RPLP1 and RPLP2 Are Essential Flavivirus Host Factors That Promote Early Viral Protein Accumulation

Abstract

The Flavivirus genus contains several arthropod-borne viruses that pose global health threats, including dengue viruses (DENV), yellow fever virus (YFV), and Zika virus (ZIKV). In order to understand how these viruses replicate in human cells, we previously conducted genome-scale RNA interference screens to identify candidate host factors. In these screens, we identified ribosomal proteins RPLP1 and RPLP2 (RPLP1/2) to be among the most crucial putative host factors required for DENV and YFV infection. RPLP1/2 are phosphoproteins that bind the ribosome through interaction with another ribosomal protein, RPLP0, to form a structure termed the ribosomal stalk. RPLP1/2 were validated as essential host factors for DENV, YFV, and ZIKV infection in two human cell lines: A549 lung adenocarcinoma and HuH-7 hepatoma cells, and for productive DENV infection of Aedes aegypti mosquitoes. Depletion of RPLP1/2 caused moderate cell-line-specific effects on global protein synthesis, as determined by metabolic labeling. In A549 cells, global translation was increased, while in HuH-7 cells it was reduced, albeit both of these effects were modest. In contrast, RPLP1/2 knockdown strongly reduced early DENV protein accumulation, suggesting a requirement for RPLP1/2 in viral translation. Furthermore, knockdown of RPLP1/2 reduced levels of DENV structural proteins expressed from an exogenous transgene. We postulate that these ribosomal proteins are required for efficient translation elongation through the viral open reading frame. In summary, this work identifies RPLP1/2 as critical flaviviral host factors required for translation.

Importance: Flaviviruses cause important diseases in humans. Examples of mosquito-transmitted flaviviruses include dengue, yellow fever and Zika viruses. Viruses require a plethora of cellular factors to infect cells, and the ribosome plays an essential role in all viral infections. The ribosome is a complex macromolecular machine composed of RNA and proteins and it is responsible for protein synthesis. We identified two specific ribosomal proteins that are strictly required for flavivirus infection of human cells and mosquitoes: RPLP1 and RPLP2 (RPLP1/2). These proteins are part of a structure known as the ribosomal stalk and help orchestrate the elongation phase of translation. We show that flaviviruses are particularly dependent on the function of RPLP1/2. Our findings suggest that ribosome composition is an important factor for virus translation and may represent a regulatory layer for translation of specific cellular mRNAs.

Keywords: flavivirus; ribosomal proteins; translation.

FIG 1

The RPLP1/2 heterodimer and RPLP0 are required for efficient DENV-2 and YFV infection of A549 and HuH-7 cells. Cells were transfected with either a nonsilencing control siRNA (NSC) or one of five independent siRNAs used to deplete RPLP1/2, three targeting RPLP1 (siP1_1, siP1_2, and siP1_6) and two targeting RPLP2 (siP2_1 and siP2_4). After 48 h, cells were infected at an MOI of 1 and infection was assessed after 24 h. (A and B) Western blotting results show knockdown of RPLP1/2 with independent siRNAs in A549 (A) and HuH-7 (B) cells. (C) Representative images showing A549 cells infected with DENV-2 (New Guinea C). Nuclei were Hoechst stained (blue), and the viral E protein was stained with 4G2 antibody (green). (D and E) Quantification of infection rates for DENV-2 and YFV (17D) are shown for A549 (D) and HuH-7 (E) cells. (F) A549 cells were transfected with the indicated siRNAs against RPLP0 and infected with YFV 48 h later at an MOI of 1. Western blotting results show knockdown of RPLP0 with two independent siRNAs. (G) Rates of infection are shown for cells transfected with NSC or siRNAs targeting RPLP0. The error bars represent standard deviations of three biological replicates. Statistical significance was assessed by a two-tailed Student's t test between NSC and experimental siRNAs. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 2

The RPLP1/2 heterodimer is required for efficient production of infectious DENV-2 and YFV. (A) Titers for supernatants from HuH-7 or A549 cells infected with DENV-2 were determined in Vero cells. (B) The same experiments as show in panel A, except with YFV. Statistical significance was assessed by a two-tailed Student's t test between NSC and experimental siRNAs. *, P < 0.05; **, P < 0.01, ***, P < 0.001.

FIG 3

Exogenous expression of RPLP1/2 rescues DENV-2 infection under conditions of endogenous RPLP1/2 knockdown. HuH-7 cells were transfected with the indicated siRNAs (NSC or siP1) and subsequently transfected with either empty vector (EV) plasmid or the siRNA-resistant RPLP1 (P1) expression plasmid. Cells were then infected with DENV-2 at an MOI of 1. (A) Western blotting results, showing RPLP1/2 levels after transfection with the indicated siRNA/plasmid combinations. (B) Representative immunofluorescence images of virus infection under different conditions. Nuclei were stained with Hoechst (blue), and the viral E protein was stained with 4G2 antibody (green). (C) Quantification of virus infection rates. The error bars represent standard deviations of three biological replicates. Statistical significance was assessed by a two-tailed Student's t test between the indicated conditions. ***, P < 0.001; ****, P < 0.0001.

FIG 4

RPLP1/2 are required for DENV-2 infection of Aedes aegypti mosquitoes. Mosquitoes were injected with dsRNA targeting RPLP1, RPLP2, or LacZ (control) and 3 days later were offered a blood meal containing DENV-2 at 1 × 10⁷ PFU/ml. Six days after oral infection, mosquitoes were homogenized and virus titers were determined in a cell-based plaque assay (yielding the number PFU per mosquito). Each data point represents an individual mosquito. (A) RNA levels of RPLP1/2 9 days after dsRNA injection. Statistical significance was assessed by a t test. *, P < 0.05. (B) The DENV-2 titer in each mosquito injected with dsRNA targeting LacZ, RPLP1, or RPLP2. The black lines represent the means and error bars represent standard errors of the means. Statistical significance was assessed by using the ranked nonparametric Mann-Whitney U test. *, P < 0.05; **, P < 0.01. N indicates the number of infected mosquitoes analyzed per condition.

FIG 5

RPLP1/2 are host factors for ZIKV. A549 and HuH- 7 cells were transfected with the indicated siRNAs and infected 48 h later with ZIKV (isolate 41525 or MEX_I_7) at an MOI of 1. (A) Representative images of infected A549 cells are shown. Nuclei were Hoechst stained (blue), and the viral E protein was stained with 4G2 antibody (green). (B and C) Quantification of infected A549 (B) and HuH-7 (C) cells is shown (top graphs) as is quantifications of infectious virus in the supernatants of A549 (B) or HuH-7 (C) cells infected with ZIKV (41525 or MEX_I_7). Error bars represent standard deviations of three biological replicates. Statistical significance was assessed by a two-tailed student's t test between NSC and experimental siRNAs, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 6

Effects of RPLP1/2 knockdown on replication of CBV3 and HCV. HuH-7 or HuH-7.5 cells were transfected with siRNAs and then infected with the indicated viruses at an MOI of 1 for 8 h (CBV3), 24 h (DENV-2), or 72 h (HCV). (A) Representative images showing HuH-7 cells infected with DENV-2 or CBV3. (B) Representative images of HuH-7.5 cells infected with DENV-2 or HCV. Nuclei were Hoechst stained (blue). Shown in green are the DENV-2 E protein, CBV3 VP1 protein, or HCV core protein. (C and D) Quantification of infection rates are shown for HuH-7 (C) and HuH-7.5 cells (D). Quantifications of virus yields are shown for DENV-2 in HuH-7 (C) or HuH-7.5 (D) cells, for CBV3 in HuH-7 cells (C), and for HCV (D) in HuH-7.5 cells. Graphs show means and standard deviations of percentages of infected cells or viral yields from three biological replicates. Statistical significance was assessed by a two-tailed Student's t test between NSC and experimental siRNAs. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 7

Depletion of RPLP1/2 results in cell-line-specific effects on global translation. Cells were transfected with siRNAs and 48 h later were incubated with methionine-free medium for 30 min before addition of [³⁵S]methionine for 30 min. Cells were subsequently lysed, and proteins were precipitated using TCA. CHX was used to control for background signal after TCA precipitation. (A) Western blotting results, showing knockdown of RPLP1/2 in HuH-7 and A549 cells. Only antibody against RPLP1 was used for the Western blot assay with HuH-7 cells. (B) Autoradiography of ³⁵S-labeled proteins fractionated by SDS-PAGE. (C) Liquid scintillation counting of incorporated ³⁵S-labeled proteins. For each cell line, the NSC results was set to 100%. The graphs show mean values of two independent experiments for HuH-7 cells and three independent experiments for A549 cells. Error bars represent standard deviation measurements. Statistical significance was assessed by a two-tailed Student's t test between NSC and other conditions. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 8

RPLP1/2 are required for early viral protein accumulation. (A) Knockdown of RPLP1/2 is shown by Western blotting of A549 cells with or without NITD008 treatment. (B) Quantification of DENV-2 RNA by RT-qPCR normalized to 18S rRNA is shown under control and RPLP1/2 knockdown conditions. (C) Luciferase measurements from infected cells harvested 4 hpi are shown for each siRNA transfection condition in the presence or absence or NITD008 treatment. (D) A549 cells were transfected with NSC siRNA or siRNA against RPLP2 and then infected with DENV-2 for 6 h at an MOI of 10 in the presence or absence of NITD008. NS3 accumulation was detected by Western blotting, and results for triplicate samples are shown for NSC and siP2 conditions. CHX-treated and uninfected samples served as controls for background signal. (E) Quantification of NS3 levels was calculated by normalizing results to those for β-actin. The NSC condition result was set to 100%. Error bars represent standard deviation measurements of three independent wells. Statistical significance was assessed by a two-tailed Student's t test between NSC and experimental siRNA conditions. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 9

RPLP1/2 knockdown impairs accumulation of DENV-2 structural proteins expressed in stable cell lines. Tetracycline-inducible HeLa and HEK-293 cells expressing C-prM-E were transfected with the indicated siRNAs, and 48 h later tetracycline was added to the medium for 24 h. Cell lysates were harvested for Western blot analysis 24 h after addition of tetracycline. (A) Representative Western blots showing expression of C-prM and E proteins under NSC or siP2_4 transfection conditions (performed in triplicate). The arrows indicate cleaved E protein, the 37-kDa C-prM minority species, and the 33-kDa C-prM majority species. Samples from uninduced cells are also indicated (-tet). (B) Quantifications of C-prM-E RNA by RT-qPCR normalized to 18S rRNA and protein bands in the Western blot assay from HeLa cells, normalized to results with β-actin. The NSC conditions were set to 100%. Graphs show means and standard deviations from quantification of two independent assays performed in triplicate. (C) The same experiment in shown in panel B, except HEK-293 cells were used. Graphs show means from the quantification of three independent assay results. Statistical significance was assessed by a two-tailed Student's t test between NSC and experimental siRNA conditions. *, P < 0.05; ****, P < 0.0001.