Interaction between hantavirus nucleocapsid protein (N) and RNA-dependent RNA polymerase (RdRp) mutants reveals the requirement of an N-RdRp interaction for viral RNA synthesis

Abstract

Viral ribonucleocapsids harboring the viral genomic RNA are used as the template for viral mRNA synthesis and replication of the viral genome by viral RNA-dependent RNA polymerase (RdRp). Here we show that hantavirus nucleocapsid protein (N protein) interacts with RdRp in virus-infected cells. We mapped the RdRp binding domain at the N terminus of N protein. Similarly, the N protein binding pocket is located at the C terminus of RdRp. We demonstrate that an N protein-RdRp interaction is required for RdRp function during the course of virus infection in the host cell.

FIG 1

Interaction of N with RdRp. (A) Pictorial representation of RdRp mutants used in this study. A conserved domain prediction software from NCBI (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) suggested that the catalytic domain is located in the region from amino acids 562 to 1286 and the upstream intervening region of unknown function corresponds to the region from amino acids 238 to 562. (B) HEK293T cells grown on 60-mm-diameter dishes were cotransfected with a plasmid expressing myc-tagged wild-type (w.t.) N protein along with another plasmid expressing the His-tagged RdRp fragment of interest (pol1–250, pol251–752, pol751–1290, pol1291–2153, pol238–562, or pol562–1286). Cells were lysed 48 h posttransfection, the resulting cell lysates were immunoprecipitated (IP) with anti-myc tag antibody, and immunoprecipitated material was examined by Western blot (WB) analysis using either anti-His tag antibody (panel i) or anti-myc tag antibody (panel ii). The light chain of anti-myc antibody is shown in panel iii. Cell lysates were also incubated with Ni-NTA beads, and the eluted material from washed beads was examined by Western blot analysis using anti-myc tag antibody (panel iv). Equal volumes of whole-cell lysates (WCL) were examined by Western blot analysis using either anti-His tag antibody (panel v) or anti-myc tag antibody (panel vi) or anti-GAPDH antibody (panel vii) or antitubulin antibody (panel viii). The band intensity for β-actin and tubulin was quantified and normalized to the last band from the left; the intensity is indicated at the bottom. Note that the plasmids used for transfection are shown in Table 1. (C) HeLa cells in a 96-well plate at 70% confluence were transfected with 2.5 μg of a plasmid expressing either the pol1–250 fragment or pol 238–562 fragment. Cell viability was measured 48 h posttransfection using the CellTox green cytotoxicity assay. The experiment was performed in duplicates. (D) Both pol1–250 and pol1291–2153 fragments were expressed in E. coli and purified using Ni-NTA beads. HEK293T cells were transfected with a plasmid (Table 1) expressing wild-type N protein. Cells were lysed 48 h posttransfection, and the resulting lysates were incubated with 2 μg of the purified RdRp fragment of interest at 4°C for 3 h. The lysates were immunoprecipitated with anti-myc tag antibody, and the immunoprecipitated material was analyzed by Western blotting using anti-His tag antibody to detect the RdRp fragments. (E) Huh7 cells in a 60-mm dish were infected with SNV at an MOI of 2. Twenty-four hours postinfection, cells were transfected with 2.5 μg of a plasmid (Table 1) expressing the Pol1291–2153 fragment. Cells were lysed, the resulting lysates were immunoprecipitated with polyclonal anti-N antibody, and the immunoprecipitated material was analyzed by Western blot analysis using anti-His antibody to detect the pol1291–2153 fragment (bottom). The cell lysates were also incubated with Ni-NTA beads, and the eluted material from washed beads was analyzed by Western blotting using anti-N antibody (top).

FIG 2

Confocal imaging of N and RdRp fragments. (A) HeLa cells were grown on a coverslip up to 70% confluence in a 35-mm-diameter dish. Cells were cotransfected with a plasmid expressing GFP-N fusion protein along with another plasmid expressing either the pol238–562 or pol1293–2153 fragment fused with mCherry. Thirty-six hours posttransfection, cells were fixed with 3.7% paraformaldehyde (PFA) and visualized with a Nikon Eclipse C1si confocal microscope. Due to the large image size, all images in each row were stacked, and the same region was excised for presentation in this figure. DAPI, 4′,6-diamidino-2-phenylindole. (B) We next examined the colocalization of N protein with pol238–562 and pol1291–2153 fragments in virus-infected cells. Vero E6 cells were grown on a coverslip as mentioned above and infected with SNV at the MOI of 2. Thirty-six hours postinfection, cells were transfected with 2.5 μg of plasmid expressing either the pol238–562 or pol1293–2153 fragment fused to mCherry. Eighteen hours posttransfection, cells were fixed and examined under confocal microscope. N protein was visualized using anti-N primary antibody and a secondary antibody conjugated with FITC. In the control experiment, rat IgG was used instead of anti-N primary antibody, and the secondary antibody was conjugated with FITC, which did not generate any signal (not shown). This demonstrates the specificity of anti-N antibody in this assay. The bar in the right bottom panel is 10 μm.

FIG 3

The RdRp binding domain is located at the N terminus of N. (A) Pictorial representation of N deletion mutants used in this work. The deleted regions are not shown. (B) HEK293T cells grown on 60-mm-diameter dishes were cotransfected with a plasmid (Table 1) expressing FLAG-tagged pol1291–2153 fragment along with another plasmid expressing the His-tagged wild type (w.t.) or N mutant (N51–428, N101–428, N151–428, N176–428 N231–428, N1–402, N1–346, N1–237, or N1–175). Forty-eight hours posttransfection, cells were lysed, the resulting cell lysates were immunoprecipitated (IP) with anti-FLAG antibody, and the immunoprecipitated material was examined by Western blotting (WB) using either anti-SNV N antibody to detect N mutants (panel i) or anti-FLAG antibody to detect pol1291–2153 fragment (panel ii). The heavy chain of anti-FLAG antibody is shown in panel iii. The cell lysates were also incubated with Ni-NTA slurry (Qiagen), and the eluted material from washed beads was examined by Western blotting using anti-FLAG antibody to detect the pol1291–2153 mutant (panel iv). Equal volumes of whole-cell lysates (WCL) were also examined by Western blot analysis using either anti-SNV N antibody (panel v) or anti-FLAG tag antibody (panel vi). Note that the plasmids used for transfection are shown in Table 1.

FIG 4

Overexpression of the pol1291–2153 mutant specifically inhibits hantavirus replication in cells. (A) Huh7 cells were transfected with 2.5 μg of plasmid expressing either the pol1291–2153 or pol751–1290 mutant. Sixteen hours posttransfection, cells were infected with SNV at an MOI of 0.5. Thirty-six hours postinfection, cells were again transfected to boost the expression of RdRp fragments. Cells were harvested at 0, 12, 24, 36, 48, and 72 h postinfection. Total RNA was extracted from half of the cells collected at each time point with the RNeasy minikit and converted to cDNA using a random primer. S-segment RNA levels were quantified by real-time PCR using β-actin as an internal control, as previously reported (4, 14). Fold changes in S-segment RNA levels related to the zero hour time point are shown. Fold changes calculated from three independent experiments were averaged and used to calculate the standard deviation, shown as error bars. The remaining half of the cells were lysed with 1× Laemmli sample buffer containing 2% SDS and analyzed by Western blotting using the monoclonal anti-His antibody to monitor the expression of RdRp mutants. (B) HeLa cells were transfected with 2.5 μg of either empty vector or a plasmid expressing the Pol1291–2153 fragment. Cells were infected with vesicular stomatitis virus 16 h posttransfection (10³ PFU/well in a 24-well plate). Cells were harvested at 0, 6, 12, 18, and 24 h postinfection. Half of the cells were used for RNA extraction to quantify the VSV genomic RNA levels using real-time PCR. Fold changes in vRNA levels were calculated as mentioned in panel A. The remaining half of the cells were lysed for Western blot analysis to check the expression of tubulin and the pol1291–2153 fragment (bottom), as described in panel A.