The DEAD-box RNA Helicase DDX6 is Required for Efficient Encapsidation of a Retroviral Genome

Viruses have to encapsidate their own genomes during the assembly process. For most RNA viruses, there are sequences within the viral RNA and virion proteins needed for high efficiency of genome encapsidation. However, the roles of host proteins in this process are not understood. Here we find that the cellular DEAD-box RNA helicase DDX6 is required for efficient genome packaging of foamy virus, a spumaretrovirus. After infection, a significant amount of DDX6, normally concentrated in P bodies and stress granules, re-localizes to the pericentriolar site where viral RNAs and Gag capsid proteins are concentrated and capsids are assembled. Knockdown of DDX6 by siRNA leads to a decreased level of viral nucleic acids in extracellular particles, although viral protein expression, capsid assembly and release, and accumulation of viral RNA and Gag protein at the assembly site are little affected. DDX6 does not interact stably with Gag proteins nor is it incorporated into particles. However, we find that the ATPase/helicase motif of DDX6 is essential for viral replication. This suggests that the ATP hydrolysis and/or the RNA unwinding activities of DDX6 function in moderating the viral RNA conformation and/or viral RNA-Gag ribonucleoprotein complex in a transient manner to facilitate incorporation of the viral RNA into particles. These results reveal a unique role for a highly conserved cellular protein of RNA metabolism in specifically re-locating to the site of viral assembly for its function as a catalyst in retroviral RNA packaging

Immunofluorescent staining of DDX6 and Dcp1.

<p>HT1080 cells were mock-transfected (A), DDX6 siRNA-transfected (B), or transfected with pcPFV/gag-gfp (C & D) for 32 hrs, and stained with anti-Dcp1 (A, B, & C) or anti-DDX6 (A, B, & D). Gag-GFP proteins were imaged as green fluorescent color (Left panels, C & D). After merged together, the yellow color indicates co-localization of green and red signals. Images were taken from a single 0.2 um Z- section using DeltaVision microscopy.</p

Exogenous expression of wild type or mutant DDX6.

<p>(A & B) HT1080 cells were transfected with control siRNA (Lanes 1) or DDX6 siRNA (Lanes 2 to 6) for 24 hrs. Cells were then transfected with pEYFP (Lanes 1 & 2), pEYFP-DDX6a-wt (Lanes 3), pEYFP-DDX6a-EQ6 (Lanes 4), pEYFP-DDX6a-EQ11 (Lanes 5), or pEYFP-DDX6-dC (Lanes 6). At 24 hr later, cells were infected with the virus at an moi of 2 for 6 hrs, washed, and incubated for another 40 hrs. Culture supernatants were assayed by FAB indicators cells to measure the virus titers. (A) Viral infectivity relative to the transfection with control siRNA and pEYFP (Lane 1). (B) Immunoblot analyses of GAPDH, endogenous DDX6, and vector-expressed YFP or YFP-DDX6 proteins in infected cell lysates. Molecular weight standards are marked for each gel. (C) HT1080 cells were transfected with pEYFP, pEYFP-DDX6a-wt, pEYFP-DDX6a-EQ6, pEYFP-DDX6a-EQ11, or pEYFP-DDX6-dC, and then infected with the virus at an moi of 2. Results are shown as relative viral infectivity compared to the transfection with the control vector pEYFP. Means and standard errors were results for each analysis from at least four independent experiments.</p

Effects of gene-specific siRNA knockdown on viral infectivity.

<p>(A) HT1080 cells were transfected twice with 120 nM control siRNA (Lanes 1) or specific siRNA (Lanes 2). 1^st siRNA transfection was performed at time 0 and 2^nd siRNA transfection at 24 h. At 24 h after the 2^nd transfection, half of the cells were infected with virus and the other half were analyzed by immunoblot for each specific protein (top panels) and GAPDH (bottom panels). (B) Culture supernatant was harvested at 48 h after infection and used to infect FAB cells to measure infectious titers. Relative infectivity of viruses derived from cells transfected with specific siRNA compared to those with control siRNA. Means and standard errors from at least four separate experiments are shown.</p

Viral proteins and RNA in cell lysates and extracellular particles after DDX6 knockdown.

<p>HT1080 cells were transfected twice with 60 nM control siRNA or DDX6 siRNA. 1^st siRNA transfection was done at time 0. 2^nd siRNA transfection was performed at 24 h, followed by the infection with foamy virus at 48 h (i.e. 24 h after 2^nd siRNA transfection). Cells and culture supernatants were harvested at 45 h after infection. (A) Immunoblot analyses of Gag, Pol, DDX6, and GAPDH in infected cell lysates. (B) Immunoblot analyses of Gag and Pol in extracellular particles. (C) Relative quantities for viral infectivity, Gag and Pol proteins, and viral RNA in extracellular particles obtained from transfection with DDX6 siRNA compared to that with control siRNA. Means and standard errors were results for each analysis from at least four independent experiments.</p

Schematic presentation of the pumilo-based BIFC system designed for pcPFV/gag-pum.

<p>(A) Two eight-nucleotide sequences (TGTAAATA & TGTAGATA) that were separated by 5 nucleotides were introduced at the 3′ end of <i>gag</i> in the proviral DNA pcPFV/gag-pum. Consequently, all viral genomic RNAs as well as unspliced <i>gag</i> and singly spliced <i>pol</i> mRNAs contained UGUAAAUA and UGUAGAUA in their sequences. (B) These motifs were target substrates for wild-type PUMHD(wt) and a variant PUMHD(3794) that had been fused separately to either the C- or N-terminal half of mCitrine, a yellow-green fluorescent protein. When pcHFV/gag-pum was co-transfected with expression vectors pcmv-PUMHD(wt)_CitC and pcmv- CitN_PUMHD(3794), viral RNA containing PUMHD-binding sequences is viewed in green fluorescent color.</p