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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2019 Oct 7;116(43):21739–21747. doi: 10.1073/pnas.1911108116

Screening Legionella effectors for antiviral effects reveals Rab1 GTPase as a proviral factor coopted for tombusvirus replication

Jun-ichi Inaba a, Kai Xu a, Nikolay Kovalev a, Harish Ramanathan b, Craig R Roy b, Brett D Lindenbach b,1, Peter D Nagy a,1
PMCID: PMC6815150  PMID: 31591191

Significance

Many bacterial pathogens infect eukaryotic cells by injecting bacterial virulence factors or effectors into the cytosol of their hosts. These effector proteins have evolved to manipulate cellular pathways and favor bacterial replication. Legionella pneumophila, the cause of Legionnaires’ disease, synthesizes >300 effector proteins. Here, we screened Legionella effectors to probe tombusvirus (TBSV)–host interactions and identify antiviral factors against TBSV in yeast surrogate host. As proof of concept that bacterial effectors can be used as tools to identify cellular pathways used by viruses, we characterized the antiviral effect of DrrA effector of Legionella. This led to the discovery that Rab1 small GTPase, the cellular target of DrrA, and COPII vesicles are coopted by TBSV for building the viral replication compartment.

Keywords: tomato bushy stunt virus, effector, host factor, viral replication, yeast

Abstract

Bacterial virulence factors or effectors are proteins targeted into host cells to coopt or interfere with cellular proteins and pathways. Viruses often coopt the same cellular proteins and pathways to support their replication in infected cells. Therefore, we screened the Legionella pneumophila effectors to probe virus–host interactions and identify factors that modulate tomato bushy stunt virus (TBSV) replication in yeast surrogate host. Among 302 Legionella effectors tested, 28 effectors affected TBSV replication. To unravel a coopted cellular pathway in TBSV replication, the identified DrrA effector from Legionella was further exploited. We find that expression of DrrA in yeast or plants blocks TBSV replication through inhibiting the recruitment of Rab1 small GTPase and endoplasmic reticulum-derived COPII vesicles into the viral replication compartment. TBSV hijacks Rab1 and COPII vesicles to create enlarged membrane surfaces and optimal lipid composition within the viral replication compartment. To further validate our Legionella effector screen, we used the Legionella effector LepB lipid kinase to confirm the critical proviral function of PI(3)P phosphoinositide and the early endosomal compartment in TBSV replication. We demonstrate the direct inhibitory activity of LegC8 effector on TBSV replication using a cell-free replicase reconstitution assay. LegC8 inhibits the function of eEF1A, a coopted proviral host factor. Altogether, the identified bacterial effectors with anti-TBSV activity could be powerful reagents in cell biology and virus–host interaction studies. This study provides important proof of concept that bacterial effector proteins can be a useful toolbox to identify host factors and cellular pathways coopted by (+)RNA viruses.

Positive-strand RNA viruses coopt numerous host components and modify several pathways to facilitate viral infections of host organisms (1–3). Replication of RNA viruses takes place in membranous intracellular replication compartments, which harbor the viral replication complexes (VRCs) (1, 4–6). Our understanding of the biogenesis of the viral replication compartment, VRC formation, and the role of coopted host factors is currently incomplete. Multiple genome-wide screens of yeast and global proteomic approaches identified numerous host proteins that affected tomato bushy stunt virus (TBSV) replication (7, 8). The coopted host proteins are required for VRC assembly or to participate in TBSV RNA synthesis (9, 10). Moreover, TBSV also usurps subcellular membranes, sterols, and phospholipids, indicating the complexity of virus–host interactions (4, 8, 11).

TBSV can replicate in the model host yeast (Saccharomyces cerevisiae) with the help of 2 viral replication proteins, p33 and p92pol (7). The p33 and p92pol replication proteins participate in the formation of VRCs, which are vesicle-like structures in peroxisomal boundary membranes (12, 13).

Bacterial virulence factors or effectors are proteins targeted into the host cells to modify or interfere with cellular proteins and pathways (14, 15). Therefore, we decided to exploit bacterial effectors as tools to probe virus–host interactions and test the effects of bacteria on RNA virus replication. We chose effectors of Legionella pneumophila, since this bacterium has the largest number of secreted effectors, which work in protozoa as well as in human macrophages (14, 15); thus, these effectors seem to be effective in diversified hosts.

Gram-negative bacterial pathogen L. pneumophila can cause severe pneumonia, called Legionnaires’ disease in humans. After phagocytosis into the host cell, Legionella uses the type IV secretion system that delivers ∼300 bacterial effectors into eukaryotic cells that are required for infection. The bacteria replicate inside the cells in Legionella-containing vacuoles (LCVs), which are phagosome derived, with the help of the effectors. The interaction among the effectors and the host factors control vesicle transport processes, and dynamics of endoplasmic reticulum (ER) membranes (14, 15). Because the Legionella effectors change evolutionarily conserved cellular processes, they might be suitable as molecular tools or probes to dissect virus–host interactions. In this paper, we screened Legionella effectors to identify those with antiviral effects against TBSV in yeast. We used a yeast surrogate host, which is a popular organism to study viral, bacterial, and fungal effectors (8, 16). Altogether, we find 28 Legionella effectors, which affect TBSV replication in yeast. These effectors target conserved cellular proteins and pathways including the secretory pathway.

To demonstrate the cellular probe potential of the identified Legionella effectors, we characterized the antiviral effects of 3 effectors. First, as an important proof of concept, 2 of the identified Legionella effectors, LegC8 and LepB, which target known TBSV host factors eEF1A and PI(3)P, respectively, were used to demonstrate the direct inhibitory effects on TBSV replication. Second, one of the identified Legionella effectors, DrrA, was then exploited to find new cellular pathways hijacked by TBSV. The DrrA effector contains a Rab1 adenynyl-transferase domain, a central Rab1 guanine nucleotide exchange factor (GEF) domain and a C-terminal PI(4)P binding domain (17, 18). DrrA modifies the cellular Rab1 small GTPase through AMPylation, which prevents deactivation of Rab1, allowing efficient recruitment of ER vesicles (COPII type) to bacterial vacuoles (19).

We demonstrate that the antiviral effect of the DrrA effector is manifested through blocking the proviral function of Rab1 small GTPase, which is the target of the DrrA effector. We show that Rab1 and COPII vesicles are recruited by TBSV into the viral replication compartment to provide an optimal membranous microenvironment for replication. Thus, the use of the DrrA effector from Legionella enabled the discovery of a cellular pathway usurped by TBSV.

Results

Screening of Legionella Effectors for Inhibitors of TBSV Replication in Yeast.

To identify Legionella effectors with antiviral effects, we cloned 302 genes of Legionella effectors (provided by C.R.R.) into a yeast expression plasmid, followed by transformation into wild-type (WT) yeast (SI Appendix, Tables S1 and S2), which also carried plasmids to launch TBSV RNA replication. We performed Northern blot analyses to measure the TBSV RNA replication level in yeast expressing each Legionella effector separately. The full screen (primary screen in high throughput format and secondary screen in low throughput format) has led to the identification of 27 effectors inhibiting and 1 (VipA) effector promoting TBSV replication in yeast (SI Appendix, Table S3). Twenty-one of the identified effectors have known cellular functions or targets (14, 15). Interestingly, several of those identified are known to target a group of cellular factors that were previously shown to be coopted host factors for TBSV. One notable example is elongation factor 1A (eEF1A), which plays a role in assembly of the TBSV VRCs and promotion of (−)RNA synthesis (9). eEF1A is targeted and its function is inhibited by LegC8 (SI Appendix, Table S3). VipA actin nucleator promotes TBSV replication, which requires stable actin filaments to support robust assembly of VRCs (20). In contrast, RavK, which cleaves actin filaments, inhibited TBSV replication. Altogether, the most frequently targeted cellular factors by the Legionella effectors, which were identified in this work, are those that 1) inhibit Rab1 GTPase function (DrrA, LepB, AnkX, Lem3, and LidA); and 2) bind to or modify phophoinositides, including PI(3)P or PI(4)P (namely, LepB, DrrA, LidA, and RavZ). Several Legionella effectors, including LepB, DrrA, LidA, LegC2, RidL, and LegA14, target endosomal trafficking, which is also important for TBSV replication (21).

The Legionella LegC8 Effector Inhibits Tombusvirus Replication In Vitro.

To validate our Legionella effector screen, first we decided to demonstrate that TBSV replication is affected by the expression of effectors that are known to target cellular proteins and pathways hijacked by TBSV. We decided to further characterize the inhibitory activities of LegC8 and LepB effectors on TBSV replication to verify our screen and provide the proof of principle (see below).

We selected the Legionella LegC8 effector because it targets and inhibits the function of eEF1A (22, 23). We used our yeast-based cell-free replicase reconstitution assay to test the direct inhibitory role of LegC8 effector (Fig. 1A). Indeed, addition of increasing amounts of purified recombinant LegC8 effector inhibited the production of both TBSV dsRNA replication intermediate and the new (+)-strand RNA progeny by 4- to 5-fold in the in vitro assay (Fig. 1B). Because we used the same amounts of purified recombinant p33 and p92pol replication proteins in the replicase reconstitution assay (Fig. 1A), the most probable interpretation of the inhibitory effect of LegC8 effector on in vitro TBSV replication is the documented inhibitory function of LegC8 effector on the coopted eEF1A host factor, which is needed for the assembly of the TBSV VRCs and promotion of (−)RNA synthesis (9).

Fig. 1.

Fig. 1.

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Cell-free TBSV replication assay supports an inhibitory effect for the Legionella LegC8 effector on TBSV replication. (A) Scheme of the CFE-based TBSV replication assay. (B) Nondenaturing PAGE analysis of the 32P-labeled TBSV repRNA products obtained in the CFE-based assay programmed with in vitro transcribed TBSV DI-72 (+)repRNA and purified recombinant MBP-p33 and MBP-p92pol replication proteins of TBSV. The affinity-purified GST-LegC8 from Escherichia coli was added 15 min before the TBSV replicase reconstitution. The CFEs were prepared from the BY4741 yeast strain. Each experiment was repeated 3 times and the data were used to calculate SD.

The Legionella LepB Effector Inhibits Tombusvirus Replication through Inhibiting the Enrichment of PI(3)P and the Recruitment of Phosphatidylethanolamine-Rich Endosomes to the Viral Replication Compartment.

The Legionella LepB effector is a PI(4)P kinase, which converts PI(3)P to PI(3,4)P2 (24). The reduction of PI(3)P in the endosomal membrane results in the loss of many cellular endosomal proteins and renders the endosomes fusion incompetent. Since enrichment of PI(3)P within the viral replication compartment and recruitment of the phosphatidylethanolamine (PE)-rich endosomes is critical for robust TBSV replication (21, 25), we tested the effect of LepB on TBSV replication in detail. First, we confirmed that expression of LepB in yeast and plant leaves led to inhibition of TBSV RNA accumulation by ∼10- and ∼5-fold, respectively (Fig. 2 A and B). In addition, the purified GST-LepB was able to inhibit TBSV replication in vitro by ∼4-fold in a replicase reconstitution assay in yeast cell-free extract (CFE) supplied with purified recombinant p33 and p92pol replication proteins and (+)repRNA (Fig. 2C). This level of inhibition of TBSV replication is comparable to that observed in a replicase reconstitution assay in yeast CFE lacking the only PI3K, Vps34 (25). Therefore, we conclude that the LepB effector is a potent inhibitor of TBSV replication.

Fig. 2.

Fig. 2.

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Expression of the Legionella LepB effector inhibits TBSV replication in yeast and plants. (A) The effect of Legionella LepB effector on TBSV repRNA accumulation was measured by Northern blot 16 h after initiation of TBSV replication in BY4741 yeast. The accumulation level of repRNA was normalized based on the ribosomal RNA (rRNA). (Bottom) Ethidium-bromide stained gel with ribosomal RNA, as a loading control. (B) Expression of LepB inhibits TBSV replication in N. benthamiana. Total RNAs were extracted from the TBSV sap-inoculated leaves 1.5 d after the inoculation, followed by Northern blot analysis. There was a lack of phenotype on the agroinfiltrated leaves at the time of sampling. Each experiment was performed 3 times. (C) Reduced TBSV replicase activity in CFE in the presence of affinity-purified GST-LepB. Denaturing PAGE analysis was used to detect the 32P-labeled TBSV repRNA products. CFEs were prepared from BY4741 yeast. The CFEs were programmed with TBSV (+)repRNA transcripts and comparable amounts of replication proteins and added purified proteins, shown in a SDS/PAGE. Purified GST-GUS was used as a negative control in the CFE assay. (D and E) Expression of LepB prevents the enrichment of PI(3)P in the viral replication compartment in N. benthamiana protoplasts infected with TBSV. Distribution of PI(3)P was detected via expression of GFP-FYVE, which specifically binds to PI(3)P. DIC, differential interference contrast. (F and G) Enrichment of PE phospholipid in the replication compartment is reduced by the expression of LepB in plant cells. PE localization was monitored with duramycin staining of N. benthamiana protoplasts infected with TBSV.

To dissect the mechanism of LepB-driven inhibition of TBSV replication, we detected the subcellular localization of PI(3)P (a LepB substrate) using GFP-FYVE protein, which can specifically recognize PI(3)P (26). PI(3)P is enriched in the large viral replication compartment in Nicotiana benthamiana cells (Fig. 2E) (25). In contrast, expression of LepB effector efficiently blocked the enrichment of PI(3)P in the viral replication compartment in N. benthamiana cells (Fig. 2D). Expression of LepB effector also efficiently blocked the enrichment of PI(3)P in the tombusviral replication compartment in yeast cells (SI Appendix, Fig. S1A).

TBSV hijacks the early endosomes to enrich PE within the viral replication compartment, needed for efficient TBSV replication (13). Testing the subcellular distribution of PE in N. benthamiana cells infected with TBSV and expressing LepB showed the lack of PE enrichment within the replication compartment harboring p33 replication protein (Fig. 2F). In contrast, PE was efficiently enriched within the replication compartment in the absence of LepB (Fig. 2G). Expression of LepB effector also efficiently blocked the enrichment of NBD-PE in the tombusviral replication compartment in yeast cells (SI Appendix, Fig. S1B). Based on these data, we propose that expression of LepB effector blocks the subversion of early endosomes by TBSV, likely by altering the protein and PI(3)P lipid composition of the early endosomes.

The Legionella DrrA Effector Inhibits Tombusvirus Replication In Vitro and in Yeast and Plant.

To explore our above effector screen in order to identify a novel cellular factor and/or pathway that might be coopted by TBSV, we chose the Legionella DrrA effector, a known inhibitor of Rab1 GTPase function. To test if TBSV replication might depend on Rab1 GTPase function, we expressed the DrrA effector in WT yeast, which resulted in inhibition of TBSV repRNA accumulation by ∼80 to 90% (SI Appendix, Table S3 and Fig. 3A). These data suggest that DrrA inhibits the functions of a host factor(s) that is also needed for TBSV replication. The fully functional DrrA is required to block TBSV replication, based on the lack of inhibition of TBSV replication by the expression of 2 DrrA mutants, namely the AMPylation domain mutant of DrrA (DrrA-ma), which cannot modify Rab1 function, or the membrane insertion domain mutant of DrrA (DrrA-mm) (19, 27) (Fig. 3A, lanes 7 through 12). The anti-TBSV effect of DrrA was further supported by the observation that the affinity-purified GST-tagged DrrA was able to block TBSV replication in vitro in a replicase reconstitution assay with purified recombinant p33 and p92pol replication proteins, TBSV (+)repRNA template and yeast CFE (Fig. 3B, lane 2 versus 1). On the contrary, purified DrrA-ma or DrrA-mm could not efficiently inhibit the in vitro replication of TBSV in the CFE-based assay (Fig. 3B, lanes 3 and 4). The in vitro data demonstrate that DrrA can directly affect TBSV replication, not through pleiotropic effect on cell viability. It is also possible that Rab1 decorated COPII vesicles, or the ER exit sites are used by TBSV for in vitro replication, which could be blocked by DrrA. However, we cannot fully exclude that the inhibitory effect of DrrA on in vitro TBSV replication is not only due to inhibition of Rab1 GTPase, but to additional cellular targets of DrrA. Based on these data, we suggest that the fully active, membrane-bound DrrA is an efficient inhibitor of TBSV replication.

Fig. 3.

Fig. 3.

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Expression of the Legionella DrrA effector inhibits TBSV replication in yeast and plants. (A) The effect of Legionella DrrA effector on TBSV repRNA accumulation was tested in BY4741 yeast. Replication of the TBSV repRNA was measured by Northern blot 16 h after initiation of TBSV replication in yeast. The accumulation level of repRNA was normalized based on the rRNA. (Middle) Ethidium-bromide stained gel with ribosomal RNA, as a loading control. The accumulation levels of His6-p33 and His6-p92pol replication proteins were tested using Western blot and anti-His antibody. (B) Reduced TBSV replicase activity in CFE in the presence of affinity-purified GST-DrrA. Denaturing PAGE analysis was used to detect the 32P-labeled TBSV repRNA products. CFEs were prepared from BY4741 yeast. The CFEs were programmed with TBSV (+)repRNA transcripts and comparable amounts of replication proteins and purified proteins. (C) Inhibition of TBSV replication in N. benthamiana by DrrA expression. Total RNAs were extracted from the TBSV sap-inoculated leaves 1 d after inoculation, followed by Northern blot analysis. DrrA and its mutants were detected via Western blotting using anti-His antibody. Each experiment was performed 3 times. (D) Lack of phenotype on the agroinfiltrated leaves at the time of sampling. Note that TBSV does not induce visible symptoms on the inoculated leaves at the early time point shown.

To test if DrrA might have a similar inhibitory activity on tombusviruses in plants, we expressed WT YFP-tagged DrrA or the above 2 DrrA mutants in N. benthamiana via agroinfiltration, followed by inoculation with infectious TBSV. Northern blot analysis of the total RNA extract obtained from the inoculated leaves 1.5 d postinoculation revealed strong inhibition of TBSV genomic RNA accumulation by DrrA (Fig. 3C, lanes 3 and 4 versus 1 and 2), whereas expression of the 2 DrrA mutants showed lesser inhibition of TBSV accumulation (Fig. 3C, lanes 5 through 8). At the time of the analysis of viral accumulation, the N. benthamiana leaves did not show visible phenotypes (Fig. 3D), although a necrotic phenotype caused by the DrrA and DrrA-mm expression, but not DrrA-ma expression, appeared ∼4 d after agroinfiltration. Altogether, the plant-based experiments demonstrated that DrrA effector is a potent inhibitor of tombusvirus replication in plants.

The Legionella DrrA Effector Inhibits Tombusvirus Replication through Interfering with the Recruitment of Rab1 Small GTPase into the Viral Replication Compartment in Yeast and Plant.

To unravel how DrrA effector could inhibit tombusvirus replication, we tested if Rab1 (called Ypt1 in yeast), the main host target of DrrA (18), is involved in TBSV replication in yeast. Rab1 is a small GTPase involved in targeting and fusing COPII vesicles from the ER to the Golgi compartment in the early secretory pathway (28, 29). To study if Rab1/Ypt1 affects TBSV replication, we used the TET::YPT1 yeast strain, in which the expression of Ypt1 could be suppressed via addition of doxycycline to the growth media (30). Analysis of viral RNA accumulation by Northern blotting revealed an ∼3-fold inhibition of TBSV repRNA accumulation in yeast with a depleted Ypt1 level (Fig. 4A). The amount of p33 replication protein was slightly decreased in yeast with the depleted Ypt1 level (Fig. 4A).

Fig. 4.

Fig. 4.

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Rab1 small GTPase is coopted to enhance TBSV replication in yeast and plants. (A) Depletion of Ypt1 (Rab1) level in TET::YPT1 yeast inhibits TBSV replication. Doxycycline (Dox) was used to down-regulate the expression of Ypt1 from the TET promoter. Replication of the TBSV repRNA in TET::YPT1 yeast coexpressing the tombusvirus p33 and p92pol replication proteins was measured by Northern blotting 24 h after initiation of TBSV replication. The accumulation level of His6-p33 replication protein was tested by Western blot and anti-His antibody. (B) Coexpression of DN mutant of AtRab-D1 and DN AtRab-D2 by agroinfiltration suppressed TBSV replication in N. benthamiana, based on Northern blot analysis of TBSV RNA accumulation. Total RNA was extracted from the inoculated leaves 1 d after the inoculation. Empty vector was used as a negative control in this experiment. (C) Lack of phenotype on the agroinfiltrated leaves at the time of sampling. (D) Copurification of GFP-Ypt1 with Flag-p33 and Flag-p92 replication proteins from the membranous fraction of yeast. (E and F) Confocal microscopy-based partial colocalization of the coopted Ypt1 with p33 replication protein in yeast not expressing or expressing DrrA. Manders’ coefficient for the quantitative analysis of colocalization of p33-RFP and GFP-Ypt1 is shown. (Scale bars, 5 μm.) (G) Partial colocalization of TBSV p33 replication protein and AtRab-D1. The RFP-tagged p33 and GFP-AtRab-D1 were expressed via agroinfiltration in plant epidermal cells, which were also infected with TBSV, followed by confocal microscope imaging. (H) Interaction between the TBSV p33 replication protein and AtRab-D1 in plant was detected via BiFC. The signal between TBSV p33-cYFP and the nYFP-AtRab-D1 protein overlaps with RFP-SKL (peroxisomal matrix marker), which suggests the p33 and AtRab-D1 interaction occurs in the TBSV replication compartment. The fluorescence-tagged proteins were expressed by agroinfiltration in N. benthamiana leaves. The infiltrated leaves were also inoculated with TBSV to induce the formation of the large replication compartment consisting of aggregated peroxisomes. The combination of p33-cYFP and nYFP-MBP was used as negative control for BiFC. (I) Inhibition of colocalization of TBSV p33 replication protein and AtRab-D1 by expression of DrrA. The p33-RFP and GFP-AtRab-D1 and DrrA were expressed via agroinfiltration in plant epidermal cells, which were also infected with TBSV, followed by confocal microscope imaging. (J) Different subcellular localization of GFP-AtRab-D1 and the peroxisomal RFP-SKL in N. benthamiana. These proteins were expressed via agroinfiltration in plant epidermal cells, which were mock infected.

Arabidopsis has 2 Rab1 subclasses, called AtRab-D1 and -D2 (31), and we coexpressed the dominant-negative (DN) mutants of AtRab-D1 and -D2 in N. benthamiana plants. We observed an ∼6-fold inhibition of TBSV genomic RNA accumulation in leaves coexpressing the dominant-negative mutants of AtRab-D1 and -D2 (Fig. 4B), which showed no visible phenotypes at the time of sampling (Fig. 4C). Altogether, these data suggest that Rab1 has proviral function in TBSV replication in both yeast and plant hosts.

To test if Rab1 is recruited into the membranous viral replication compartment, first, we FLAG-affinity purified the tombusvirus replicase complex containing Flag-p33 and Flag-p92pol replication proteins from yeast membranes, which resulted in the copurification of GFP-Ypt1 (Fig. 4D, lane 2 versus 1). This indicates that the p33 replication protein interacts with Rab1/Ypt1 in yeast cell membranes. Second, colocalization studies between RFP-tagged p33 replication protein and the GFP-tagged Ypt1 by confocal microscopy revealed partial colocalization in punctate structures in yeast cells (Fig. 4E), suggesting that Ypt1 is recruited to the site of replication (peroxisomal membranes). Interestingly, expression of DrrA prevented the colocalization of RFP-p33 and GFP-Ypt1 in yeast cells (Fig. 4F). Expression of DrrA led to markedly reduced-sized punctate structures formed by p33 replication protein. Moreover, the distribution of Ypt1 was more diffused (lacked punctate structures) in yeast cells expressing DrrA (Fig. 4F). Third, confocal microscopy-based analysis also showed colocalization of p33-RFP and GFP-AtRab-D1 within the characteristic TBSV replication compartment in N. benthamiana cells (Fig. 4G). Fourth, we performed bimolecular fluorescence complementation (BiFC) experiments in N. benthamiana, which showed the interaction between p33 replication protein and AtRab-D1, and that the interaction occurred within the replication compartment (visualized through RFP-SKL peroxisome matrix marker) (Fig. 4H). Expression of DrrA prevented the colocalization of p33-RFP and GFP-tagged AtRab-D1 (Fig. 4I). Similar to yeast data, expression of DrrA led to markedly reduced-sized punctate structures formed by the p33 replication protein in plant cells. Also, the distribution of AtRab-D1 was more diffused in N. benthamiana cells expressing DrrA (Fig. 4I) in comparison with the negative control (Fig. 4J). Altogether, these data suggest that TBSV recruits Ypt1/Rab-D1 to the replication compartment in yeast and plant cells. In addition, expression of DrrA effector interferes with the recruitment of Rab1 homologs into the viral replication compartment both in yeast and plant cells.

The Legionella DrrA Effector Inhibits Tombusvirus Replication through Blocking the Recruitment of COPII Vesicles into the Viral Replication Compartment in Yeast and Plant.

Since the major cellular function of Rab1 in eukaryotic cells is supporting forward (anterograde) transport from the ER to Golgi via COPII vesicles (28, 29), we tested if TBSV might hijack COPII vesicles via interaction between p33 replication protein and Rab1.

COPII vesicle formation depends on the ER-resident Sar1 small GTPase, which is involved in cargo selection and COPII complex assembly (32, 33). We expressed the dominant-negative Sar1 (H77L mutant) in WT yeast, which led to an ∼4-fold reduction in TBSV repRNA accumulation (Fig. 5A). Similarly, expression of dominant-negative Sar1 (H74L mutant) in N. benthamiana leaves reduced TBSV RNA accumulation by ∼30-fold (Fig. 5B). These data support an important role for Sar1 GTPase, and likely the COPII vesicles, in TBSV replication in yeast and plant cells.

Fig. 5.

Fig. 5.

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Expression of dominant-negative mutant of Sar1 inhibits TBSV replication in yeast and plants. (A) The effect of Sar1-DN on TBSV repRNA accumulation was tested in BY4741 yeast by Northern blot 24 h after initiation of TBSV replication. The accumulation level of repRNA was normalized based on the rRNA. (Middle) Northern blot analysis of ribosomal RNA level, as a loading control. The accumulation levels of His6-p33 and His6-p92pol replication proteins were tested using Western blot and anti-His antibody. (B) Inhibition of TBSV replication in N. benthamiana by AtSar1-DN expression. Total RNAs were extracted from the TBSV sap-inoculated leaves 1 d after the inoculation, followed by Northern blot analysis. Each experiment was performed 3 times.

Expression of the GFP-HDEL ER luminal marker with the retrieval signal, which travels from the ER to the Golgi via COPII vesicles in plants (32), revealed colocalization of a portion of GFP-HDEL with the viral replication compartment (containing the p33-BFP within the peroxisomal membranes in N. benthamiana protoplasts) (Fig. 6A). Similarly, robust recruitment of GFP-HDEL into the viral replication compartment was observed in the epidermal cells of N. benthamiana infected with TBSV (Fig. 6B). The subcellular distribution of GFP-HDEL and RFP-SKL was different in protoplasts not infected with TBSV (Fig. 6A). Moreover, the peroxisomal marker RFP-SKL showed a different localization pattern from GFP-HDEL in mock-inoculated plants (Fig. 6B).

Fig. 6.

Fig. 6.

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Recruitment of COPII cargo to the large replication compartment in plants infected with TBSV. (A) Partial colocalization of TBSV p33 replication protein and GFP-HDEL, in TBSV-infected protoplasts. RFP-SKL marks the aggregated peroxisomes, the site of TBSV replication. The Bottom shows the lack of colocalization of GFP-HDEL with the peroxisomal RFP-SKL in the absence of TBSV. (Scale bars, 5 μm.) (B) Partial colocalization of TBSV p33 replication protein and GFP-HDEL, in TBSV-infected N. benthamiana plants. The p33-BFP and GFP-HDEL were expressed via agroinfiltration in plant epidermal cells, which were also infected with TBSV, followed by confocal microscope imaging. The Bottom shows the lack of colocalization of GFP-HDEL with the peroxisomal RFP-SKL in the absence of TBSV. (C and D) Expression of AtSar1-DN or DrrA effector interferes with the recruitment of GFP-HDEL to the large replication compartment in plants infected with TBSV. See further details in B. (E and F) Expression of DrrA-ma and DrrA-mm mutants only partially interferes with the recruitment of GFP-HDEL to the large replication compartment in plants infected with TBSV. See further details in B.

To inhibit COPII formation, we expressed Sar1-DN mutant in N. benthamiana replicating TBSV, which led to efficient inhibition of retargeting of GFP-HDEL into the viral replication compartment (Fig. 6C). Blocking Rab1 function through expression of DrrA in N. benthamiana replicating TBSV resulted in reduced retargeting of GFP-HDEL into the viral replication compartment (Fig. 6D). However, expression of DrrA-ma did not inhibit, whereas expression of DrrA-mm partially inhibited the retargeting of GFP-HDEL to the replication compartment (Fig. 6 E and F).

To obtain further evidence for the recruitment of COPII vesicles into the TBSV replication compartment, we expressed GFP-Sec13, a coat component of COPII vesicles (32), in N. benthamiana infected with TBSV. Confocal microscopy imaging showed the efficient retargeting of GFP-Sec13 to the large replication compartment (Fig. 7A). On the contrary, the peroxisomal marker RFP-SKL showed different localization from GFP-Sec13 in mock-inoculated plants (Fig. 7 A, Bottom). Inhibition of Rab1 function through expression of DrrA inhibited the retargeting of GFP-Sec13 into the viral replication compartment in N. benthamiana replicating TBSV (Fig. 7B). Expression of DrrA-ma did not inhibit the retargeting of GFP-Sec13 into the viral replication compartment (Fig. 7C), whereas expression of DrrA-mm partially inhibited the retargeting of GFP-Sec13 to the replication compartment (Fig. 7D). Therefore, we suggest that TBSV recruits Rab1 GTPase and COPII vesicles into the replication compartment and this process can be greatly inhibited by expression of the Sar1-DN mutant or DrrA effector in plants.

Fig. 7.

Fig. 7.

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DrrA blocks the recruitment of COPII vesicles to the replication compartment in plants infected with TBSV. (A) Partial colocalization of TBSV p33 replication protein and GFP-Sec13 coat protein of COPII vesicles, in TBSV-infected N. benthamiana plants. The p33-BFP and GFP-Sec13 were coexpressed via agroinfiltration in plant epidermal cells, which were also infected with TBSV, followed by confocal microscope imaging. The Bottom shows the lack of colocalization of GFP-Sec13 with the peroxisomal RFP-SKL in the absence of TBSV. (B) Expression of DrrA blocks the recruitment of GFP-Sec13 to the replication compartment in plants infected with TBSV. See further details in A. (C and D) Expression of DrrA-ma does not block, whereas DrrA-mm only partially interferes with the recruitment of GFP-Sec13 to the replication compartment in plants infected with TBSV. See further details in A. (E and F) The effect of induction or repression of the expression of syntaxin-18 like Ufe1 SNARE protein on the colocalization of GFP-Ypt1 and p33-RFP in GALS::UFE1 yeast strain, documented via confocal microscopy. Manders’ coefficient for the quantitative analysis of colocalization of p33-RFP and GFP-Ypt1 is shown. (G and H) The effect of induction or repression of the expression of the ER-resident Use1 SNARE protein on the colocalization of GFP-Ypt1 and p33-RFP in the GAL1::USE1 yeast strain, documented via confocal microscopy. Each experiment was performed 2 times.

TBSV usurps the ERAS (ER arrival site) subdomain in the ER as an assembly hub for the biogenesis of the TBSV replication compartment (34). The ERAS consists of the ER-resident SNARE proteins, namely the syntaxin 18-like Ufe1, Use1, and Sec20, and the Dsl1 tethering complex (35). Through binding to Ufe1 and Use1 SNARE proteins (34), the p33 replication protein might utilize the SNARE and tethering proteins to capture Rab1 and COPII vesicles. This was tested by depleting Ufe1 or Use1 levels through repressing GAL1 promoter-based expression of either Ufe1 or Use1 mRNAs in GALS::UFE1 and GAL1::USE1 yeast cells, respectively (34). Confocal microscopy revealed the lack of colocalization of Rab1 with p33 replication protein in yeasts with depleted levels of either Ufe1 or Use1 (Fig. 7 F and H), whereas Rab1 was partially colocalized with p33 replication protein in yeast cells when Ufe1 and Use1 expression was induced (Fig. 7 E and G). These data indicate that efficient subversion of Rab1 into the tombusvirus replication compartment depends on these coopted ER SNARE proteins.

Discussion

Many bacterial pathogens infect and survive within eukaryotic cells by injecting minute quantities of bacterial virulence factors or effector proteins, typically enzymes that posttranslationally modify key cellular proteins, into the cytosol of their hosts. These effector proteins have evolved to manipulate cellular pathways, prevent bacterial degradation, and favor bacterial replication (14–16). For instance, L. pneumophila, the cause of Legionnaires’ disease, synthesizes >300 effector proteins, some of which encode biochemical activities to reprogram endolysosomal membrane transport, potently inhibit cellular autophagy, and divert innate immune responses (14, 15).

We exploited Legionella effectors to probe tombusvirus–host interactions and identify host factors subverted by TBSV in a yeast surrogate host. Among the 303 Legionella effectors tested, we found that 28 effectors affected TBSV replication in yeast (SI Appendix, Table S3). The identified bacterial effectors target conserved cellular proteins and pathways including the endosomal pathway, which are also targeted by TBSV during replication (21).

As an important proof of concept that bacterial effectors could be utilized as cellular probes to dissect viral processes, we tested the effect of the LegC8 effector, which is an inhibitor of eEF1A translation elongation factor, on TBSV replication in a cell-free assay. The use of purified recombinant TBSV replication proteins and LegC8 in the replication assay allowed for the demonstration of direct inhibitory effect of LegC8 on TBSV replication (Fig. 1).

Also studying LepB helped to confirm the critical roles of PI(3)P and the endosomal compartment in the biogenesis of tombusvirus VRCs. LepB interferes with the endosomal functions by converting the endosomal PI(3)P to PI(3,4)P2 (24). The reduction of PI(3)P in the endosomal membrane leads the loss of many cellular endosomal proteins and makes the endosomes fusion incompetent. Accordingly, TBSV was unable to enrich PI(3)P and PE phospholipid, which is present in the endosomal membranes (21), within the replication compartment when LepB was expressed in plant or yeast cells. The critical role of PI(3)P in tombusvirus replication has been shown recently (25). Altogether, the obtained data on LegC8 and LepB effectors confirmed their direct inhibitory effects on TBSV replication, strongly supporting the idea that Legionella effectors could be used as probes in TBSV–host interactions. Moreover, our study provides proof of concept that bacterial effector proteins can be used as molecular tools to identify cellular pathways used by (+)RNA viruses.

One of the intriguing findings in our Legionella effector screen in the TBSV–yeast system is the targeting of Rab1 small GTPase function by the largest number of identified bacterial effectors. The list includes DrrA/SidM with Rab1 GEF activity, LepB with Rab1 GAP activity, AnkX, which through phosphorylcholination, inactivates Rab1, whereas Lem3 has the opposite function as AnkX (SI Appendix, Table S3). Another effector LidA also binds to Rab1 (14). The identification of 5 Legionella effectors in our antiviral screen that affect Rab1 functions strongly supports the proviral role of Rab1 small GTPase in TBSV replication.

Accordingly, expression of DrrA effector or depletion of Rab1/Ypt1 had silimilar inhibitory effect on TBSV replication in yeast. Similarly, expression of dominant-negative mutants of Rab1 (Rab-D1 and Rab-D2) or DrrA showed a strong inhibitory effect on tombusvirus replicaton in plants. Rab1 was recruited into the viral replication compartment based on copurification of Ypt1 with the tombusvirus replicase, colocalization of Rab1 with p33 replication protein in plant cells, and BiFC assay in plants (Fig. 4). Expression of DrrA effector interfered with the recruitment of Rab1 into the viral replication compartment. Altogether, these data support the proviral role of Rab1 in tombusvirus replication.

What function(s) does Rab1 provide for TBSV replication? The cellular function of Rab1 is involved in targeting and fusing COPII vesicles to the Golgi compartment (28, 29). We find that through binding to p33 replication protein, Rab1 facilitates the recruitment of COPII vesicles into the viral replication compartment. Accordingly, GFP with the HDEL retrieving signal and Sec13, which is a coat protein component of COPII vesicles, are efficiently retargeted to the large viral replication compartment in plant cells. Expression of DrrA effector or dominant-negative Sar1 small GTPase, which blocks formation of COPII vesicles in the ER, prevented the retargeting of GFP-HDEL and Sec13 into the viral replication compartment in plant cells. Interference with the recruitment of Rab1 and subversion of COPII vesicles led to reduced-sized viral replication compartment (Figs. 5 and 6), suggesting that COPII vesicles are utilized by tombusviruses for enlargement of membrane surfaces available for VRC biogenesis. It is also likely that the lipid composition of COPII vesicles might be useful to obtain optimal lipid composition within the VRCs. Previous work with isolated subcellular organelles and in vitro assembled TBSV replicase complexes indicated that the ER-like membranes are the most suited for TBSV replication (36). Altogether, we propose that TBSV hijacks Rab1 and COPII vesicles to create enlarged membrane surfaces and optimal lipid composition within the viral replication compartment. Altogether, the use of Legionella effectors helped us to find proviral functions for cellular proteins and pathways.

The LCV is an intracellular membranous structure induced during the infection and serves as the site for bacterial replication (14, 15). The biogenesis of LCV requires major membrane remodelling after cellular phagocytic entry of the Legionella bacteria and is driven by the translocated effectors via recruitment of cellular proteins and membranes and modifying the lipid composition of the LCV membrane to prevent fusion with the lysosome and block being targeted by the autophagosome. Overall, the LCV membrane becomes similar to ER–Golgi membranes. We recognize that the TBSV replication compartment, albeit forming in the peroxisomal compartment, shows many similarities with LCV. For example, TBSV also recruits COPII vesicles with the help of p33 replication protein–Rab1 small GTPase interaction (Fig. 4). Both LCV and the viral replication compartment are enriched with PE (21). Moreover, the formation of membrane contact sites (MCSs) between the ER and the LCV and between the ER and the tombusvirus replication compartment is another feature shared by these pathogens. In the case of TBSV infection, the MCS serves as a site to enrich sterols within the replication compartment with the help of coopted VAP and OSBP-like proteins (12, 37). Other interesting features for both pathogens is the subversion of the actin network and the dependence on the TORC1 and eEF1A activities during replication (9, 20, 38). Thus, we observe similarities between the formation/maturation of the LCV and the TBSV replication compartment at multiple levels that could facilitate future research in understanding the assembly of these parasitic replication structures inside infected cells. Overall, the remarkable antiviral effects of selected Legionella effectors might be due to the observed molecular similarities in remodelling host membranes and targeting cellular pathways by these pathogens.

Rab1 and COPII vesicles are also coopted by different viruses for various purposes, from replication, intracellular trafficking of viral proteins, to particle assembly (39–42). For example, Rab1 facilitates the interaction of the hepatitis C virus (HCV) NS5A protein with lipid droplets to alter lipid metabolism that favors HCV replication (43). Sar1 GTPase and COPII components interact with the 6K2 protein of potyviruses, which are needed for the formation of 6K2 vesicles and virus replication (44, 45). COPII components also interact with the 1a replication protein of brome mosaic virus (BMV) to facilitate ER localization of 1a and the formation of the viral replication compartment (46).

Summary.

Our study provides an important proof of concept that bacterial effector proteins can be used as tools to identify host proteins and cellular pathways used by (+)RNA viruses. The Legionella DrrA effector was used to identify and dissect the proviral function of the previously uncharacterized Rab1, and COPII vesicles, which are coopted by the viral replication protein through interaction with Rab1 small GTPase. Altogether, the identified bacterial effectors with anti-TBSV activity could be useful as reagents in cell biology and virus–host interaction studies.

Experimental Procedures

These are presented in SI Appendix, Supplementary Materials.

Supplementary Material

Supplementary File
pnas.1911108116.sapp.pdf (516.9KB, pdf)

Acknowledgments

We thank Dr. Judit Pogany for designing experiments and effector library construction for expression in yeast and for comments on the manuscript and Ms. Jannine Baker for her assistance in screening the Legionella effector library in yeast. Dr. Christopher T. Beh (Simon Fraser University) and Dr. Daniel J. Klionsky (University of Michigan) provided yeast plasmids. This work was supported by the National Science Foundation’s Division of Molecular and Cellular Biosciences (1517751) awarded to P.D.N. and NIH-NIAID (AI120113) awarded to B.D.L.

Footnotes

The authors declare no competing interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1911108116/-/DCSupplemental.

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Supplementary Materials

Supplementary File
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