Identification and Characterization of the Host Protein DNAJC14 as a Broadly Active Flavivirus Replication Modulator

Viruses in the Flavivirus genus of the Flaviviridae family are arthropod-transmitted and contribute to staggering numbers of human infections and significant deaths annually across the globe. To identify cellular factors with antiviral activity against flaviviruses, we screened a cDNA library using an iterative approach. We identified a mammalian Hsp40 chaperone protein (DNAJC14) that when overexpressed was able to mediate protection from yellow fever virus (YFV)-induced cell death. Further studies revealed that DNAJC14 inhibits YFV at the step of viral RNA replication. Since replication of bovine viral diarrhea virus (BVDV), a member of the related Pestivirus genus, is also known to be modulated by DNAJC14, we tested the effect of this host factor on diverse Flaviviridae family members. Flaviviruses, including the pathogenic Asibi strain of YFV, Kunjin, and tick-borne Langat virus, as well as a Hepacivirus, hepatitis C virus (HCV), all were inhibited by overexpression of DNAJC14. Mutagenesis showed that both the J-domain and the C-terminal domain, which mediates self-interaction, are required for anti-YFV activity. We found that DNAJC14 does not block YFV nor HCV NS2-3 cleavage, and using non-inhibitory mutants demonstrate that DNAJC14 is recruited to YFV replication complexes. Immunofluorescence analysis demonstrated that endogenous DNAJC14 rearranges during infection and is found in replication complexes identified by dsRNA staining. Interestingly, silencing of endogenous DNAJC14 results in impaired YFV replication suggesting a requirement for DNAJC14 in YFV replication complex assembly. Finally, the antiviral activity of overexpressed DNAJC14 occurs in a time- and dose-dependent manner. DNAJC14 overexpression may disrupt the proper stoichiometry resulting in inhibition, which can be overcome upon restoration of the optimal ratios due to the accumulation of viral nonstructural proteins. Our findings, together with previously published work, suggest that the members of the Flaviviridae family have evolved in unique and important ways to interact with this host Hsp40 chaperone molecule

DNAJC14 inhibits YFV in a temporal and dose-dependent manner.

<p>(A) SW13 cells were mock transduced (Mock) or transduced with Trip-RFP-hNZAP (NZAP), Trip-RFP-hDNAJC14-FL (FL) or Trip-RFP-hDNAJC14-NT1 (NT1) and infected 2 d later with YFV-Venus (moi = 5). Cells were fixed at 1.5 d post infection and analyzed by flow cytometry. The Venus (y-axis) and RFP (x-axis) fluorescence intensities of the cells are shown; gates to indicate expression of the transduced protein or productive infection were set on Mock transduced, uninfected cells (not shown). (B) SW13 cells were transduced with Trip-RFP-hDNAJC14-NT1 and infected 2 d later with YFV-Venus (moi = 5) and analyzed as in (A) at the indicated days after infection. (C) SW13 cells were left untransduced (closed circles) or were transduced with Trip-RFP-hDNAJC14-NT1 (RFP-NT1, open circles) and infected 2 d later with YFV-Venus (moi = 5). At the indicated times, the medium was removed and YFV present in the medium was quantified by plaque assay. Each data point represents the mean titer obtained from duplicate wells; error bars indicating the range are obscured by the symbols. The dotted line indicates the sensitivity of the plaque assay.</p

DNAJC14 is recruited to YFV replication complexes.

<p>(A) SW13 cells were left untransduced (top row) or were transduced (lower 2 rows) with the noninhibitory V1-hDNAJC14 mutants FL-H471Q or CT1 as indicated. Two d later the cells were mock treated (left panels) or were challenged with YFV (moi = 5, right 3 panels). After an additional 2 d, the cells were fixed and immunostained with rabbit anti-YFV NS3 polyclonal antibodies (NS3), and mouse anti-calnexin antibody (calnexin) or mouse anti-myc monoclonal antibody (myc) as indicated. AF488-conjugated anti-mouse IgG and AF594-conjugated anti-rabbit IgG antibodies were used as secondary antibodies. The cells were analyzed by confocal microscopy and representative images are shown. Calnexin or DNAJC14 mutants are shown in green, YFV NS3 is shown in red, and the merged images are shown on the right. (B) SW13 cells were left untransduced or were transduced with the V1-hDNAJC14-CT1 mutant (CT1-myc) as indicated and were infected 2 d later with YFV (moi = 1). After 2 d of infection, myc-tagged DNAJC14-CT1 was immunoprecipitated using anti-myc antibody. Western blots were performed using antibodies against NS3, calnexin and the myc epitope tag as indicated. (C) SW13 cells were left uninfected or were infected with YFV (moi = 1) as indicated. The cells were fixed 1 d later and analyzed by confocal microscopy for endogenous DNAJC14 (red) and double stranded RNA (dsRNA, green). The merged image is shown on the right. Arrows indicate several areas of colocalized DNAJC14 and dsRNA.</p

DNAJC14 confers resistance to YFV-induced cell death.

<p>(A) Photographs 7 d after YFV challenge (moi = 1) of SW13 cells transduced with Round 3 of the selected lentiviral cDNA constructs compared to cells transduced with V1-GFP vector control. (B) The cells transduced with the Round 3 lentivirus pool and surviving YFV infection (Rd 3) were expanded and reinfected with YFV at the indicated moi. Crystal violet staining was performed 3 d later. Cells transduced with vector alone serve as a control (V1-GFP). (C) DNA was isolated from naïve SW13 or Round 3 (Rd 3) cells, and the lentiviral insert amplified by PCR. The major band was identified as encoding a truncated hamster DNAJC14. Sizes of the DNA markers (kb) are indicated to the left. (D) A schematic of human DNAJC14 is shown, with the putative transmembrane (TM) domains (gray), J domain (red) with conserved HPD sequence, zinc finger motifs (blue) and Jiv90 domain (orange) indicated. A schematic of the isolated hamster clone, showing homology to amino acids 305 to 702 of human DNAJC14, is shown below.</p

DNAJC14 does not inhibit NS2/3 cleavage of YFV and HCV.

<p>YFV replication is inhibited in cells inducibly expressing the NT5 mutant form of DNAJC14. (A) T-REx-293-NT5 cells were left uninduced or induced by treatment with doxycycline (Dox) for the indicated h and lysates were analyzed by Western blot using anti-myc and anti-actin (loading control) antibodies. Migration of size standards (in kDa) is indicated to the left. (B) T-REx-293-NT5 (NT5) or T-REx-293-LacZ (LacZ) cells were induced to express DNAJC14-NT5 or β-galactosidase, respectively, by 24 h treatment with doxycycline. The cells were then infected with YFV (moi = 5) and the medium was harvested and replaced at each timepoint. Virion production since the prior time point was enumerated by plaque assay. Pfu, plaque forming units. (C) YFV and HCV NS2/3 cleavage in T-REx-293-NT5 cells. T-REx-293-NT5 cells were left untreated or were induced to express DNAJC14-NT5 by 24 h treatment with doxycycline (Dox) as indicated. The cells were then cotransfected with pEGFP (loading control) and either pFlag-HCV-NS2/3(181) or pFlag-YFV-NS2/3(181). As controls, NS2-3 proteins containing active site mutations in the HCV NS2 (H143A) or YFV NS3 (S138A) proteases and incapable of cleavage activity were also expressed as indicated. Cells were harvested 1 day later and analyzed by Western blot using anti-Flag antibody (top panel) or anti-myc and anti-GFP antibodies (bottom panel). Arrows indicate the migration of the relevant proteins and migration of size standards (in kDa) is indicated on the left.</p

DNAJC14 inhibits a post entry step.

<p>(A) and (B) V1-GFP- (GFP) or V1-hDNAJC14-FL (FL) transduced SW13 cells were electroporated 2 d later with in vitro transcribed YF-17D RNA to bypass the entry step. Cells and media were harvested at the indicated times after electroporation. (A) Western blot analysis was performed on equal volumes of the cell extracts using the antibodies indicated to the right; actin serves as a loading control and antibodies to myc detect the tagged DNAJC14 protein. Migration of size standards (in kDa) is indicated to the left. (B) Virus present in the medium from V1-GFP- (GFP, black circles) and V1-hDNAJC14-FL (FL, red triangles) transduced cells was enumerated by plaque assay. A single separate well was utilized for each time point. Pfu, plaque forming units. (C) Schematic of the YFV replicon construct, which expresses Renilla luciferase (RLuc) in place of the structural proteins. The locations of the YFV nonstructural proteins are indicated in the polyprotein, which is targeted to the ER by a signal sequence (red bar). UTR, untranslated region; 2A (black bar), the foot and mouth disease virus 2A autoproteolytic peptide. (D) V1-GFP- (GFP, circles) and V1-hDNAJC14-FL- (FL, triangles) transduced SW13 cells were electroporated with the wild type YF replicon RNA (filled symbols, solid lines) or with replication incompetent RNA containing a mutation in the RNA-dependent RNA polymerase (ΔDD, open symbols, dashed lines). At various times after electroporation, the cells were harvested and luciferase activity was determined. Data represents the mean luciferase value of triplicate samples; error bars indicate the standard deviation and are sometimes obscured by the symbol. RLU, relative light units. Similar results were obtained in an independent experiment utilizing Huh7.5 cells.</p

Modulation of DNAJC14 levels by siRNA alters YFV replication.

<p>SW13 cells transduced with V1-GFP (Vector) or V1-hDNAJC14-FL (DNAJC14) were treated with irrelevant control siRNA or siRNA targeting DNAJC14 as indicated and were infected with YFV (moi = 5). After 24 h the medium from triplicate samples was collected for virus titration, while 2 samples were pooled for RNA isolation and cells in the remaining sample were harvested for Western analysis. (A) DNAJC14 RNA levels were determined by quantitative RT-PCR. For each sample, DNAJC14 RNA levels were normalized to levels of GAPDH RNA and the ratio present in the vector control cells treated with the irrelevant control siRNA was set to 1. Bars represent mean relative levels obtained from triplicate RT reactions; error bars indicate the standard deviation. Asterisks indicate a significant difference from the cells transduced with control vector and receiving the control siRNA (students t test; *p<0.05, **p<0.01, ***p<0.001). (B) Virus present in the medium was titered by plaque assay. Bars represent mean titers from triplicate samples; error bars indicate the standard deviation. Pfu, plaque forming units. Asterisks indicate a significant difference in virus production compared to cells transduced with control vector and receiving the control siRNA (students t test; ***p<0.001). Virus titers obtained after silencing DNAJC14 in the vector control versus DNAJC14 transduced cells were not statistically different (ns). (C) Western blot analysis was performed on the silenced samples using the indicated antibodies. Whether the cells were transduced with V1-hDNAJC14-FL (DNAJC14-myc+) or control vector (−) is indicated. Migration of size markers (in kDa) is indicated to the left. The asterisk indicates a non-specific band.</p

Mutagenesis of DNAJC14.

<p>(A) A schematic of DNAJC14 is shown with the truncation mutants indicated below. Arrows above the schematic indicate the location of point mutants engineered in the NT5 truncation mutant backbone. Each mutant contained a C-terminal myc epitope tag, indicated in green. (B and C) SW13 cells were transduced with lentivirus expressing the indicated mutants and 2 d later were challenged with YFV (moi = 5). Cells and media were harvested 1 d later. V, control V1 lentivirus; FL, lentivirus expressing full-length hDNAJC14; WT, the NT5 truncation mutant without any point mutations. (B) Western blot analysis was performed on equal volumes of the cell extracts using anti-myc antibody to detect the tagged DNAJC14 or mutant protein. Migration of size standards (in kDa) is indicated to the left. (C) YFV present in the medium was enumerated by plaque assay. Data represent mean values obtained from triplicate wells; error bars indicate the standard deviation. Pfu, plaque forming units.</p

DNAJC14 inhibits YFV replication.

<p>(A). YFV replication is inhibited in Round 3 (Rd 3) cells. Rd 3 or naïve cells were infected with YFV (moi = 1) and at the indicated days after infection the amount of virus released into the medium was determined by plaque assay. A single well was used for each cell type, with replacement of the medium at each timepoint. (B). Hamster DNAJC14 inhibits YFV protein expression. SW13 cells were transduced with control lentivirus (V1-GFP) or lentivirus containing the truncated hamster DNAJC14 insert (clone 1-2, 1-3, 1-4 and 1-5) and were infected 2 d later with YFV (moi = 0.5). Cells were analyzed by Western blot for NS3 expression 3 d after infection. Naïve cells serve as a negative control and actin serves as a loading control. (C). Full-length and truncated human DNAJC14 inhibit YFV replication. SW13 cells were mock transduced (Mock) or transduced with the indicated lentiviral vectors and 2 d later were infected with YFV (moi = 0.5) as indicated. Cells were harvested and subjected to Western blot analysis 3 d after infection using antibodies as indicated to the right. V1-FL expresses myc epitope tagged full-length human DNAJC14 while V1-NT1 expresses myc tagged truncated human DNAJC14 (aa 305–702). Naïve cells were left untransduced and uninfected and serve as a negative control. For B and C migration of size standards (in kDa) is indicated.</p