogenesis whereby SS nucleic acid within the NC negatively regulates NC envelopment, which is relieved upon second strand DNA synthesis.

Viral DNA-Dependent Induction of Innate Immune Response to Hepatitis B Virus in Immortalized Mouse Hepatocytes

“…To detect viral RNA packaged inside NCs, NCs in MNase-treated cytoplasmic lysate were resolved on native agarose gels and detected by Northern blotting, without NaOH denaturation, before transfer to membranes, using a 32 P-labeled RNA probe specific for the HBV pgRNA, as previously described (9,45). Under these conditions, we have shown previously that only pgRNA, and not plus-strand DNA, in NCs is detected (46,47). The signal from 32 P-labeled viral RNA was detected using a Typhoon 9400 imager and analyzed with Quantity One software.…”

“…The signal from 32 P-labeled viral RNA was detected using a Typhoon 9400 imager and analyzed with Quantity One software. HBV capsid proteins were detected using a mouse monoclonal antibody (MAb) specific for the N terminus of HBc (47). The chemiluminescent signal of core protein on Western blots was detected with the ChemiDoc MP system and analyzed with BioLab software.…”

Alteration of Mature Nucleocapsid and Enhancement of Covalently Closed Circular DNA Formation by Hepatitis B Virus Core Mutants Defective in Complete-Virion Formation

“…Briefly, intact NCs from MNase-digested cell lysate were analyzed by native agarose gel electrophoresis followed by detection using a 32 P-labeled HBV plus-strand [(ϩ)-strand]-specific riboprobe without the NaOH denaturation step prior to transfer to membrane. Under these conditions, we have shown previously that only pgRNA, but not (ϩ)-strand DNA, in HBV NCs is detected (44,46). The same membrane was subsequently probed with an anti-HBV core polyclonal antibody (Dako) to detect the core protein (9,44).…”

Cyclin-Dependent Kinase 2 Phosphorylates S/T-P Sites in the Hepadnavirus Core Protein C-Terminal Domain and Is Incorporated into Viral Capsids

“…Bacterial induction and lysate preparation were carried out as described previously (21). Purification of the capsids was then performed similarly to capsid purification from HepG2 and LMH cells by sucrose gradient ultracentrifugation (40).…”