The conserved serine 177 in the delta antigen of hepatitis delta virus is one putative phosphorylation site and is required for efficient viral RNA replication

Hepatitis delta virus (HDV) small delta antigen (S-HDAg) plays a critical role in virus replication. We previously demonstrated that the S-HDAg phosphorylation occurs on both serine and threonine residues. However, their biological significance and the exact phosphorylation sites of S-HDAg are still unknown. In this study, phosphorylated S-HDAg was detected only in the intracellular compartment, not in viral particles. In addition, the number of phosphorylated isoforms of S-HDAg significantly increased with the extent of viral replication in transfection system. Site-directed mutagenesis showed that alanine replacement of serine 177, which is conserved among all the known HDV strains, resulted in reduced phosphorylation of S-HDAg, while the mutation of the other two conserved serine residues (2 and 123) had little effect. The S177A mutant dramatically decreased its capability in assisting HDV RNA replication, with a preferential and profound impairment of the antigenomic RNA replication. Furthermore, the viral RNA editing, a step relying upon antigenomic RNA replication, was also abolished by this mutation. These results suggested that phosphorylation of S-HDAg, with serine 177 as a presumable site, plays a critical role in viral RNA replication, especially in augmenting the replication of antigenomic RNA.     

Characterization of the phosphorylated forms and the phosphorylated residues of hepatitis delta virus delta antigens

Hepatitis delta virus (HDV) replication requires both the cellular RNA polymerase and one virus-encoded protein, small delta antigen (S-HDAg). S-HDAg has been shown to be a phosphoprotein, but its phosphorylation status is not yet clear. In this study, we employed three methods to address this question. A special two-dimensional gel electrophoresis, namely, nonequilibrium pH gradient electrophoresis, was used to separate the very basic S-HDAg. By carefully adjusting the pH of solubilization solution, the ampholyte composition, and the appropriate electrophoresis time periods, we were able to clearly resolve S-HDAg into two phosphorylated isoforms and one unphosphorylated form. In contrast, the viral large delta antigen (L-HDAg) can only be separated into one phosphorylated and one unphosphorylated form. By metabolic (32)P labeling, both immunoprecipitated S-HDAg and L-HDAg were found to incorporate radioactive phosphate. The extent of S-HDAg phosphorylation was increased upon 12-O-tetradecanoylphorbol-13-acetate treatment, while that of L-HDAg was not affected. Finally, phosphoamino acid analysis identified serine and threonine as the phospho residues in the labeled S-HDAg and only serine in the L-HDAg. Therefore, HDV S- and L-HDAgs differ in their phosphorylation patterns, which may account for their distinct biological functions.     

Hepatitis delta virus antigen is methylated at arginine residues, and methylation regulates subcellular localization and RNA replication

Hepatitis delta virus (HDV) contains a circular RNA which encodes a single protein, hepatitis delta antigen (HDAg). HDAg exists in two forms, a small form (S-HDAg) and a large form (L-HDAg). S-HDAg can transactivate HDV RNA replication. Recent studies have shown that posttranslational modifications, such as phosphorylation and acetylation, of S-HDAg can modulate HDV RNA replication. Here we show that S-HDAg can be methylated by protein arginine methyltransferase (PRMT1) in vitro and in vivo. The major methylation site is at arginine-13 (R13), which is in the RGGR motif of an RNA-binding domain. The methylation of S-HDAg is essential for HDV RNA replication, especially for replication of the antigenomic RNA strand to form the genomic RNA strand. An R13A mutation in S-HDAg inhibited HDV RNA replication. The presence of a methylation inhibitor, S-adenosyl-homocysteine, also inhibited HDV RNA replication. We further found that the methylation of S-HDAg affected its subcellular localization. Methylation-defective HDAg lost the ability to form a speckled structure in the nucleus and also permeated into the cytoplasm. These results thus revealed a novel posttranslational modification of HDAg and indicated its importance for HDV RNA replication. This and other results further showed that, unlike replication of the HDV genomic RNA strand, replication of the antigenomic RNA strand requires multiple types of posttranslational modification, including the phosphorylation and methylation of HDAg.     

ERK1/2-mediated phosphorylation of small hepatitis delta antigen at serine 177 enhances hepatitis delta virus antigenomic RNA replication

The small hepatitis delta virus (HDV) antigen (SHDAg) plays an essential role in HDV RNA double-rolling-circle replication. Several posttranslational modifications (PTMs) of HDAgs, including phosphorylation, acetylation, and methylation, have been characterized. Among the PTMs, the serine 177 residue of SHDAg is a phosphorylation site, and its mutation preferentially abolishes HDV RNA replication from antigenomic RNA to genomic RNA. Using coimmunoprecipitation analysis, the cellular kinases extracellular signal-related kinases 1 and 2 (ERK1/2) are found to be associated with the Flag-tagged SHDAg mutant (Ser-177 replaced with Cys-177). In an in vitro kinase assay, serine 177 of SHDAg was phosphorylated directly by either Flag-ERK1 or Flag-ERK2. Activation of endogenous ERK1/2 by a constitutively active MEK1 (hemagglutinin-AcMEK1) increased phosphorylation of SHDAg at Ser-177; this phosphorylation was confirmed by immunoblotting using an antibody against phosphorylated S177 and mass spectrometric analysis. Interestingly, we found an increase in the HDV replication from antigenomic RNA to genomic RNA but not in that from genomic RNA to antigenomic RNA. The Ser-177 residue was critical for SHDAg interaction with RNA polymerase II (RNAPII), the enzyme proposed to regulate antigenomic RNA replication. These results demonstrate the role of ERK1/2-mediated Ser-177 phosphorylation in modulating HDV antigenomic RNA replication, possibly through RNAPII regulation. The results may shed light on the mechanisms of HDV RNA replication.     

Control of simian virus 40 DNA replication by the HeLa cell nuclear kinase casein kinase I.

The initiation of simian virus 40 (SV40) DNA replication is regulated by the phosphorylation state of the viral initiator protein, large T antigen. We describe the purification from HeLa cell nuclei of a 35-kDa serine/threonine protein kinase that phosphorylates T antigen at sites that are phosphorylated in vivo and thereby inhibits its ability to initiate SV40 DNA replication. The inhibition of both origin unwinding and DNA replication by the kinase is reversed by protein phosphatase 2A. As determined by molecular weight, substrate specificity, autophosphorylation, immunoreactivity, and limited sequence analysis, this kinase appears to be identical to casein kinase I, a ubiquitous serine/threonine protein kinase that is closely related to a yeast kinase involved in DNA metabolism. The HeLa cell phosphorylation cycle that controls the initiation of SV40 DNA replication may also play a role in cellular DNA metabolism.     

Hepatitis delta virus ribonucleoproteins shuttle between the nucleus and the cytoplasm

Hepatitis delta virus (HDV) infection of individuals infected with hepatitis B virus (HBV) is associated with more severe liver damage and an increased risk of fulminant disease. HDV is a single-stranded RNA virus that encodes a single protein, the delta antigen, which is expressed in two forms, small (S-HDAg) and large (L-HDAg). Here we show that although HDV ribonucleoproteins are mainly detected in the nucleus, they are also present in the cytoplasm of cells infected with HDV or transfected with HDV cDNA. Making use of an heterokaryon assay, we demonstrate that HDV ribonucleoproteins shuttle continuously between the nucleus and the cytoplasm. In the absence of HDV RNA, both forms of the delta antigen are retained in the nucleus, whereas in the absence of the delta antigen, HDV RNA is predominantly detected in the cytoplasm. Coexpression of HDV RNA and S-HDAg (which binds to the viral RNA and contains a nuclear localization signal) results in nuclear accumulation of the viral RNA. This suggests that HDV RNA mediates export of viral particles to the cytoplasm whereas the delta antigen triggers their reimport into the nucleus.     

The large delta antigen of hepatitis delta virus potently inhibits genomic but not antigenomic RNA synthesis: a mechanism enabling initiation of viral replication

Hepatitis delta virus (HDV) contains two types of hepatitis delta antigens (HDAg) in the virion. The small form (S-HDAg) is required for HDV RNA replication, whereas the large form (L-HDAg) potently inhibits it by a dominant-negative inhibitory mechanism. The sequential appearance of these two forms in the infected cells regulates HDV RNA synthesis during the viral life cycle. However, the presence of almost equal amounts of S-HDAg and L-HDAg in the virion raised a puzzling question concerning how HDV can escape the inhibitory effects of L-HDAg and initiate RNA replication after infection. In this study, we examined the inhibitory effects of L-HDAg on the synthesis of various HDV RNA species. Using an HDV RNA-based transfection approach devoid of any artificial DNA intermediates, we showed that a small amount of L-HDAg is sufficient to inhibit HDV genomic RNA synthesis from the antigenomic RNA template. However, the synthesis of antigenomic RNA, including both the 1.7-kb HDV RNA and the 0.8-kb HDAg mRNA, from the genomic-sense RNA was surprisingly resistant to inhibition by L-HDAg. The synthesis of these RNAs was inhibited only when L-HDAg was in vast excess over S-HDAg. These results explain why HDV genomic RNA can initiate replication after infection even though the incoming viral genome is complexed with equal amounts of L-HDAg and S-HDAg. These results also suggest that the mechanisms of synthesis of genomic versus antigenomic RNA are different. This study thus resolves a puzzling question about the early events of the HDV life cycle.     

A diminished activation capacity of the interferon-inducible protein kinase PKR in human T lymphocytes.

The double-stranded (ds) RNA activated protein kinase PKR is an interferon (IFN)-inducible serine/threonine protein that regulates protein synthesis through the phosphorylation of the alpha subunit of translation initiation factor 2 (eIF-2alpha). PKR activation in cells is induced by virus infection or treatment with dsRNA and is modulated by a number of viral and cellular factors. To better understand the mechanisms of PKR action we have analyzed and compared the mode of PKR activation in a number of cell lines of different histological origin. Here we show that PKR activation and phosphorylation of eIF-2alpha are both diminished in various virus-transformed and nontransformed human T cells. Priming of T cells with IFN does not restore PKR activation. In vitro kinase assays show that the diminished PKR activation in T cells correlates with the presence of a 60-kDa (p60) phosphoprotein coimmunoprecipitated with PKR. P60 is absent from PKR immunoprecipitates from non T cells. Incubation of active PKR with T cell extracts results in inhibition of PKR autophosphorylation, which is proportional to the amount of phosphorylated p60 in the kinase reactions. Treatment of T cells with proteasome inhibitors or incubation of PKR immunoprecipitates with phosphatase inhibitors does not restore PKR activation. However, phosphorylation of p60 is enhanced upon treatment with the phosphatase inhibitor microcystin. These data show that the impaired activation capacity of PKR in human T cells is exerted at the post-translational levels in a manner that is independent of cell transformation or virus infection.     

Dynamic phosphorylation of Autographa californica nuclear polyhedrosis virus pp31.

Autographa californica nuclear polyhedrosis virus (AcMNPV) pp31 is a nuclear phosphoprotein that accumulates in the virogenic stroma, which is the viral replication center in the infected-cell nucleus, binds to DNA, and serves as a late expression factor. Considering that reversible phosphorylation could influence its functional properties, we examined phosphorylation and dephosphorylation of pp31 in detail. Our results showed that pp31 is posttranslationally phosphorylated by both cellular and virus-encoded or -induced kinases. Threonine phosphorylation of pp31 by the virus-specific kinase activity was sensitive to aphidicolin, indicating that it requires late viral gene expression. We also found that pp31 is dephosphorylated by a virus-encoded or -induced phosphatase(s), indicating that phosphorylation of pp31 is a dynamic process. Analysis of pp31 fusion proteins showed that pp31 contains at least three phosphorylation sites. The amino-terminal 100 amino acids of pp31 include at least one serine residue that is phosphorylated by a cellular kinase(s). The C-terminal 67 amino acids of pp31 include at least one threonine residue that is phosphorylated by the virus-specific kinase(s). Finally, this C-terminal domain of pp31 includes at least one serine that is phosphorylated by either a host or viral kinase(s). Interestingly, site-directed mutagenesis of the consensus threonine phosphorylation sites in the C-terminal domain of pp31 failed to prevent threonine phosphorylation, suggesting that the virus-specific kinase is unique and has an undetermined recognition site.     

The 58,000-dalton cellular inhibitor of the interferon-induced double-stranded RNA-activated protein kinase (PKR) is a member of the tetratricopeptide repeat family of proteins.

PKR is a serine/threonine protein kinase induced by interferon treatment and activated by double-stranded RNAs. As a result of activation, PKR becomes autophosphorylated and catalyzes phosphorylation of the alpha subunit of protein synthesis eukaryotic initiation factor 2 (eIF-2). While studying the regulation of PKR in virus-infected cells, we found that a cellular 58-kDa protein (P58) was recruited by influenza virus to downregulate PKR and thus avoid the kinase's deleterious effects on viral protein synthesis and replication. We now report on the cloning, sequencing, expression, and structural analysis of the P58 PKR inhibitor, a 504-amino-acid hydrophilic protein. P58, expressed as a histidine fusion protein in Escherichia coli, blocked both the autophosphorylation of PKR and phosphorylation of the alpha subunit of eIF-2. Western blot (immunoblot) analysis showed that P58 is present not only in bovine cells but also in human, monkey, and mouse cells, suggesting the protein is highly conserved. Computer analysis revealed that P58 contains regions of homology to the DnaJ family of proteins and a much lesser degree of similarity to the PKR natural substrate, eIF-2 alpha. Finally, P58 contains nine tandemly arranged 34-amino-acid repeats, demonstrating that the PKR inhibitor is a member of the tetratricopeptide repeat family of proteins, the only member identified thus far with a known biochemical function.     

Phosphorylation sites in polyomavirus large T antigen that regulate its function in viral, but not cellular, DNA synthesis.

Polyomavirus large T antigen (large T) is a highly phosphorylated protein that can be separated by proteolysis into two domains that have independent function. A cluster of phosphorylation sites was found in the protease-sensitive region connecting the N-terminal and C-terminal domains. Edman degradation of 32P-labeled protein identified serines 267, 271, and 274 and threonine 278 as sites of phosphorylation. Analysis of site-directed mutants confirmed directly that residues 271, 274, and 278 were phosphorylated. Threonine 278, shown here to be phosphorylated by cyclin/cyclin-dependent kinase activity, is required for viral DNA replication in either the full-length large T or C-terminal domain context. The serine phosphorylations are unimportant in the C-terminal domain context even though their mutations activates viral DNA replication in full-length large T. This finding suggests that these sites may function in relating the two domains to each other. Although the phosphorylation sites were involved in viral DNA replication, none was important for the ability of large T to drive cellular DNA replication as measured by bromodeoxyuridine incorporation, and they did not affect large T interactions with the Rb tumor suppressor family.     

Involvement of protein serine and threonine phosphorylation in human sperm capacitation.

The involvement of serine and threonine phosphorylation in human sperm capacitation was investigated. Anti-phosphoserine monoclonal antibody (mAb) recognized six protein bands in the 43-55-kDa, 94 +/- 2-kDa, 110-kDa, and 190-kDa molecular regions, in addition to a faint band each in the 18-kDa and 35-kDa regions. Anti-phosphothreonine mAb recognized protein bands in six similar regions, except that the 18-kDa, 35-kDa, and 94 +/- 2-kDa protein bands were sharper and thicker, and an additional band was observed in the 110-kDa molecular region. In the 43-55-kDa molecular region, there was a well-characterized glycoprotein, designated fertilization antigen, that showed a further increase in serine/threonine phosphorylation after exposure to solubilized human zona pellucida. In a cell-free in vitro kinase assay carried out on beads or in solution, four to eight proteins belonging to similar molecular regions, namely 20 +/- 2 kDa, 43-55 kDa, 94 +/- 2 kDa, and 110 +/- 10 kDa, as well as in 80 +/- 4 and 210 +/- 10 kDa regions, were phosphorylated at dual residues (serine/tyrosine and threonine/tyrosine). Capacitation increased the intensity of serine/threonine phosphorylation per sperm cell, increased the number of sperm cells that were phosphorylated, and induced a subcellular shift in the serine/threonine-specific fluorescence. These findings indicate that protein serine/threonine phosphorylation is involved and may have a physiological role in sperm capacitation.     

Changes in the proteome of Huh7 cells induced by transient expression of hepatitis D virus RNA and antigens

Hepatitis delta virus (HDV) infection of human hepatocytes infected with the hepatitis B virus (HBV) is associated with increased liver damage and risk of fulminant disease. Although considerable progress has been made towards the elucidation of the mechanisms of HDV replication and pathogenesis, little is still known about the host factors involved in the different steps of the replication cycle. Here, we made use of a proteomic approach to analyse the global alterations in protein expression that arise in human hepatocytes separately transfected with each of the HDV components. Huh7 cells were transiently transfected with plasmids that code for the small delta antigen (S-HDAg), large delta antigen (L-HDAg), genomic RNA (gRNA), and antigenomic RNA (agRNA), respectively. Total protein extracts were separated by 2-DE and differentially expressed spots were identified by MALDI-TOF followed by database searching. We identified 32 proteins known to be involved in different pathways namely nucleic acid metabolism, protein metabolism, transport, signal transduction, apoptosis, and cell growth. Moreover, the down regulation of hnRNP D, HSP105, and triosephosphate isomerase was further confirmed by Real time PCR.     

trans-dominant inhibition of human hepatitis delta virus genome replication.

Infection with hepatitis delta virus (HDV) is an important cause of acute and chronic liver disease and can be rapidly fatal. Sequencing of the HDV RNA genome has revealed variability at the C-terminal end of the delta antigen reading frame. One genome type (termed the S genome) synthesizes a 24-kDa protein thought to be required for genome replication. Another genome type (termed the L genome) extends the reading frame by 19 amino acids as a result of a single base change. Replication of the S and L genomes was studied in cultured fibroblasts. While the S genome efficiently initiated genome replication, the L genome did not. Moreover, in a codelivery experiment, L genome RNA inhibited replication of the S genome. Potent trans inhibition was also observed following cotransfection of the S genome and a plasmid encoding the larger delta antigen. Mutational analysis indicated that the inhibitory activity was not a simple function of the large delta antigen reading frame's extra length. Implications for the viral life cycle, clinical infection, and potential treatment are discussed.     

Casein kinase II and protein kinase C modulate hepatitis delta virus RNA replication but not empty viral particle assembly.

Hepatitis delta virus (HDV) contains two virus-specific delta antigens (HDAgs), large and small forms, which are identical in sequence except that the large one contains 19 extra amino acids at the C terminus. HDAgs are nuclear phosphoproteins with distinct biological functions; the small form activates HDV RNA replication, whereas the large form suppresses this process but is required for viral particle assembly. In this study, we have characterized the phosphorylative property of HDAg in a human hepatoma cell line (HuH-7) and examined the role of phosphorylation in HDAg function. As demonstrated by in vivo labeling and kinase inhibitor experiments, the phosphorylation levels of both HDAgs were diminished by the inhibitor of casein kinase II (CKII). Nevertheless, phosphorylation of only the small form could be markedly reduced by the protein kinase C (PKC) inhibitor, suggesting different phosphorylation properties between the two HDAgs. When these two kinase inhibitors were added separately to the transient-expression system, HDV RNA replication was profoundly suppressed. In contrast, the inhibitors did not affect the assembly of empty HDAg particle from HDAgs and hepatitis B virus surface antigen. To further examine the role of phosphorylation in HDAg function, two conservative CKII recognition sites at Ser-2 and Ser-123 of both HDAgs and one potential PKC recognition site at Ser-210 of the large HDAg were altered to alanine by site-directed mutagenesis. Transfection experiments indicated that mutation at Ser-2, but not Ser-123, significantly impaired the activity of the small HDAg in assisting HDV RNA replication. This property is in accordance with our observation that Ser-2, not Ser-123, was the predominant CKII phosphorylation site in the small HDAg. Our studies also excluded the possibility that the phosphorylation of Ser-2, Ser-123, or Ser-210, had roles in the trans-suppression activity of the large HDAg, in the assembly of empty virus-like HDAg particle, and in the nuclear transport of HDAgs. In conclusion, our results indicate that both CKII and PKC positively modulate HDV RNA replication but not the assembly of empty HDAg particle. The role of CKII in HDV replication may at least in part be accounted for by the phosphorylation of Ser-2 in the small HDAg. The effect of PKC on HDV RNA replication is, however, not to mediate the phosphorylation of the conservative Ser-210 in the large HDAg but rather to act on as-yet-unidentified Ser or Thr residues in the small HDAg or cellular factors. These findings provide the first insight into the roles of phosphorylation of the two HDAgs in the HDV replication cycle.     

Tyrosine Phosphorylation of A17 during Vaccinia Virus Infection: Involvement of the H1 Phosphatase and the F10 Kinase

Vaccinia virus encodes two protein kinases (B1 and F10) and a dual-specificity phosphatase (VH1), suggesting that phosphorylation and dephosphorylation of substrates on serine/threonine and tyrosine residues are important in regulating diverse aspects of the viral life cycle. Using a recombinant in which expression of the H1 phosphatase can be regulated experimentally (vindH1), we have previously demonstrated that repression of H1 leads to the maturation of noninfectious virions that contain several hyperphosphorylated substrates (K. Liu et al., J. Virol. 69:7823-7834). In this report, we demonstrate that among these is a 25-kDa protein that is phosphorylated on tyrosine residues in H1-deficient virions and can be dephosphorylated by recombinant H1. We demonstrate that the 25-kDa phosphoprotein represents the product of the A17 gene and that A17 is phosphorylated on serine, threonine, and tyrosine residues during infection. Detection of phosphotyrosine within A17 is abrogated when Tyr(203) (but not Tyr(3), Tyr(6), or Tyr(7)) is mutated to phenylalanine, suggesting strongly that this amino acid is the site of tyrosine phosphorylation. Phosphorylation of A17 fails to occur during nonpermissive infections performed with temperature-sensitive mutants defective in the F10 kinase. Our data suggest that this enzyme, which was initially characterized as a serine/threonine kinase, might in fact have dual specificity. This hypothesis is strengthened by the observation that Escherichia coli induced to express F10 contain multiple proteins which are recognized by antiphosphotyrosine antiserum. This study presents the first evidence for phosphotyrosine signaling during vaccinia virus infection and implicates the F10 kinase and the H1 phosphatase as the dual-specificity enzymes that direct this cycle of reversible phosphorylation.     

A tightly associated serine/threonine protein kinase regulates phosphoinositide 3-kinase activity.

We identified a serine/threonine protein kinase that is associated with and phosphorylates phosphoinositide 3-kinase (PtdIns 3-kinase). The serine kinase phosphorylates both the 85- and 110-kDa subunits of PtdIns 3-kinase and purifies with it from rat liver and immunoprecipitates with antibodies raised to the 85-kDa subunit. Tryptic phosphopeptide maps indicate that p85 from polyomavirus middle T-transformed cells is phosphorylated in vivo at three sites phosphorylated in vitro by the associated serine kinase. The 85-kDa subunit of PtdIns 3-kinase is phosphorylated in vitro on serine at a stoichiometry of approximately 1 mol of phosphate per mol of p85. This phosphorylation results in a three- to sevenfold decrease in PtdIns 3-kinase activity. Dephosphorylation with protein phosphatase 2A reverses the inhibition. This suggests that the association of protein phosphatase 2A with middle T antigen may function to activate PtdIns 3-kinase.