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

Abstract

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.

FIG. 1

Kinetic study of HDAg's in replicating cells and in viral particles. pCD2G (HDV dimeric cDNA) cotransfected with pS1X (a construct expressing HBV surface antigen) into HuH-7 cells. After transfection, there was HDV replication, HDAg expression, and HDV viral particle secretion. Proteins were extracted from the replicating cells on days 2 (A), 3 (B), 5 (C), and 12 (D) posttransfection, and a longer exposure time of cells on day 12 is also shown (E). (F) Secreted viral particles were collected from medium on day 12. These materials were subjected to the NEPHGE–SDS-PAGE system and detected by Western blotting. The unphosphorylated form of HDAg is the most basic spot to the right end. The asterisk above each panel indicates the position of trypsinogen with known pI of 9.3, and the star indicates the position of lysozyme with a pI of 10.5 to 11 as pH markers.

FIG. 2

(A) Conserved serine residues among variant HDV strains. On the left are the geographical origins of the HDV isolates: A, American (26); I, Italian (48); N, Nauru (10); F, French (39); L, Lebanon (21); T, Taiwan (11); J, Japan-2 (17); C, Central African (42); J′, Japan-1 (16); P, Peru (5). The numbered residues are the three conserved serine residues among the HDV strains, and PESP encompassing serine 177 is the predicted consensus sequence of the MAP kinase substrate (34). (B) In vivo phosphorylation of HDAg. pCDAg-S (wild type; lane 1), serine-substituted mutants (lanes 2 to 4; the numbers indicate mutated serine residues), and pCDAg-L (L-HDAg, lane 5) were transfected into HuH-7 cells. On day 2 posttransfection, transfected cells were metabolically labeled with [³²P]-orthophosphate and immunoprecipitated with monoclonal anti-HDAg antibody. Seven-eighths of the immunoprecipitates were subjected to SDS-PAGE and analyzed by autoradiography. (C) One-eighth of the labeled immunoprecipitates were detected by Western blotting with human polyclonal anti-HDAg antibody. The positions of S-HDAg (24 kDa) and L-HDAg (27 kDa) are indicated.

FIG. 3

Serine 177 of S-HDAg is dispensable for HDV antigenomic RNA production. pCDm2G was cotransfected with constructs expressing wild-type S-HDAg (lane 2), S-HDAg mutants (lanes 3 to 5; the number above each lanes indicates the location of the mutated residue), or salmon sperm DNA (lane 6, negative control). On day 6 posttransfection, total cellular RNA was extracted and subjected to Northern blotting for detection of genomic RNA (A), antigenomic RNA (B), and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (C) by using different probes. (B) The star represents mRNA transcribed from S-HDAg-expressing plasmids. The positions of monomeric (1.7-kb) HDV RNA are indicated. (D) The intracellular S-HDAg was detected by Western blotting with anti-HDAg monoclonal antibody. The position of 24-kDa S-HDAg is indicated. Lane 1, a positive control.

FIG. 4

Serine 177 of S-HDAg is required for HDV genomic RNA production. Wild-type (lane 2) and mutant (lanes 3 to 5) S-HDAg or salmon sperm DNA (lane 6, negative control) was cotransfected with pCDm2AG. Antigenomic RNA (A), genomic RNA (B), and GAPDH (C) were detected by Northern blotting. (A) The star represents mRNA transcribed from S-HDAg-expressing plasmids, and the asterisk represents primary transcripts from the CMV promoter that were terminated at the poly(A) site for the mRNA. (D) The expression of S-HDAg was analyzed by Western blotting. Samples are organized identically to those in Fig. 3.

FIG. 5

The importance of serine 177 of S-HDAg in supporting HDV genomic RNA production was demonstrated by using a cDNA-free RNA transfection system. (A) Northern blot analysis of antigenomic RNA production from HuH-7 cells cotransfected with in vitro-transcribed genomic RNA and S-HDAg-encoding mRNA (lanes 2 and 3, wild type and serine 177 mutants). (B) Northern blot analysis of genomic RNA production from HuH-7 cells transfected with in vitro-transcribed antigenomic RNA and S-HDAg-encoding mRNA (lanes 2 and 3, wild type and serine 177 mutants). Lanes 1, positive controls which were from cotransfection with pCDm2G and pCDAg-S (A) and cotransfection with pCDm2AG and pCDAg-S (B); lanes 4, negative controls from transfection with only nonreplicating genomic (A) and antigenomic (B) RNA. The S-HDAg expression from genomic RNA transfection (C) or antigenomic RNA transfection (D) was analyzed by Western blotting.

FIG. 6

The expression of L-HDAg, a consequence of RNA editing, was influenced by the serine 177 mutation. The wild-type dimer of HDV, pCD2G and pCD2AG (2G_wt and 2AG_wt, respectively), and the Ser 177 dimer mutants, pCD2G₁₇₇ and pCD2AG₁₇₇ (2G₁₇₇ and 2AG₁₇₇, respectively), were transfected into HuH-7 cells. The HDV RNA and HDAg's were examined on days 6, 9, and 12 posttransfection (D6, D9, and D12). For detection of the antigenomic RNA production from 2G_wt and 2G₁₇₇, genomic RNA (A) and antigenomic RNA (C) were analyzed by Northern blotting. For detection of the genomic RNA production from 2AG_wt and 2AG₁₇₇, antigenomic RNA (B) and genomic RNA (D) were also analyzed by Northern blotting. The expression of S-HDAg (E) and L-HDAg (F) from these dimer constructs were analyzed by Western blotting and are shown. The positions of L-HDAg and S-HDAg are indicated.

FIG. 7

Effect of serine 177 phosphorylation on RNA editing. Total cellular RNA from transfection of wild-type dimers (pCD2G and pCD2AG) and mutant dimers (pCD2G₁₇₇ and pCD2AG₁₇₇) as described for Fig. 6 was subjected to an RT-PCR-based editing assay (see Materials and Methods for details) and followed by autoradiography. Arrowheads represent edited RNA.