Phosphorylation of specific serine residues within the acidic domain of the phosphoprotein of vesicular stomatitis virus regulates transcription in vitro

152

We previously demonstrated that the core protein of hepatitis C virus (HCV) can suppress gene expression and replication of hepatitis B virus (HBV) in a human hepatoma cell line (HuH-7). In this study, we have characterized the phosphorylation property of HCV core protein and examined the effect of phosphorylation on its suppressive activity of HBV. Our results indicated that both the full-length HCV core protein (22 kDa) and its processed or degraded forms (14 to 18 kDa) were phosphorylated in insect cells. As demonstrated by using the glutathione S-transferase fusion protein expression system and in vitro transcription and translation system, the phosphorylation of HCV core protein was carried out by protein kinase A (PKA) and protein kinase C (PKC) in vitro. In both kinase reactions, it was determined that the phosphorylated amino acid was a serine residue. The potential phosphorylated sites in core protein were identified as residues Ser-53 and Ser-116 for PKA and Ser-53 and Ser-99 for PKC. Comparison of the phosphorylation intensities of the wild type and Ser mutants suggested that Ser-99 and Ser-116 were the major phosphorylation sites for PKC and PKA, respectively. The phosphorylation of Ser-99 and Ser-116, but not Ser-53, in HCV core protein was essential for the suppressive activity of HCV core protein on HBV gene expression and replication in HuH-7 cells. Mutation of the former two serine residues to alanine or aspartate residues led to a drastic loss of the inhibitory effects of HCV core protein on HBV gene expression (both transcription and antigen production) and pregenomic RNA encapsidation, as well as the release of HBV virus particles. In contrast, the Ser-53 mutant conferred the same level of suppressive activity as the wild type did. This property is in accordance with the observation that Ser-99 and Ser-116 are the predominant phosphorylation sites in the HCV core construct. All serine mutants (including those with mutations in PKA, PKC, and both kinase recognition sites) of HCV core protein retained the ability to translocate into the nucleus. Furthermore, wild-type HCV core protein diminished its suppressive activity when cells were treated with PKA or PKC inhibitor. In conclusion, HCV core protein is a phosphoprotein and in HuH-7 cells, its trans suppression of HBV gene expression and replication is positively regulated by PKA and PKC. The role of phosphorylation in the control of trans-suppressive activity cannot be reproduced by introducing an acidic residue. In addition, our results imply that phosphorylation of serine residues by these two kinases is not a prerequisite for HCV core protein entrance into the nucleus.

Section: Discussionmentioning

confidence: 70%

Modulation of the trans-suppression activity of hepatitis C virus core protein by phosphorylation

Shih¹,

Chen²,

Chen³

et al. 1995

152

“…Some studies suggest that CKII-mediated phosphorylation of the VSV P protein also takes place in the context of mammalian cells in vivo. Ala substitution at Ser-59 and Ser-61 in the New Jersey VSV P protein expressed in COS cells eliminates essentially all constitutive phosphorylation, and when this mutant construct is translated in wheat germ lysates, the protein is inactive in transcription reconstitution (31). Likewise, we showed recently that Ser-60 and Thr-62 of Indiana P are phosphorylated in vivo when expressed in BHK cells via the vaccinia virus-T7 system.…”

mentioning

confidence: 80%

“…P protein produced in bacteria lacks phosphates and does not reconstitute transcription in vitro when mixed with purified L and N-RNA template unless first modified by casein kinase II (CKII) (2,3,17). The acceptor sites for this in vitro modification have been mapped to Ser-59 and Ser-61 in the New Jersey VSV serotype P protein (31) and to Ser-60 and Thr-62 in the Indiana serotype (17). Without phosphorylation of at least one of these sites, the bacterial P is unable to multimerize in vitro (15,17) and, at least for the Indiana protein, cannot bind to L protein and N-RNA template (18).…”

mentioning

confidence: 99%

Constitutive phosphorylation of the vesicular stomatitis virus P protein modulates polymerase complex formation but is not essential for transcription or replication

Spadafora

Canter

Jackson

et al. 1996

As a subunit of both the P-L polymerase complex and the P-N assembly complex, the vesicular stomatitis virus (VSV) P protein plays a pivotal role in transcription and replication of the viral genome. Constitutive phosphorylation of this protein is currently thought to be essential for formation of the P-L complex. We recently identified the three relevant phosphate acceptor sites in the VSV Indiana serotype P protein (R. L.

“…The bacterially expressed inactive P protein, however, becomes transcriptionally active if it is first phosphorylated by casein kinase II (CKII) (4,26). By various mutagenesis and biochemical studies, the acceptor sites for CKII-mediated phosphorylation of P protein have been mapped to Ser-59 and Ser-61 for New Jersey serotype (51) and Ser-60, Thr-62, and Ser-64 for Indiana serotype of VSV (13,50). Phosphorylation of these residues which are located in the amino-terminal acidic domain I of the protein (see Fig.…”

mentioning

confidence: 99%

Phosphorylation within the amino-terminal acidic domain I of the phosphoprotein of vesicular stomatitis virus is required for transcription but not for replication

Pattnaik

Hwang

et al. 1997

Self Cite

Phosphorylation by casein kinase II at three specific residues (S-60, T-62, and S-64) within the acidic domain I of the P protein of Indiana serotype vesicular stomatitis virus has been shown to be critical for in vitro transcription activity of the viral RNA polymerase (P-L) complex. To examine the role of phosphorylation of P protein in transcription as well as replication in vivo, we used a panel of mutant P proteins in which the phosphate acceptor sites in domain I were substituted with alanines or other amino acids. Analyses of the alanine-substituted mutant P proteins for the ability to support defective interfering RNA replication in vivo suggest that phosphorylation of these residues does not play a significant role in the replicative function of the P protein since these mutant P proteins supported replication at levels >70% of the wild-type P-protein level. However, the transcription function of most of the mutant proteins in vivo was severely impaired (2 to 10% of the wild-type P-protein level). The level of transcription supported by the mutant P protein (P 60/62/64 ) in which all phosphate acceptor sites have been mutated to alanines was at best 2 to 3% of that of the wild-type P protein. 81% of the wild-type level), substitution with phenylalanine (P FFF ) rendered the protein much less active in transcription (<5%). Substitution with arginine residues led to significantly reduced activity in replication (6%), whereas glutamic acid substituted P protein (P EEE ) supported replication (42%) and transcription (86%) well. In addition, the mutant P proteins that were defective in replication (P RRR ) or transcription (P 60/62/64 ) did not behave as transdominant repressors of replication or transcription when coexpressed with wild-type P protein. From these results, we conclude that phosphorylation of domain I residues plays a major role in in vivo transcription activity of the P protein, whereas in vivo replicative function of the protein does not require phosphorylation. These findings support the contention that different phosphorylated states of the P protein regulate the transcriptase and replicase functions of the polymerase protein, L. Increasing the amount of P 60/62/64 expression in transfected cells did not rescue significant levels of transcription. Substitution with other amino acids at these sites had various effects on replication and transcription. While substitution with threonine residues (P TTT ) had no apparent effect on transcription (113% of the wild-type level) or replication (