RNA Helicase A Is an Important Host Factor Involved in Dengue Virus Replication

Abstract

Dengue virus (DENV) utilizes host factors throughout its life cycle. In this study, we identified RNA helicase A (RHA), a member of the DEAD/H helicase family, as an important host factor of DENV. In response to DENV2 infection, nuclear RHA protein was partially redistributed into the cytoplasm. The short interfering RNA-mediated knockdown of RHA significantly reduced the amounts of infectious viral particles in various cells. The RHA knockdown reduced the multistep viral growth of DENV2 and Japanese encephalitis virus but not Zika virus. Further study showed that the absence of RHA resulted in a reduction of both viral RNA and protein levels, and the data obtained from the reporter replicon assay indicated that RHA does not directly promote viral protein synthesis. RHA bound to the DENV RNA and associated with three nonstructural proteins, including NS1, NS2B3, and NS4B. Further study showed that different domains of RHA mediated its interaction with these viral proteins. The expression of RHA or RHA-K417R mutant protein lacking ATPase/helicase activity in RHA-knockdown cells successfully restored DENV2 replication levels, suggesting that the helicase activity of RHA is dispensable for its proviral effect. Overall, our work reveals that RHA is an important factor of DENV and might serve as a target for antiviral agents.IMPORTANCE Dengue, caused by dengue virus, is a rapidly spreading disease, and currently there are no treatments available. Host factors involved in the viral replication of dengue virus are potential antiviral therapeutic targets. Although RHA has been shown to promote the multiplication of several viruses, such as HIV and adenovirus, its role in the flavivirus family, including dengue virus, Japanese encephalitis virus, and emerging Zika virus, remains elusive. The current study revealed that RHA relocalized into the cytoplasm upon DENV infection and associated with viral RNA and nonstructural proteins, implying that RHA was actively engaged in the viral life cycle. We further provide evidence that RHA promoted the viral yields of DENV2 independent of its helicase activity. These findings demonstrated that RHA is a new host factor required for DENV replication and might serve as a target for antiviral drugs.

Keywords: dengue virus; flavivirus; host factor; viral replication.

FIG 1

RHA was relocalized into cytoplasm upon DENV2 infection and was involved in the viral production. (A) A549 cells were mock infected or infected with DENV2 (MOI of 3). Cells were harvested at 0, 12, and 24 h p.i. for protein extraction. Proteins derived from cytoplasmic fraction and nuclear fraction of the cells were subjected to Western blotting. GAPDH and Lamin B served as the loading control for the cytoplasmic fraction and nuclear fraction. Calnexin served as an indicator of ER. Data are representative of at least three independent experiments. (B) Confirmation of siRNA efficiency. A549 cells were transfected with 16 nmol siNC, siRHA1, siRHA2, or siRHAm (mixture of siRHA1 and siRHA2). At 48 h posttransfection, cells were harvested for Western blotting. GAPDH was probed as an internal control. (C) Cell growth curve of RHA-KD cells. At 1, 2, and 3 days posttransfection with siNC or siRHAm, cell numbers were counted by trypan blue staining. (D) Single-step virus growth assay. A549 cells were transfected with siNC or siRHAm for 48 h, followed by DENV2 infection (MOI of 3). At 24 h p.i., cell supernatants were collected for focus-forming assay. (E) Western blot to examine the siRNA efficacy in monocyte-derived macrophage (MDM) and HepG2 cells. Cells were transfected with 16 nmol siNC, siRHA1, siRHA2, or siRHAm. At 48 h posttransfection, cells were harvested for Western blotting. (F) Single-step virus growth assay. MDM and HepG2 cells were transfected with siNC, siRHA1, siRHA2, or siRHAm for 48 h, followed by DENV2 infection (MOI of 3). At 24 h p.i., cell supernatants were collected for focus-forming assay. (G) Multistep virus growth assay. A549 cells were transfected with siRNA for 48 h, followed by infection of DENV2 NGC, DENV2 16681, JEV, and ZIKV (MOI of 0.01). Samples were collected at 24, 48, 72, and 96 h p.i. The viral titers were measured by standard plaque or focus-forming assay. Data were shown as means ± SD from at least three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (each by unpaired, two-tailed Student's t test).

FIG 2

RHA knockdown reduced the viral RNA and protein levels. A549 cells were transfected with siNC or siRHAm for 48 h and applied for the following detections. (A) Entry assay. Cells were inoculated with DENV2 (MOI of 3), followed by incubation at 4°C or 37°C for 1 h. Cells were washed with PBS and harvested for RNA extraction and real-time PCR to measure viral RNA levels. (B) Cells were mock infected or infected with DENV2 and harvested at 6, 12, and 24 h p.i. The viral RNA level was measured by real-time PCR. (C and D) Cells were infected with DENV2 and harvested at 8, 16, and 24 h p.i. for Western blotting to detect NS3 (C) or at 24 h p.i. for IFA to detect prM (D). Actin served as an internal control. (E) At least 200 cells from each sample in three independent experiments were counted. (F) Effect of RHA knockdown on translation of the DENV2 reporter replicon. Cells were transfected with pDENrep-FH (16681) replicon (WT) and the NS5 GVD mutant (NS5^GVD) RNAs. The cells were harvested at 6 h posttransfection for luciferase assay. Data are shown as means ± SD from at least three independent experiments. NS, no statistical significance; ***, P < 0.001 by unpaired, two-tailed Student's t test.

FIG 3

RHA was associated with viral RNA. A549 cells were infected with DENV2 (A and B) or ZIKV (C and D) at an MOI of 3 and harvested at 6 and 12 h p.i. for RIP assay. Whole-cell lysates were prepared for immunoprecipitation using anti-RHA antibody (Ab) or control IgG. Total RNAs in the precipitates were isolated, followed by reverse transcription. Conventional PCR (A and C) or real-time PCR (B and D) using primers specific for the DENV2 or ZIKV genome were performed to measure the relative levels of RHA-associated viral RNA. Data are shown as means ± SD from three independent experiments. ***, P < 0.001 by unpaired, two-tailed Student's t test.

FIG 4

RHA interacted with viral NS1, NS3, and NS4B. (A) 293T cells were transfected with plasmids expressing RHA-FLAG and DENV2 NS proteins for 48 h. Whole-cell extracts were prepared for co-IP assay using anti-FLAG antibody. IB, immunoblot. (B) A549 cells were infected with DENV2 (MOI of 3). Whole-cell extracts were prepared at 24 h p.i. for co-IP assay using anti-RHA antibody. Protein complexes were separated by SDS-PAGE and detected by Western blotting with antibodies against the indicated proteins. Data were representative of three independent experiments.

FIG 5

Mapping of RHA domains that interacted with viral NS proteins. (A) Schematic representation of full-length RHA-FLAG and three truncated forms of RHA. 293T cells were transfected with plasmids encoding different regions of RHA and DENV2 NS1-HA (B), NS2B3-HA (C), or NS4B-HA (D). The whole-cell extracts were prepared at 48 h posttransfection and immunoprecipitated with FLAG antibody. Western blot was probed with FLAG or HA antibody. RHA-FLAG, full-length RHA tagged with FLAG at C terminus; RHAN-FLAG, N-terminal fragment of RHA tagged with FLAG at C terminus; RHAM-FLAG, intermediate fragment of RHA tagged with FLAG at C terminus; RHAC-FLAG, C-terminal fragment of RHA tagged with FLAG at C terminus. Data are representative of three independent experiments. Asterisks indicate nonspecific bands.

FIG 6

Expression of RHA^RSC or RHA-K417R rescued viral replication levels. (A and B) Control cells or RHA^RSC- or RHA-K417R (K417R)-expressing cells were transfected with siNC or siRHA1. At 48 h posttransfection, cells were infected by DENV2 at an MOI of 3. Cells and supernatants were collected at 24 h p.i. for Western blotting (A) and plaque assay (B). Western blot was probed with antibodies against RHA, FLAG, NS3, and GAPDH. (C) Co-IP assay. The RHA^RSC- or RHA-K417R-expressing cells were infected by DENV2 at an MOI of 3. At 24 h p.i., cells were harvested for co-IP assay using anti-FLAG antibody. Protein complexes were separated by SDS-PAGE and detected by Western blotting with antibodies against the indicated proteins. Data are representative of three independent experiments. ***, P < 0.001 by unpaired, two-tailed Student's t test.

FIG 7

RHA proviral function was independent of its regulation of IFN production. A549 cells were transfected with siNC or siRHAm. At 48 h posttransfection, cells were mock infected or infected with DENV2 (MOI of 3). Cells were harvested at 12 and 24 h p.i. for real-time PCR to detect the IFN-β level. (A) Actin was measured as a control. (B) Control and IFNAR1^KO A549 cells were transfected with siNC or siRHAm for 48 h, followed by DENV2 infection (MOI of 3). Supernatants were collected at 24 h. The viral titers were measured by focus-forming assay. Data are shown as means ± SD from three independent experiments. NS, no statistical significance. **, P < 0.01; ***, P < 0.001 (both by unpaired, two-tailed Student's t test).