Co-Opting ATP-Generating Glycolytic Enzyme PGK1 Phosphoglycerate Kinase Facilitates the Assembly of Viral Replicase Complexes

The intricate interactions between viruses and hosts include exploitation of host cells for viral replication by using many cellular resources, metabolites and energy. Tomato bushy stunt virus (TBSV), similar to other (+)RNA viruses, induces major changes in infected cells that lead to the formation of large replication compartments consisting of aggregated peroxisomal and ER membranes. Yet, it is not known how TBSV obtains the energy to fuel these energy-consuming processes. In the current work, the authors discovered that TBSV co-opts the glycolytic ATP-generating Pgk1 phosphoglycerate kinase to facilitate the assembly of new viral replicase complexes. The recruitment of Pgk1 into the viral replication compartment is through direct interaction with the viral replication proteins. Altogether, we provide evidence that the ATP generated locally within the replication compartment by the co-opted Pgk1 is used to fuel the ATP-requirement of the co-opted heat shock protein 70 (Hsp70) chaperone, which is essential for the assembly of new viral replicase complexes and the activation of functional viral RNA-dependent RNA polymerase. The advantage of direct recruitment of Pgk1 into the virus replication compartment could be that the virus replicase assembly does not need to intensively compete with cellular processes for access to ATP. In addition, local production of ATP within the replication compartment could greatly facilitate the efficiency of Hsp70-driven replicase assembly by providing high ATP concentration within the replication compartment

Coordinated Function of Cellular DEAD-Box Helicases in Suppression of Viral RNA Recombination and Maintenance of Viral Genome Integrity

The intricate interactions between viruses and hosts include an evolutionary arms race and adaptation that is facilitated by the ability of RNA viruses to evolve rapidly due to high frequency mutations and genetic RNA recombination. In this paper, we show evidence that the co-opted cellular DDX3-like Ded1 DEAD-box helicase suppresses tombusviral RNA recombination in yeast model host, and the orthologous RH20 helicase functions in a similar way in plants. In vitro replication and recombination assays confirm the direct role of the ATPase function of Ded1p in suppression of viral recombination. We also present data supporting a role for Ded1 in facilitating the switch from minus- to plus-strand synthesis. Interestingly, another co-opted cellular helicase, the eIF4AIII-like AtRH2, enhances TBSV recombination in the absence of Ded1/RH20, suggesting that the coordinated actions of these helicases control viral RNA recombination events. Altogether, these helicases are the first co-opted cellular factors in the viral replicase complex that directly affect viral RNA recombination. Ded1 helicase seems to be a key factor maintaining viral genome integrity by promoting the replication of viral RNAs with correct termini, but inhibiting the replication of defective RNAs lacking correct 5\u27 end sequences. Altogether, a co-opted cellular DEAD-box helicase facilitates the maintenance of full-length viral genome and suppresses viral recombination, thus limiting the appearance of defective viral RNAs during replication

The Proteasomal Rpn11 Metalloprotease Suppresses Tombusvirus RNA Recombination and Promotes Viral Replication via Facilitating Assembly of the Viral Replicase Complex

RNA viruses co-opt a large number of cellular proteins that affect virus replication and, in some cases, viral genetic recombination. RNA recombination helps viruses in an evolutionary arms race with the host\u27s antiviral responses and adaptation of viruses to new hosts. Tombusviruses and a yeast model host are used to identify cellular factors affecting RNA virus replication and RNA recombination. In this study, we have examined the role of the conserved Rpn11p metalloprotease subunit of the proteasome, which couples deubiquitination and degradation of proteasome substrates, in tombusvirus replication and recombination in Saccharomyces cerevisiae and plants. Depletion or mutations of Rpn11p lead to the rapid formation of viral RNA recombinants in combination with reduced levels of viral RNA replication in yeast or in vitro based on cell extracts. Rpn11p interacts with the viral replication proteins and is recruited to the viral replicase complex (VRC). Analysis of the multifunctional Rpn11p has revealed that the primary role of Rpn11p is to act as a matchmaker that brings the viral p92pol replication protein and the DDX3-like Ded1p/RH20 DEAD box helicases into VRCs. Overexpression of Ded1p can complement the defect observed in rpn11 mutant yeast by reducing TBSV recombination. This suggests that Rpn11p can suppress tombusvirus recombination via facilitating the recruitment of the cellular Ded1p helicase, which is a strong suppressor of viral recombination, into VRCs. Overall, this work demonstrates that the co-opted Rpn11p, which is involved in the assembly of the functional proteasome, also functions in the proper assembly of the tombusvirus VRCs. IMPORTANCE: RNA viruses evolve rapidly due to genetic changes based on mutations and RNA recombination. Viral genetic recombination helps viruses in an evolutionary arms race with the host\u27s antiviral responses and facilitates adaptation of viruses to new hosts. Cellular factors affect viral RNA recombination, although the role of the host in virus evolution is still understudied. In this study, we used a plant RNA virus, tombusvirus, to examine the role of a cellular proteasomal protein, called Rpn11, in tombusvirus recombination in a yeast model host, in plants, and in vitro. We found that the cellular Rpn11 is subverted for tombusvirus replication and Rpn11 has a proteasome-independent function in facilitating viral replication. When the Rpn11 level is knocked down or a mutated Rpn11 is expressed, then tombusvirus RNA goes through rapid viral recombination and evolution. Taken together, the results show that the co-opted cellular Rpn11 is a critical host factor for tombusviruses by regulating viral replication and genetic recombination

Dynamic Interplay between the Co-Opted Fis1 Mitochondrial Fission Protein and Membrane Contact Site Proteins in Supporting Tombusvirus Replication

Plus-stranded RNA viruses have limited coding capacity and have to co-opt numerous pro-viral host factors to support their replication. Many of the co-opted host factors support the biogenesis of the viral replication compartments and the formation of viral replicase complexes on subverted subcellular membrane surfaces. Tomato bushy stunt virus (TBSV) exploits peroxisomal membranes, whereas the closely-related carnation Italian ringspot virus (CIRV) hijacks the outer membranes of mitochondria. How these organellar membranes can be recruited into pro-viral roles is not completely understood. Here, we show that the highly conserved Fis1 mitochondrial fission protein is co-opted by both TBSV and CIRV via direct interactions with the p33/p36 replication proteins. Deletion of FIS1 in yeast or knockdown of the homologous Fis1 in plants inhibits tombusvirus replication. Instead of the canonical function in mitochondrial fission and peroxisome division, the tethering function of Fis1 is exploited by tombusviruses to facilitate the subversion of membrane contact site (MCS) proteins and peroxisomal/mitochondrial membranes for the biogenesis of the replication compartment. We propose that the dynamic interactions of Fis1 with MCS proteins, such as the ER resident VAP tethering proteins, Sac1 PI4P phosphatase and the cytosolic OSBP-like oxysterol-binding proteins, promote the formation and facilitate the stabilization of virus-induced vMCSs, which enrich sterols within the replication compartment. We show that this novel function of Fis1 is exploited by tombusviruses to build nuclease-insensitive viral replication compartment

A rapid and simple quantitative method for specific Detection of Smaller Co-terminal RNA by PCR (DeSCo-PCR): Application to the detection of viral subgenomic RNAs

RNAs that are 5’-truncated versions of a longer RNA, but share the same 3’ terminus can be generated by alternative promoters in transcription of cellular mRNAs or by replicating RNA viruses. These truncated RNAs cannot be distinguished from the longer RNA by a simple two-primer RT-PCR because primers that anneal to the cDNA from the smaller RNA also anneal to - and amplify - the longer RNA-derived cDNA. Thus, laborious methods, such as northern blot hybridization, are used to distinguish shorter from longer RNAs. For rapid, low-cost and specific detection of these truncated RNAs, we report Detection of Smaller Co-terminal RNA by PCR (DeSCo-PCR). DeSCo-PCR employs a non-extendable blocking primer (BP), which outcompetes a forward primer (FP) for annealing to longer RNA-derived cDNA, while FP outcompetes BP for annealing to shorter RNA-derived cDNA. In the presence of BP, FP and the reverse primer, only cDNA from the shorter RNA is amplified in a single-tube reaction containing both RNAs. Many positive strand RNA viruses generate 5’-truncated forms of the genomic RNA (gRNA) called subgenomic RNAs (sgRNA), which play key roles in viral gene expression and pathogenicity. We demonstrate that DeSCo-PCR is easily optimized to selectively detect relative quantities of sgRNAs of red clover necrotic mosaic virus from plants and Zika virus from human cells, each infected with viral strains that generate different amounts of sgRNA. This technique should be readily adaptable to other sgRNA-producing viruses, and for quantitative detection of any truncated or alternatively spliced RNA

Coordinated function of cellular DEAD-box helicases in suppression of viral RNA recombination and maintenance of viral genome integrity.

The intricate interactions between viruses and hosts include an evolutionary arms race and adaptation that is facilitated by the ability of RNA viruses to evolve rapidly due to high frequency mutations and genetic RNA recombination. In this paper, we show evidence that the co-opted cellular DDX3-like Ded1 DEAD-box helicase suppresses tombusviral RNA recombination in yeast model host, and the orthologous RH20 helicase functions in a similar way in plants. In vitro replication and recombination assays confirm the direct role of the ATPase function of Ded1p in suppression of viral recombination. We also present data supporting a role for Ded1 in facilitating the switch from minus- to plus-strand synthesis. Interestingly, another co-opted cellular helicase, the eIF4AIII-like AtRH2, enhances TBSV recombination in the absence of Ded1/RH20, suggesting that the coordinated actions of these helicases control viral RNA recombination events. Altogether, these helicases are the first co-opted cellular factors in the viral replicase complex that directly affect viral RNA recombination. Ded1 helicase seems to be a key factor maintaining viral genome integrity by promoting the replication of viral RNAs with correct termini, but inhibiting the replication of defective RNAs lacking correct 5' end sequences. Altogether, a co-opted cellular DEAD-box helicase facilitates the maintenance of full-length viral genome and suppresses viral recombination, thus limiting the appearance of defective viral RNAs during replication

Opposite effects of subverted cellular DEAD-box helicases on TBSV recombination in yeast and in the CFE assay.

<p>(A) TBSV co-opt two groups of cellular helicases that become part of the VRCs: the DDX3-like Ded1/AtRH20 and the eIF4AIII-like AtRH2. These helicases bind to different <i>cis</i>-acting replication elements present at the 3’ and 5’ ends of the viral (-)RNA as shown. Note that RI(-) carries the promoter for (+)-strand synthesis, while the RIII(-) is a replication enhancer element (REN). (B) Over-expression of AtRH2 enhances recRNAs and degRNA accumulation, while over-expression of AtRH20 decreases the occurrence of these RNAs in wt or ded1–199^ts yeasts. The Northern blot analysis was done as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004680#ppat.1004680.g001" target="_blank">Fig. 1</a>. (C) Western blot analysis to detect the expression level of His₆-tagged cellular helicases and the His₆-tagged viral replication proteins in the WT and ded1–199^ts yeasts. Asterisk marks the SDS-resistant p33 dimer. (D) The scheme of the CFE-based TBSV replication assay. Note that the level of Ded1p was depleted in TET::DED1 yeast prior to CFE isolation. (E) Denaturing PAGE analysis of the accumulation of 5’-truncated DI-RIIΔ70 degRNA used as the original template in the CFE assay and the <i>in vitro</i> generated recRNA. See further details in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004680#ppat.1004680.g003" target="_blank">Fig. 3</a>.</p

Suppression of TBSV recRNA accumulation by Ded1p in <i>in vitro</i> replication assays.

<p>(A) The membrane-enriched fraction (MEF) was isolated from wt or ded1–199^ts yeast expressing the tombusvirus p33 and p92 replication proteins in combination with the DI-AU-FP repRNA, followed by <i>in vitro</i> tombusvirus replication assay. The denaturing PAGE analysis shows the emergence of recRNAs and degRNA as indicated. (B–C) Northern blot analysis of the polarity of the TBSV RNAs synthesized in the MEF assay (see panel A, except the assay was done with nonlabeled ribonucleotides) using ³²P-labeled probes to detect (-)-strands and (+)-strands, respectively. The ratios between various TBSV RNAs were calculated as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004680#ppat.1004680.g002" target="_blank">Fig. 2</a>. (D) Scheme of the yeast cell-free (CFE) assay for TBSV replication. Note that the assembly of the TBSV replicase takes place in the CFE using recombinant viral components as shown. The TBSV p33 and p92 replication proteins and Ded1p and its mutants (D1 is inactive, D11 has enhanced ATPase activity) were purified from <i>E</i>. <i>coli</i>. (E) Reduced replication of the full-length DI-72 repRNA in TET::DED1 yeast CFEs with high or depleted levels of Ded1p (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004680#ppat.1004680.g001" target="_blank">Fig. 1</a>). (F-G) Denaturing PAGE analysis of the effect of Ded1p and its mutants on the replication of the full-length recombinogenic DI-AU-FP and the 5’-truncated DI-RIIΔ70 degRNA, respectively, in the CFE assay. The experiments were repeated twice.</p

Reduced TBSV repRNA accumulation in yeast with depleted Pgk1 level.

<p>(A) Northern blot analysis shows decreased TBSV repRNA accumulation in a yeast strain (GALS::PGK1) when Pgk1 was depleted. To launch TBSV repRNA replication, we expressed His₆-p33 and His₆-p92^pol from the copper-inducible <i>CUP1</i> promoter, and DI-72(+) repRNA from the <i>ADH1</i> promoter in the parental (BY4741) and in GALS::PGK1 yeast strains. Note that GALS::PGK1 yeast strain expresses HA-tagged Pgk1 from the galactose-inducible <i>GALS</i> promoter from chromosomal location (i.e., HA-Pgk1 replaces the wt Pgk1 in this haploid yeast strain). The yeast cells were cultured for 16 hours at 29°C in either 2% galactose +2% raffinose [(Raf+Gal), inducing condition for HA-Pgk1] or 2% raffinose (lack of induction of HA-Pgk1 expression) SC minimal media supplemented with 50 μM CuSO₄. The accumulation level of DI-72(+) repRNA was normalized based on 18S rRNA levels (second panel from top). Middle panel: Northern blot analysis of <i>PGK1</i> mRNA levels in total RNA samples. Bottom two panels: Western blot analysis of the accumulation level of HA-tagged Pgk1, His₆-tagged p33, His₆-p92^pol proteins using anti-HA and anti-His antibodies, respectively. Each experiment was performed three times. (B) Complementation assay with plasmid-borne expression of His₆-Pgk1 in GALS::PGK1 yeast strain. His₆-Pgk1 was expressed from the <i>TEF1</i> constitutive promoter, whereas the expression of the chromosomal HA-tagged Pgk1 was suppressed via 2% glucose in the growth media. Top panels: Northern blot analysis of repRNA level, middle panel: ethidium-bromide stained gel with ribosomal RNA, as a loading control, whereas bottom panels show Western blot analysis using anti-His antibody. Panels on the right represent samples obtained from yeast grown on the nonfermentable glycerol media. (C) The effect of over-expression of yeast Pgk1 on TBSV repRNA accumulation. The plasmid-borne His₆-Pgk1 was expressed from <i>TEF1</i> promoter in BY4741 (wt) yeast. See further details in panel B. (D) The effect of heterologous expression of NbPgk1 on TBSV repRNA accumulation in yeast. The plasmid-borne His₆-NbPgk1 was expressed from <i>TEF1</i> promoter in BY4741 (wt) yeast. See further details in panel B.</p

Suppression of viral RNA recombination by Ded1 helicase is independent of the Xrn1 pathway.

<p>(A) Schematic representation of the previously characterized Xrn1-driven TBSV RNA recombination pathway in yeast. Plasmid-driven expression of the DI-(RI in the presence of p33/p92 replication proteins leads to partial 5’ truncations by the cellular Xrn1p 5’-to-3’ exoribonuclease generating DI-RIIΔ70-like degRNAs as shown. DI-RIIΔ70-like degRNAs then participate in RNA recombination as shown. (B) Recombination profile of DI-(RI RNA in <i>xrn1Δ</i>, <i>met22Δ</i> (as a control) and in ded1–199 yeasts. The original expressed DI-(RI degRNA, DI-RIIΔ70-like degRNAs and recRNAs are depicted with arrowheads and arrows, respectively. Note that the recRNA profile is dramatically different in <i>xrn1Δ</i> yeast when compared to ded1–199 yeast. (C) Half-like of DI-RIIΔ70 degRNA in WT, ded1–95 and ded1–199 yeasts. Note that yeast did not express p33/p92 proteins.</p