Full-Length Hepatitis B Virus Core Protein Packages Viral and Heterologous RNA with Similarly High Levels of Cooperativity

Porterfield, J Zachary; Dhason, Mary Savari; Loeb, Daniel D.; Nassal, Michael; Stray, Stephen J.; Zlotnick, Adam

doi:10.1128/jvi.00586-10

Cited by 144 publications

(252 citation statements)

References 73 publications

(94 reference statements)

Supporting

Mentioning

236

Contrasting

Order By: Relevance

“…The thermodynamic framework developed in this study should be useful for interpreting results from experimental studies of in vitro assembly of viruses and virus-like particles (22)(23)(24)(25)(26), as well as for the design of viral vectors for gene therapy, where, in addition to minimizing toxicity, a major goal is to successfully deliver the appropriate amount of therapeutic gene (27). More generally, because the presence of charged macromolecules in cells is ubiquitous, we expect the associated Donnan effects, much like the well known crowding effects (28), to significantly influence a wide range of biomacromolecular behaviors and cellular processes.…”

Section: Discussionmentioning

confidence: 99%

Thermodynamic basis for the genome to capsid charge relationship in viral encapsidation

Ting

Wang

2011

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

We establish an appropriate thermodynamic framework for determining the optimal genome length in electrostatically driven viral encapsidation. Importantly, our analysis includes the electrostatic potential due to the Donnan equilibrium, which arises from the semipermeable nature of the viral capsid, i.e., permeable to small mobile ions but impermeable to charged macromolecules. Because most macromolecules in the cellular milieu are negatively charged, the Donnan potential provides an additional driving force for genome encapsidation. In contrast to previous theoretical studies, we find that the optimal genome length is the result of combined effects from the electrostatic interactions of all charged species, the excluded volume and, to a very significant degree, the Donnan potential. In particular, the Donnan potential is essential for obtaining negatively overcharged viruses. The prevalence of overcharged viruses in nature may suggest an evolutionary preference for viruses to increase the amount of genome packaged by utilizing the Donnan potential (through increases in the capsid radius), rather than high charges on the capsid, so that structural stability of the capsid is maintained.polyelectrolyte | viral assembly T he most prevalent viruses in nature are single-stranded RNA viruses with the genetic material enclosed in icosahedral-shaped capsids made up of 60 T protein units, where the T-number is a small integer index. The protein units (capsomers) often contain highly basic peptide arms that extend into the capsid interior and, under physiological conditions, are positively charged. Electrostatic attraction provides the driving force for encapsidating the negatively charged RNA, which, in turn, helps overcome the electrostatic repulsion among the capsomers during the capsid assembly.In a series of classic experiments, Bancroft and coworkers (1, 2) demonstrated that certain viruses can encapsidate nonnative RNA and even generic polyanions. Dominance of the electrostatics as the driving force for viral assembly has led to the expectation of a simple relationship between the total capsid charge Q P and the genome charge Q R , as every nucleotide carries one unit of negative charge. Belyi and Muthukumar (3) compiled data for 19 wild type viruses from several viral families and found an apparent "universal" charge ratio of Q R ∕Q P ≈ 1.6, which they explained by combining the ground-state dominance approximation for polyelectrolyte binding to an oppositely charged polymer brush with the Manning condensation theory (4). The former predicts a 1∶1 charge ratio and the latter is used as a qualitative argument for the actual charge on the RNA being less than the nominal charge. Hu, et al. (5), however, assumed that the RNA winds around individual peptide arms and found that the viruses are most stable when the total contour length of the RNA is close to the total length of the peptide arms; this roughly gives a charge ratio of 2. In other works that ignore the peptide arms completely, there is additional disagreemen...

show abstract

Section: Discussionmentioning

confidence: 99%

Thermodynamic basis for the genome to capsid charge relationship in viral encapsidation

Ting

Wang

2011

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…In all these cases, the site(s) of core phosphorylation was never defined, except that SRPK1 and -2 were shown to phosphorylate the HBc CTD in vitro (10,45) and in Escherichia coli (8). However, the SRPKs appear to have rather relaxed substrate specificity in these systems, phosphorylating mostly S176 and S178 in the HBc CTD and only weakly at the three S-P sites.…”

mentioning

confidence: 99%

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

Ludgate

Ning

Nguyen

et al. 2012

J Virol

View full text Add to dashboard Cite

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...

show abstract

“…Specific packaging of pgRNA into the capsid is mediated by binding of the primer region of the viral polymerase to the  stem-loop in the 5' region of pgRNA (Hirsch et al, 1990;JunkerNiepmann et al, 1990;Knaus & Nassal, 1993;Nassal, 1992;Porterfield et al, 2010). The pgRNA is then reverse transcribed by the reverse transcriptase domain of the polymerase within the capsid in the cytoplasm of the infected cell.…”

Section: Overview Of the Hepatitis B Virus Life Cyclementioning

confidence: 99%

Hepatitis B Virus X Protein: A Key Regulator of the Virus Life Cycle

Protzer¹

2012

Viral Genomes - Molecular Structure, Diversity, Gene Expression Mechanisms and Host-Virus Interactions

View full text Add to dashboard Cite

Full-Length Hepatitis B Virus Core Protein Packages Viral and Heterologous RNA with Similarly High Levels of Cooperativity

Cited by 144 publications

References 73 publications

Thermodynamic basis for the genome to capsid charge relationship in viral encapsidation

Thermodynamic basis for the genome to capsid charge relationship in viral encapsidation

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

Hepatitis B Virus X Protein: A Key Regulator of the Virus Life Cycle

Contact Info

Product

Resources

About