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.
Phosphorylation of the hepadnavirus core protein C-terminal domain (CTD) is important for viral RNA packaging, reverse transcription, and subcellular localization. Hepadnavirus capsids also package a cellular kinase. The identity of the host kinase that phosphorylates the core CTD or gets packaged remains to be resolved. In particular, both the human hepatitis B virus (HBV) and duck hepatitis B virus (DHBV) core CTDs harbor several conserved serine/threonine-proline (S/T-P) sites whose phosphorylation state is known to regulate CTD functions. We report here that the endogenous kinase in the HBV capsids was blocked by chemical inhibitors of the cyclin-dependent kinases (CDKs), in particular, CDK2 inhibitors. The kinase phosphorylated the HBV CTD at the serine-proline (S-P) sites. Furthermore, we were able to detect CDK2 in purified HBV capsids by immunoblotting. Purified CDK2 phosphorylated the S/T-P sites of the HBV and DHBV CTD in vitro. Inhibitors of CDKs, of CDK2 in particular, decreased both HBV and DHBV CTD phosphorylation in vivo. Moreover, CDK2 inhibitors blocked DHBV CTD phosphorylation, specifically at the S/T-P sites, in a mammalian cell lysate. These results indicate that cellular CDK2 phosphorylates the functionally critical S/T-P sites of the hepadnavirus core CTD and is incorporated into viral capsids.T he human hepatitis B virus (HBV) continues to pose a significant health risk worldwide, causing more than one million deaths annually (52). Chronic HBV infection, estimated to affect 350 million people globally, dramatically elevates the risk for developing serious liver diseases, including cirrhosis and hepatocellular carcinoma. HBV is a member of the Hepadnaviridae family, which includes hepatotropic DNA viruses that consist of an enveloped icosahedral capsid enclosing an approximately 3-kb DNA genome in a partially double-stranded, relaxed circular (RC) form. These DNA viruses are also retroid viruses and encode a reverse transcriptase (RT) enzyme that converts a so-called pregenomic RNA (pgRNA) template to the RC DNA through reverse transcription within cytoplasmic capsids. Capsids are composed of multiple copies (180 or 240) of one virally encoded protein, the core or capsid protein (9,63,65,71).Phosphorylation of the hepadnavirus core protein is important for RNA packaging, DNA synthesis, and subcellular localization. The HBV core protein (HBc) contains three major serine-proline (S-P) phosphorylation sites in its C-terminal domain (CTD) (32). The duck hepatitis B virus (DHBV) core protein (DHBc) contains six known phosphorylation sites, four of which also have the serine/threonine-proline (S/T-P) motifs (43, 68). Mutational analyses indicate that phosphorylation of the core protein at these S/T-P sites is required for RNA packaging and DNA synthesis in HBV (29, 31). For DHBV, dynamic CTD phosphorylation at the S/T-P sites is required for complete DNA synthesis such that the S/T-P phosphorylation is needed for first-strand DNA synthesis and dephosphorylation is required for second-strand DNA s...
A positive relationship between genetic diversity at neutral markers and juvenile survival has been demonstrated for many vertebrate populations, although the correlation is typically weak and the explanation for it remains controversial. We assessed variation at 9-12 microsatellite loci in 65 juvenile harp seals (Phoca groenlandica) that stranded in poor condition around Long Island, NY, from 2001 to 2004. Compared with seals that died, surviving individuals had slightly higher measures of mean d(2), which reflects the size difference between alleles within an individual and provides an index of outbreeding. In contrast, there were no significant differences between survivors and nonsurvivors in heterozygosity or estimates of internal relatedness. This pattern is attributed to the fact that these microsatellite markers were exceptionally variable in this species (9-22 alleles per locus), and all individuals were heterozygous at most loci. Under these circumstances, mean d(2) may provide a powerful measure for assessing diversity-fitness correlations.
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