Central Role of a Serine Phosphorylation Site within Duck Hepatitis B Virus Core Protein for Capsid Trafficking and Genome Release

Assembly of hepatitis B virus (HBV) begins with packaging of the pregenomic RNA (pgRNA) into immature nucleocapsids (NC), which are converted to mature NCs containing the genomic relaxed circular (RC) DNA as a result of reverse transcription. Mature NCs have two alternative fates: (i) envelopment by viral envelope proteins, leading to secretion extracellularly as virions, or (ii) disassembly (uncoating) to deliver their RC DNA content into the host cell nucleus for conversion to the covalently closed circular (CCC) DNA, the template for viral transcription. How these two alternative fates are regulated remains to be better understood. The NC shell is composed of multiple copies of a single viral protein, the HBV core (HBc) protein. HBc mutations located on the surface of NC have been identified that allow NC maturation but block its envelopment. The potential effects of some of these mutations on NC uncoating and CCC DNA formation have been analyzed by transfecting HBV replication constructs into hepatoma cells. All envelopment-defective HBc mutations tested were competent for CCC DNA formation, indicating that core functions in envelopment and uncoating/nuclear delivery of RC DNA were genetically separable. Some of the envelopment-defective HBc mutations were found to alter specifically the integrity of mature, but not immature, NCs such that RC DNA became susceptible to nuclease digestion. Furthermore, CCC DNA formation could be enhanced by NC surface mutations that did or did not significantly affect mature NC integrity, indicating that the NC surface residues may be closely involved in NC uncoating and/or nuclear delivery of RC DNA. IMPORTANCEHepatitis B virus (HBV) infection is a major health issue worldwide. HBV assembly begins with the packaging into immature nucleocapsids (NCs) of a viral RNA pregenome, which is converted to the DNA genome in mature NCs. Mature NCs are then selected for envelopment and secretion as complete-virion particles or, alternatively, can deliver their DNA to the host cell nucleus to maintain the viral genome as nuclear episomes, which are the basis for virus persistence. Previous studies have identified mutations on the capsid surface that selectively block NC envelopment without affecting NC maturation. We have now discovered that some of the same mutations result in preferential alteration of mature NCs and increased viral nuclear episomes. These findings provide important new insights into the regulation of the two alternative fates of mature NCs and suggest new ways to perturb viral persistence by manipulating levels of viral nuclear episomes.T he human pathogen hepatitis B virus (HBV) belongs to the family of Hepadnaviridae, a group of small, hepatotropic DNA viruses that also include closely related animal viruses, such as the duck hepatitis B virus (DHBV) (1). Hepadnaviruses contain a small (ca. 3-kb), partially double-stranded (ds-), relaxed circular (RC) DNA genome enclosed within an icosahedral capsid that is, in turn, formed by multiple copies (240 or 180) of ...

Section: Resultssupporting

confidence: 74%

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

Cui

Luckenbaugh

Bruss

et al. 2015

“…In support of the latter view, Gazina et al found no significant impact of site-specific mutations within the DHBV core C terminus on pgRNA packaging (8). Further, previous work on mutations of the DHBV core C terminus has revealed a destabilizing effect at a later stage of nucleocapsid maturation, with selective nuclease sensitivity of capsid-associated rcDNA but not ssDNA (15,16). It should be noted, however, that mutational studies introduce permanent modifications that cannot reflect dynamic changes in the phosphorylation state during nucleocapsid assembly and disassembly.…”

Section: Discussionsupporting

confidence: 51%

“…In a recent study it was shown that DHBV core protein mutants lacking parts of the C-terminal basic domain or carrying an alanine or aspartate residue instead of one of the phosphorylatable serines formed capsids that rendered rcDNA selectively sensitive to nuclease. Hence, its absence from nuclease-treated samples was not caused by a defect in DNA synthesis but by the inability of the mutant capsid structure to effectively shield the DNA from nuclease attack (14,15).…”

mentioning

confidence: 99%

Hepatitis B Virus Nucleocapsids Formed by Carboxy-Terminally Mutated Core Proteins Contain Spliced Viral Genomes but Lack Full-Size DNA

Köck

Nassal

Deres

et al. 2004

Self Cite

125

The carboxy-terminal sequence of the hepatitis B virus (HBV) core protein constitutes a nucleic acid binding domain that is rich in arginine residues and contains three serine phosphorylation sites. While dispensable for capsid assembly, this domain is involved in viral replication, as demonstrated by the effects of mutations on RNA packaging and/or reverse transcription; however, the underlying mechanisms are poorly understood. Here we tested a series of core protein mutants in which the three serine phosphorylation sites were replaced by glutamic acid, in parallel with a previously described deletion variant lacking the 19 C-terminal amino acid residues, for their ability to support viral replication in transfected hepatoma cells. Replacement of all serines and the deletion gave rise to nucleocapsids containing a smaller than wild-type DNA genome. Rather than a single-stranded DNA intermediate, as previously thought, this was a 2.0-kbp double-stranded DNA molecule derived from spliced pregenomic RNA (pgRNA). Interestingly, full-length pgRNA was associated with nucleocapsids but was found to be sensitive to nuclease digestion, while encapsidated spliced RNA and 3 truncated RNA species were nuclease resistant. These findings suggest that HBV pgRNA encapsidation is directional and that a packaging limit is determined by the C-terminal portion of the core protein.Hepatitis B virus (HBV) is an important human pathogen accounting for about one million deaths each year (13). The viral genome, as present in infectious virions, is a 3.2-kbp circular, partially double-stranded DNA (dsDNA) molecule that contains overlapping reading frames encoding the core protein, a reverse transcriptase (P), three surface proteins, and the X protein (20,25).Viral replication involves reverse transcription of a pregenomic RNA (pgRNA) intermediate inside nucleocapsids, which are formed by 180 or 240 core protein subunits (4-6, 30). Specific encapsidation of pgRNA occurs via binding of P protein to a 5Ј-proximal RNA stem-loop structure, termed ε. Subgenomic RNAs lack the 5Ј ε and are excluded from encapsidation. Notably, however, spliced RNA species containing all sequence elements necessary for packaging and for reverse transcription have been observed in liver tissue and in transfected cells (9,23,(26)(27)(28)(29). Viral DNA synthesis is a highly complex process involving three translocation events of P to priming sites located at the respective 5Ј and 3Ј ends of the template (17). Thus, both ends must be accessible to the encapsidated P protein. The final product is a partially doublestranded, relaxed circular DNA (rcDNA). Occasionally, one of the translocation steps fails (in situ priming), resulting in a double-stranded linear molecule (17,19,24,25).The C-terminal domain of the core protein plays a crucial role in viral replication. It is rich in arginine residues and contains three serine phosphorylation sites. Though dispensable for particle assembly, it is required for pgRNA packaging (2,3,7,10). Further, the phenotypes of core pr...

“…Since an accumulative effect in the inhibition mediated by the p300(S384A) mutant and A238L was not observed, it can be proposed that A238L might block the phosphorylation of serine 384, thus supporting the hypothesis that involves this serine in the A238L-mediated inhibition. To further confirm this hypothesis, we generated mutant constructs of p300 in which serine 384 has been substituted by aspartic acid, which is a phosphoserine mimicking substitution that can produce constitutively active mutants (37). The S384D mutant construct was then used in experiments similar to those described above, showing that in ASFVinfected Vero cells, the ectopic expression of the S384D mutant construct not only reversed the inhibition induced by the A238L protein (expressed during the Ba71Vwt virus infection) on the activity of the COX-2 (Fig.…”

Section: Vol 83 2009 A238l Inhibits P300-mediated Inflammatory Respmentioning

confidence: 99%

African Swine Fever Virus Blocks the Host Cell Antiviral Inflammatory Response through a Direct Inhibition of PKC-θ-Mediated p300 Transactivation

Granja

Sánchez

Sabina

et al. 2009

During a viral infection, reprogramming of the host cell gene expression pattern is required to establish an adequate antiviral response. The transcriptional coactivators p300 and CREB binding protein (CBP) play a central role in this regulation by promoting the assembly of transcription enhancer complexes to specific promoters of immune and proinflammatory genes. Here we show that the protein A238L encoded by African swine fever virus counteracts the host cell inflammatory response through the control of p300 transactivation during the viral infection. We demonstrate that A238L inhibits the expression of the inflammatory regulators cyclooxygenase-2 (COX-2) and tumor necrosis factor alpha (TNF-␣) by preventing the recruitment of p300 to the enhanceosomes formed on their promoters. Furthermore, we report that A238L inhibits p300 activity during the viral infection and that its amino-terminal transactivation domain is essential in the A238L-mediated inhibition of the inflammatory response. Importantly, we found that the residue serine 384 of p300 is required for the viral protein to accomplish its inhibitory function and that ectopically expressed PKCcompletely reverts this inhibition, thus indicating that this signaling pathway is disrupted by A238L during the viral infection. Furthermore, we show here that A238L does not affect PKC-enzymatic activity, but the molecular mechanism of this viral inhibition relies on the lack of interaction between PKC-and p300. These findings shed new light on how viruses alter the host cell antiviral gene expression pattern through the blockade of the p300 activity, which represents a new and sophisticated viral mechanism to evade the inflammatory and immune defense responses.