A Co-Opted DEAD-Box RNA helicase enhances tombusvirus plus-strand synthesis

Abstract

Replication of plus-strand RNA viruses depends on recruited host factors that aid several critical steps during replication. In this paper, we show that an essential translation factor, Ded1p DEAD-box RNA helicase of yeast, directly affects replication of Tomato bushy stunt virus (TBSV). To separate the role of Ded1p in viral protein translation from its putative replication function, we utilized a cell-free TBSV replication assay and recombinant Ded1p. The in vitro data show that Ded1p plays a role in enhancing plus-strand synthesis by the viral replicase. We also find that Ded1p is a component of the tombusvirus replicase complex and Ded1p binds to the 3'-end of the viral minus-stranded RNA. The data obtained with wt and ATPase deficient Ded1p mutants support the model that Ded1p unwinds local structures at the 3'-end of the TBSV (-)RNA, rendering the RNA compatible for initiation of (+)-strand synthesis. Interestingly, we find that Ded1p and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is another host factor for TBSV, play non-overlapping functions to enhance (+)-strand synthesis. Altogether, the two host factors enhance TBSV replication synergistically by interacting with the viral (-)RNA and the replication proteins. In addition, we have developed an in vitro assay for Flock house virus (FHV), a small RNA virus of insects, that also demonstrated positive effect on FHV replicase activity by the added Ded1p helicase. Thus, two small RNA viruses, which do not code for their own helicases, seems to recruit a host RNA helicase to aid their replication in infected cells.

Figure 1. Co-purification of Ded1p with the p33 replication protein from yeast.

The FLAG/6×His-tagged p33HF or 6×His-tagged p33H was purified from yeast extracts using a FLAG-affinity column (lanes 3–4). Top panel: Western blot analysis of Ded1p tagged with 6×HA, expressed from the natural promoter in the chromosome, with anti-HA antibody in the purified p33 preparations. Bottom panel: Western blot analysis of HF-tagged p33 or 6×His-tagged p33 with anti-His antibody. Note that “total” represents the total protein extract from yeast expressing the shown proteins. Each experiment was repeated three times.

Figure 2. Cell-free TBSV replication assay supports a role for Ded1p helicase in plus-strand synthesis.

(A) Scheme of the CFE-based TBSV replication assay. Purified recombinant p33 and p92^pol replication proteins of TBSV and in vitro transcribed TBSV DI-72 (+)repRNA were added to the whole cell extract prepared from Ded1p-depleted yeast strain. The assay also contained purified recombinant Ded1p (0.3 µg) or MBP (the same molar amount as Ded1p) as a control. (B) Left panel: Detection of single- and double-stranded RNA products produced in the cell-free TBSV replication assay. The dsRNA products are also shown on the right subpanel with high contrast for better visualization. Odd numbered lanes represent replicase products, which were not heat treated (thus both ssRNA and dsRNA products are present), while the even numbered lanes show the heat-treated replicase products (only ssRNA is present). The % of dsRNA and ssRNA in the samples are shown. Note that, in the nondenatured samples, the dsRNA product represents the annealed (−)RNA and the (+)RNA, while the ssRNA products represents the newly made (+)RNA products. Right panel: A higher contrast image of portion of the left panel to aid the visualization of the dsRNA products. Each experiment was repeated three times. (C) Denaturing PAGE analysis of the ³²P-labeled TBSV repRNA products obtained in the CFE assay in the presence of ATPase inactive D1 mutant of Ded1p (0.3 µg), wt Ded1p (0.3 µg) or MBP. Each experiment was repeated three times.

Figure 3. Step-wise cell-free TBSV replication assay does not support a role for Ded1p helicase in the assembly of the TBSV VRC.

(A) Scheme of the CFE-based TBSV replicase assembly and replication assays. Purified recombinant p33 and p92^pol replication proteins of TBSV and in vitro transcribed TBSV DI-72 (+)repRNA were added to the whole cell extract prepared from Ded1p-depleted yeast strain in step 1. The assay either contained or lacked the purified recombinant Ded1p (0.3 µg) or MBP during step 1. Note that the assay was performed in the presence of ATP/GTP to facilitate TBSV VRC assembly, but prevent RNA synthesis in step 1. After step 1, centrifugation was used to collect the membrane fraction of the CFE, and after washing the membranes, step 2 was performed in the presence of ATP/CTP/GTP and ³²P-UTP to allow TBSV RNA replication. In the samples presented in right panel, Ded1p or MBP were added at the beginning of step 2. (B) Denaturing PAGE analysis of the ³²P-labeled TBSV repRNA products obtained in the CFE assays in the presence of wt and various Ded1p mutants (0.3 µg each) (Figure S3C) or MBP. See further details in Figure 2. Note that the various preps of D3 mutant of Ded1p showed high variation in in vitro activity (for reasons currently unknown)- see Figure 4B. Each experiment was repeated at least three times and the data were used to calculate standard deviation.

Figure 4. Ded1p promotes plus-strand synthesis by the affinity-purified tombusvirus and FHV replicases.

(A) Scheme of the tombusvirus replicase assay. Yeast with depleted Ded1p co-expressing p33 and p92^pol replication proteins and DI-72 (+)repRNA were used to affinity-purify the RNA-free tombusvirus replicase. The in vitro assays were programmed with DI-72 (−)repRNA, and they also contained purified recombinant Ded1p, mutants or MBP in addition to ATP/CTP/GTP and ³²P-UTP. (B) Representative denaturing gel of ³²P-labeled RNA products synthesized by the purified tombusvirus replicase in vitro in the presence of 0.5 µg or 1.0 µg of purified recombinant Ded1p or its mutants is shown. The level of complementary RNA synthesis producing “repRNA” (marked as “FL”, the full-length product, made via de novo initiation from the 3′-terminal promoter) in each sample was compared to that of the replicase activity obtained in the absence of added recombinant protein (lane 1). Note that this replicase preparation also synthesizes de novo internal initiation products (“ii”) and 3′-terminal extension products (“3′TEX”). Each experiment was repeated three times. (C) Representative denaturing gel of ³²P-labeled RNA products synthesized by the purified FHV replicase in vitro in the presence of 1.0 µg of purified recombinant Ded1p or its mutants is shown. Note that the FHV replicase can use TBSV (−)repRNA as a template in vitro. Each experiment was repeated three times. (D) Representative denaturing gel of ³²P-labeled RNA products synthesized by the purified FHV replicase in vitro in the presence of 1.0 µg of purified recombinant Ded1p or its mutants is shown. Note that the FHV DI-634 (−)repRNA was used as a template in the FHV replicase assay.

Figure 5. Ded1p binds to the 3′ end of the TBSV (−)RNA.

(A) Schematic representation of the four regions in DI-72 repRNA used in the binding assay. (B) In vitro binding assay with purified Ded1p using ³²P-labeled ssRNA templates. The assay contained the (−) or (+) DI-72 repRNA (∼0.1 pmol) plus increasing amount [0.4 µg (lanes 2–3 and 8–9), 0.8 µg (lanes 4–5 and 10–11) and 1.2 µg (lanes 6 and 12)] of purified recombinant Ded1p. The free or Ded1p-bound ssRNA was separated on nondenaturing 5% acrylamide gels. (C) RNA gel shift analysis shows that Ded1p binds the most efficiently to RI(−). ³²P-labeled DI-72 (−)repRNA template (∼0.1 pmol) and unlabeled competitor RNAs (3 and 6 pmol) representing one of the four regions (see panel A) were used in the competition assay. The Ded1p - ³²P-labeled ssRNA complex was visualized on nondenaturing 5% acrylamide gels. Each experiment was repeated at least three times.

Figure 6. AtRH20 DEAD-box helicase promotes TBSV repRNA replication in the CFE-based assay.

(A) Representative denaturing gel of ³²P-labeled TBSV repRNA products obtained in the CFE assay prepared from Ded1p-depleted yeast strain in the presence of 0.2 µg (lane 3) or 0.3 µg (lane 4) of purified recombinant AtRH20 is shown. The experiment was repeated three times. (B) AtRH20 DEAD-box helicase promotes plus-strand synthesis by the affinity-purified tombusvirus replicase. Representative denaturing gel of ³²P-labeled RNA products synthesized by the purified tombusvirus replicase in vitro in the presence of 0.5 µg (lane 3) or 1.0 µg (lanes 2, 4) of purified recombinant AtRH20 is shown. Yeast with depleted Ded1p co-expressing p33 and p92^pol replication proteins and DI-72 (+)repRNA were used to affinity-purify the RNA-free tombusvirus replicase. The in vitro assays were programmed with DI-72 (−)repRNA, and they also contained purified recombinant AtRH20 (Lanes 2–4) in addition to ATP/CTP/GTP and ³²P-UTP. Lane 4 represents the sample with no tombusvirus replicase added. The experiment was repeated three times.

Figure 7. Ded1p facilitates the RNA synthesis by the tombusvirus and FHV replicases on short partial DNA/RNA duplex.

(A) Schematic representation of the DNA/RNA duplex used in the replicase assays. The template consists of DI-72 (−)repRNA, whose 3′ end forms a 21 nt duplex with a complementary DNA oligo as shown. The following cis-acting sequences involved in (+)-strand synthesis are shown: cPR is an 11 nt long sequence used as a (+)-strand initiation promoter; PPE REN is a promoter proximal replication enhancer; while RIII(−)REN is a strong replication enhancer within RIII(−) sequence. (B) Representative denaturing gel of ³²P-labeled RNA products synthesized by the purified tombusvirus replicase in vitro in the presence of 1.0 µg of purified recombinant Ded1p or D1 mutant. The level of complementary RNA synthesis using the DNA/RNA duplex (panel A) in each sample was compared to that of the replicase activity obtained in the absence of added recombinant protein (lane 1). See further details in Figure 4. (C) Representative denaturing gel of ³²P-labeled RNA products synthesized by the purified FHV replicase in vitro in the presence of 1.0 µg of purified recombinant Ded1p or D1 mutant on the DNA/RNA duplex (panel A). Each experiment was repeated three times.

Figure 8. Ded1p and GAPDH (Tdh2p) act synergistically to enhance RNA synthesis by the tombusvirus replicase.

(A) Schematic representation of the replicase assay used. The purified recombinant proteins were added at the same time to the assay. (B) Representative denaturing gel of ³²P-labeled RNA products synthesized by the purified tombusvirus replicase in vitro in the presence of 1.0 µg of purified recombinant MBP-Ded1p and/or 0.4 µg of GST-Tdh2p on DI-72 (−)repRNA template. The level of complementary RNA synthesis in each sample was compared to that of the replicase activity obtained in the presence of GST and MBP recombinant proteins (lane 5) (used in comparable molar amounts to MBP-Ded1p and GST-Tdh2p). The ³²P-labeled RNA products were RNase One treated to visualize the 3′terminal extension products (“3′TEX”) better. Note that the RNAse One treatment causes a shift in migration of the 3′TEX product (compare with Figure 4B, untreated samples). See further details in Figure 4. (C) Representative denaturing gel of ³²P-labeled RNA products synthesized by the purified tombusvirus replicase in vitro in the presence of 1.0 µg of purified recombinant MBP-Ded1p and/or 0.4 µg of GST-Tdh2p on the DNA/RNA duplex (Figure 7 panel A). Each experiment was repeated three times.

Figure 9. A model describing the functions of Ded1p and GAPDH during tombusvirus replication.

The (−)repRNA, which is shown with a “closed” 3′ end due to either a secondary structure within the promoter (cPR) in the (−)repRNA or part of a dsRNA structure, could be unwound by Ded1p around the 3′ end, making the (−)repRNA “open”. The open ssRNA structure would facilitate (+)RNA synthesis by the tombusvirus p92 RdRp protein. GAPDH could facilitate (+)RNA synthesis via binding to a 3′ AU-rich sequence (indicated as a box) and also bringing in the p92 protein and positioning it over the cPR promoter as shown. Thus, Ded1p and GAPDH might play a complementary role, leading to synergistic effect on (+)RNA synthesis. In the absence of Ded1p, GAPDH could promote both 3′-terminal extension and (+)RNA synthesis by facilitating the positioning of p92 over the 3′ end.