Human parainfluenza virus type 2 L protein regions required for interaction with other viral proteins and mRNA capping

Abstract

The large RNA polymerase (L) protein of human parainfluenza virus type 2 (hPIV2) binds the nucleocapsid, phosphoprotein, and V protein, as well as itself, and these interactions are essential for transcription and replication of the viral RNA genome. Although all of these interactions were found to be mediated through the domains within the N terminus of L, the C terminus of the L protein was also required for minigenome reporter gene expression. We have identified a highly conserved rubulavirus domain near the C terminus of the L protein that is required for mRNA synthesis but not for genome replication. Remarkably, this region of L shares homology with a conserved region of cellular capping enzymes that binds GTP and forms a lysyl-GMP enzyme intermediate, the first step in the cellular capping reaction. We propose that this conserved region of L also binds GTP (or GDP) to carry out the second step of the unconventional nonsegmented negative-strand virus capping reaction.

FIG. 1.

Analysis of interactions between the C-terminally truncated L and P, V, NP, or L proteins by immunoprecipitation (IP) and Western blot (WB) assay. (A) Schematic diagram of the C-terminally truncated L proteins. Binding to P, V, NP, or L protein is summarized at right. (B to E) The proteins from transfected BSR T7/5 cell lysates were analyzed by Western blotting for anti-P/V MAb (B and C), anti-NP MAb (D), or anti-Flag polyclonal antibody (E) (top panels) and anti-L MAb (middle panels). Immunoprecipitates with anti-P/V MAb (B and C), anti-NP MAb (D), or anti-Flag antibody (E) were probed by anti-L MAb in a Western blot assay (bottom panels). Numbers on the bottom correspond to the L proteins depicted in panel A. Sizes of molecular mass markers (in kDa) are shown. The asterisk at the left indicates the immunoglobulin heavy chain.

FIG. 2.

Analysis of interactions between the N- and C-terminally truncated L and P, V, NP, or L proteins by immunoprecipitation and Western blot assay. (A) Schematic diagram of the N- and C-terminally truncated L proteins. Binding to P, V, NP, or L protein is summarized at right. (B to E) The proteins from transfected BSR T7/5 cell lysates were analyzed by Western blotting for anti-P/V MAb (B and C), anti-NP MAb (D), or anti-L MAb (E) (top panels) and for anti-Flag polyclonal antibody (middle panels). Immunoprecipitates with anti-P/V MAb (B and C), anti-NP MAb (D), or anti-Flag polyclonal antibody (E) were probed by anti-Flag polyclonal antibody or anti-L MAb in Western blot assays (bottom panels). Numbers on the bottom correspond to the L proteins depicted in panel A. Sizes of molecular mass markers (in kDa) are shown.

FIG. 3.

GFP expression from the minigenome system by C-terminally truncated L proteins. (A) Schematic diagram of the minigenome system. Plasmids pTM1-NP, pTM1-P, and pTM1-L were used to express NP, P, and L proteins in BSR T7/5 cells, a cell line that constitutively expresses T7 RNA polymerase (RNAP). The negative-strand minigenome can be generated from transcription of primary T7 transcript in BSR T7/5 cells. This template is assembled and transcribed into the reporter gene mRNA, resulting in expression of GFP as well as being used as a template for replication by a complex of NP, P, and L proteins. (B) The plasmids pPIV2-GFP (1 μg), pTM1-NP (0.75 μg), pTM1-P (0.4 μg), and pTM1-L or mutants (0.75 μg) were transfected into BSR T7/5 cells. At 48 h posttransfection, cells were lysed and assayed by Western blotting using antibodies against NP, P, and L proteins and GFP.

FIG. 4.

GFP expression from the minigenome system by L proteins that have mutations in the amino acids conserved among paramyxoviruses. (A) Comparison of sequences at the C termini of paramyxovirus L proteins. hPIV2, human parainfluenza virus type 2 (GenBank accession number AB176531); SV41, simian virus 41 (NC 006428); PIV5, parainfluenza virus type 5 (NC 006430); MuV, mumps virus (D10575); PIV4A or -4B, parainfluenza virus type 4A (AB543336) or 4B (AB543337); MPRV, Mapuera virus (EF095490); PoRV, porcine rubulavirus (NC 009640); MenV, Menangle virus (AF326114); TiV, Tioman virus (AF298895); NDV, Newcastle disease virus; HeV, Hendra virus; NiV, Nipah virus; MeV, measles virus; RPV, rinderpest virus; SeV, Sendai virus; PIV3, parainfluenza virus type 3. The amino acids are numbered from the amino terminus of each L protein. The asterisks indicate amino acids conserved among rubulaviruses. (B) Schematic diagram of L proteins that have mutations in the conserved motif. (C) GFP expression from the minigenome system by the L mutants as shown in Fig. 3. Numbers on the bottom correspond to each L mutant protein listed in panel B.

FIG. 5.

GFP expression from the minigenome system by L proteins that have mutations in the conserved amino acids. (A) Schematic diagram of the L mutants. (B) GFP expression from minigenome system by the L mutants as shown in Fig. 3. Numbers on the bottom correspond to each L mutant protein listed in panel A.

FIG. 6.

Alignment of negative-stranded RNA virus polymerase domains displaying homology with the catalytic site of eukaryotic mRNA guanylyltransferases. The catalytic lysine is marked in bold. SeV, Sendai virus (UniProtKB accession number Q9DUD8); PIV3, parainfluenza virus type 3 (P12577); MeV, measles virus (P12576); RPV, rinderpest virus (P41357); CDV, canine distemper virus (P24658); HRSVA, human respiratory syncytial virus A (strain A2) (P28887); HRSVL, human respiratory syncytial virus A (long strain) (Q9IWW8); HRSVB, human respiratory syncytial virus B (strain B1) (O36635); BRSV, bovine respiratory syncytial virus (O91940); MPV, murine pneumonia virus (Q50EW2); NDV, Newcastle disease virus (Q9DLD3); HeV, Hendra virus (O89344); NiV, Nipah virus (Q997F0); hPIV2, human parainfluenza virus type 2 (P26676); SV41, simian virus 41 (P35341); PIV5, parainfluenza virus type 5 (Q03396); MuV, mumps virus (P30929); mouse (O55236); rat (B5DFA8); bovine (Q2KHX7); human (O60942); Xenopus laevis (Q6NWX1); zebrafish (Q6NY98); tick (B7PBC2); Drosophila melanogaster (Q9VY44); yeast, Saccharomyces cerevisiae (Q01159); PBCV, Paramecium bursaria Chlorella virus (Q84424); VARV, variola major virus (P33057). ScanProsite software (http://www.expasy.org/tools/scanprosite/) was used to scan the experimentally defined C-terminal motif against the UniProt/Swiss-Prot database. The alignment of paramyxovirus polymerases was made by the Muscle method; cellular motifs were added manually.

FIG. 7.

Minigenome RNA expression and L mutants. (A) RT-PCR. Total RNAs were purified from BSR T7/5 cells transfected with pPIV2-GFP, pTM1-P, pTM1-NP, and pTM1-L or mutants. RT-PCRs were carried out as described in Materials and Methods. Ethidium bromide staining of products from the PCRs is shown. Lane 1, untransfected; lane 2, without pTM1-L; lane 3, PIV2 wt; lane 4, C-terminal-50-aa-truncated L (Fig. 3B, CΔ50); lane 5, K2218/2222/G2225A (Fig. 4B, row 4); lane 6, G2225M (Fig. 5A, row 4); lane 7, H1298/R1299A (Fig. 5A, row 7); lane 8, without the RT reaction. (B) Primer extension. The same RNAs were analyzed by primer extension, using a ³²P-oligonucleotide representing positions 178 to 160 (Materials and Methods). Lane 1, untransfected; lane 2, without pTM1-L; lane 3, PIV2 wt; lane 4, C-terminal-50-aa-truncated L; lane 5, K2218/2222/G2225A; lane 6, G2225M; lane 7, H1298/R1299A.