Introduction

RNA interference (RNAi) pathways play a pivotal role in regulating the virulence of viruses across both animal and plant kingdoms1,2,3,4. These pathways act as defense mechanisms, limiting pathogen replication and thereby prompting the evolution of countermeasures by pathogens5,6. Viral suppressors of RNA silencing (VSRs) have been detected in various plant virus families and several insect viruses6,7. Additionally, certain sequences and proteins present in arthropod-borne human pathogens are speculated to function as VSRs5,8. More recently, the nonstructural protein NS2A of dengue-2 virus was shown to act as a VSR9. These ongoing evolutionary arms races have independently led to the emergence of similar VSR strategies on multiple occasions5,10. Consequently, while VSRs may share functional characteristics at the biochemical level, they often display minimal genetic conservation10.

Research on potential VSRs typically focuses on the analysis of individual proteins, which are expressed either transiently or stably within host cells, alongside a secondary reporter construct to measure their activity11,12,13,14. The absence of a uniform reporter system, however, has led to the use of a broad array of cell types, promoters, and transgene reporters to evaluate VSR function. This diversity in experimental approaches can lead to false-positive or -negative results, as well as inconsistent and non-replicable findings across different research systems. Although stable genetic integration (transgenic expression) offers certain benefits over temporary transient expression, including the potential to establish a uniform testing protocol, the expression levels of VSRs may still be influenced by the genomic context of their insertion site in the host DNA, known as position effects15,16.

The aim of our study was to examine how genomic insertion sites influence the expression of candidate VSR proteins17,18,19 using a transgenic Drosophila melanogaster reporter system12. Our findings reveal that silencing varies depending on the phiC31 docking site20,21,22, suggesting that insufficient expression from specific genomic locations within D. melanogaster, and potentially other organisms, may impede the detection of VSR activity. However, we demonstrate that integrating gypsy insulators23,24, which are capable of significantly enhancing gene expression in comparison to the uninsulated site25, can mitigate these issues. These results pave the way for establishing a standardized reporter system in D. melanogaster with consistent levels of transgene expression, facilitating the initial assessment of viral proteins and enabling meaningful comparisons of VSR activity. Moreover, this information may be used to develop better reporter systems in other organisms.

Results

Tissue-specific induction of a VSR and position effects at attP docking sites

We generated transgenic flies that express the well-characterized VSR protein, Drosophila C virus (DCV)-1A19, using the binary GAL4-UAS26 system (Fig. 1 and S1). Conditional expression in the Drosophila eye was achieved (Fig. 1) with a GMR-GAL4 driver27,28. To systematically evaluate the effects of host chromatin position on VSR protein expression, a UAS-DCV-1A cassette (Fig. 1 and S1) was integrated into three different attP landing sites: VK122, attP4020, or attP1821.

Fig. 1
Fig. 1The alternative text for this image may have been generated using AI.
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A transgenic reporter system designed to silence the D. melanogaster white gene using a dsRNA hairpin sequence. In the sensor lines, both the white inverted repeat sequence and the yeast GAL4 protein are regulated by the GMR enhancer, which is active in immature and adult retinal tissue where the endogenous white gene is also expressed. The tissue-specific expression of the inverted repeats produces hairpin-loop RNA, inducing RNAi that targets the D. melanogaster white gene. In the test lines, VSR proteins or controls (such as EGFP) are under the transcriptional control of a D. melanogaster promoter linked to GAL4-responsive upstream activating sequence (UAS) repeats. When the test lines are crossed with reporter lines, GAL4 drives the tissue-specific expression of VSR or control sequences. Additionally, some test lines were created with gypsy insulator sequences positioned either upstream, downstream, or flanking the GAL4-responsive elements.

In order to assess position effects at these attP landing sites using a biologically relevant assay, the VSR-expressing lines were crossed with an RNAi-sensor line (Fig. 1). The RNAi-sensor contains two transgenic cassettes, GMR-white inverted repeat (IR) on the X chromosome and GMR-GAL4 on chromosome 2 (Fig. 1). The D. melanogaster white gene encodes an ABC transporter crucial for eye pigmentation29, allowing its expression to be phenotypically monitored and indirectly quantified in terms of eye pigment levels. The white (+) eye phenotype is dark red (Fig. 2), while a white (-) or null mutant displays white eyes. Silencing of the white target gene mediated through the expression of a double-stranded RNA hairpin from the GMR-white-IR transgene12 results in an intermediate or orange phenotype, with paleness (e.g., in presence of VSR) depending on the level of silencing (Fig. 2).

Fig. 2
Fig. 2The alternative text for this image may have been generated using AI.
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Position effects and DCV-1A mediated suppression of silencing by the white GMR-hairpin transgene in the presence or absence of gypsy insulators. Far left, (1) eye color phenotype of GMR-GAL4; UAS-GFP flies (VK1). (2) Eye color of flies bearing GMR-whiteIR; GMR-GAL4; UAS-GFP transgenes (VK1). (3–6) Suppression of white silencing by DCV-1A expressed from the VK1 landing site with upstream, downstream, flanking (both), or no gypsy insulators. (7–10) Suppression from the attP40 landing site with upstream, downstream, flanking, or no gypsy insulators. (11) Eye color phenotype of GMR-whiteIR; GMR-GAL4; UAS-GFP (attP18) flies. (12–15) Suppression from attP18 landing site with upstream, downstream, flanking, or no gypsy insulators. Eye pigment levels were measured in three separate experiments using age-matched 4-day-old adults, and are expressed as a percentage relative to the pigment levels measured in the GMR-GAL4; UAS-GFP (VK1) controls. Error bars represent standard error. Significance was calculated by Student’s t-test.

In order to determine the landing site permitting suppression of target gene silencing to the greatest degree, we quantified eye pigment levels in RNAi-sensor adults expressing DCV-1A (GMR-whiteIR; GMR-Gal4; UAS-DCV-1A) from the various attP loci (Fig. 2). Notably, we found that expression from the VK1 locus on chromosome 2 suppressed silencing of the target gene by the greatest amount (Fig. 2). Expression of DCV-1A from the other two landing-site loci- attP40 orattP18- resulted in less robust suppression than was observed at the VK1 locus (Fig. 2). These results suggest that position effects may influence the results of phenotypic assays measuring VSR activity.

Gypsy insulators facilitate optimal VSR expression at multiple attP dockingsites

Although the above results suggest that the VK1 attP site exhibits the highest level of VSR expression among the landing sites evaluated (Fig. 2), position effects might still prevent optimal expression from this locus. Flanking transgenes with insulators has previously been shown to block the effects of neighboring enhancers, silencers, as well as heterochromatin (i.e., position effects), in order to ensure that expression of the transgenes is sufficient to produce a detectable phenotype23. The gypsy retrovirus insulator in particular has been previously found to boost gene expression to levels several fold greater than un-insulated loci25. Thus, in order to test if VSR expression could be further optimized, we created additional transgenic UAS-DCV-1A lines with flanking gypsy retrovirus insulators.

Flanking gypsy insulators did not affect VSR activity at the VK1 locus (Fig. 2), suggesting that the expression of DCV-1A at this landing site cannot be optimized further. However, we found that flanking the UAS-DCV-1A construct with gypsy insulators equalized VSR activity across all three landing sites, significantly increasing suppression at attP40 and attP18 loci (Fig. 2). These results suggest that gypsy insulators can be used to express VSR proteins at consistent and high levels across permissive loci.

Transgenic RNAi sensor reporter systems sometimes fail to identify well characterized VSRs

Consistent levels of VSR expression from transgene constructs with flanking gypsy insulators (Fig. 1) at specific genomic integration sites (Fig. 2) suggest that it is possible to compare the activity of VSR proteins from different viruses (Fig. 3). Therefore, we compared the VSR activity of DCV-1A at the VK1 attP site with that of another well-characterized VSR protein, flock house virus (FHV) B217,30,31,32,33, expressed from a transgene (UAS-FHV B2) integrated at the same locus. Both transgenes contained flanking gypsy insulators to ensure optimal expression25, as position effects may also depend on the inserted transgene sequence. Surprisingly, FHV-B2 did not exhibit any evidence of VSR activity (Fig. 3). After confirming the sequences of both B2 and the regulatory elements present in the integrated UAS-FHV B2 cassette (Fig. S1 and S2), we assessed VSR activity at the attP18 landing site. Consistent with the results reported above, no evidence of VSR activity was observed at the attP18 locus either (Fig. 3). To confirm that our transgenic reporter system could identify VSR proteins other than DCV-1A, we generated transgenic flies expressing a third well-characterized VSR, Cricket Paralysis virus (CrPV) 1A18. Significant suppression of target gene silencing was observed in RNAi-sensor adults expressing CrPV-1A (Fig. 3). Finally, expression of the B2 protein from the UAS-FHV B2 transgene integrated at both the VK1 and attP18 landing sites was confirmed through western blotting (Fig. S3, S4 and S5). The specificity of the B2 antibody was confirmed with a recombinant B2 protein (Fig. S3, S4 and S5). While the absolute expression levels of the VSRs tested were not quantified, it seems unlikely that the observed lack of activity resulted from insufficient FHV-B2 expression. All VSRs were expressed from identical promoter elements at the same genomic loci. Moreover, the attP landing sites were specifically chosen for their expression levels, and the flanking gypsy insulators employed here are a well-established method in D. melanogaster for alleviating position effects to further optimize robust expression. Thus, these results illustrate the limitations of studying VSRs as isolated proteins with reporter systems.

Fig. 3
Fig. 3The alternative text for this image may have been generated using AI.
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Suppression of GMR-hairpin mediated silencing of white by DCV-1A, CrPV-1A, or FHV-B2. (A) Far left, (1) Eye pigmentation of GMR-GAL4; UAS-GFP flies (VK1). (2) Eye color observed in flies with GMR-whiteIR; GMR-GAL4; UAS-GFP transgenes (VK1). (3) Suppression of white silencing mediated by DCV-1A expressed from the VK1 landing site with flanking gypsy insulators. (4–5) Lack of white silencing inhibition by FHV-B2 expressed from the VK1 or attP18 landing sites with flanking gypsy insulators. (6) Suppression of white silencing by CrPV-1A expressed from the attP18 landing site with flanking gypsy insulators. Eye pigment levels were measured in three separate experiments using age-matched 4-day-old adults, and are expressed as a percentage relative to the pigment levels measured in the GMR-GAL4; UAS-GFP (VK1) controls. Error bars represent standard error. Significance was calculated by Student’s t-test.

Discussion

The attP landing sites used in this study were chosen because they have in previous work been associated with high levels of gene expression20,21,22,34. Thus, position effects may prevent optimal expression to an even greater degree at other loci or tissues, suggesting that each locus must be selected on a case-by-case basis after testing. Additionally, the proteins used in this study have been associated with robust VSR functions17,18,19,30,31,32,33. Lower-level expression of proteins with less robust VSR activity could further hinder positive identification with reporter assays. We show here that consistently high levels of suppression can be achieved at permissive loci (Fig. 2) by flanking VSR expressing transgenes with gypsy retrovirus insulators25. However, our demonstration of consistent high-level suppression using insulator-flanked transgenes at permissive loci (Fig. 2) is primarily based on DCV-1A, and insulator effectiveness should also be evaluated on a case-by-case basis for each VSR tested.

Although position effects are a universal phenomenon, in that genomic location invariably exerts some influence, their practical impact ranges from negligible in permissive regions to profound in repressive contexts. Our results suggest that position effects are minimal at the VK1 locus (i.e., expression was already at or near maximum levels), explaining why flanking gypsy insulators did not significantly alter VSR activity at this genomic location (Fig. 2). However, inclusion of the gypsy insulators at other genomic loci, which our results suggest were in more repressive contexts, brought VSR activity to similarly high levels, presumably by increasing expression levels. These results and interpretation are consistent with previous descriptions of gypsy insulator function25. While ideally genomic insertion sites would be standardized, genetic crossing schemes sometime require that transgenes be located on specific chromosomes. Thus, the ability to create different genetic lines with consistently high and equivalent levels of transgene expression is important for using reporter systems to identify, study and compare VSR proteins. An advantage of this system is that it ensures consistent levels of VSR expression across well characterized loci on multiple chromosomes (Fig. 2).

However, our studies also highlighted the limitations of studying isolated proteins with reporter systems. The VSR function of B2 has been conclusively demonstrated in genetic rescue experiments, considered the gold standard for identifying such proteins. The replication of a B2-defective FHV has been rescued in both dcr-2 and ago-2 null mutants, unequivocally implicating the protein as a VSR17,33. However, examining the protein in isolation with a trans-acting reporter assay did not identify the VSR function previously associated with B2 (Fig. 3). Notably, prior work demonstrated that co-transfecting GFP dsRNA with pB2-GFP resulted in the effective destruction of B2-GFP mRNA via RNAi35. The authors concluded that for suppression to occur, B2 expression must precede the introduction of dsRNA and the initiation of RNAi, a temporal constraint that may similarly explain why the D. melanogaster reporter system described here failed to detect the VSR activity. Thus, the inability of this assay to detect B2’s VSR function is likely attributable to mechanistic differences with the VSRs of DCV-1A and CrPV 1A. While some studies have previously shown the VSR activity of B2 in reporter assays13,36, our inability to detect this function highlights the inherent difficulties of comparing results from different reporter assays. Our study is also not the first to show that reporter assays sometimes fail to detect the VSR activity of proteins with VSR functions, further reinforcing the notion that these systems are not universally applicable for identifying VSRs with diverse mechanisms of action10,36.

Nevertheless, viral proteins are often multifunctional, and it is not always possible to obtain viable mutants knocking out only VSR functions, precluding genetic rescue experiments37,38. Therefore, reporter assays will continue to be important tools for identifying and studying VSR proteins, particularly, as preliminary screens. However, our findings underscore the importance of taking a comprehensive, holistic approach to definitively identify or rule out authentic VSR functions. The results of reporter assays should not be considered in isolation as definitive determinations of VSR function or lack thereof.

Materials and methods

Transgenic flies and genetic crosses

A balanced transgenic RNAi-sensor line (P{GMR-w.IR}; P{w[+ mC] GMR-GAL4}/CyO) was generated through genetic cross of two different transgenic D. melanogaster linesobtained from the Bloomington stock center (P{GMR-w.IR}13D; P{ry[+ t7.2] = neoFRT}40A r2d2[S165fsX]12 and (w[*]; P{w[+ mC] = GAL4-ninaE.GMR}). All transgenic test lines were engineered with transgenes (EGFP, DCV-1A, FHV-B2 or CrPV-1A) expressed under a Gal4-inducible UAS promoter26, with upstream, downstream, flanking, or without, gypsy elements (Fig. 1). UAS constructs (Fig. S1) were integrated into D. melanogaster via ϕC31-integrase mediated recombination34,39 at VK122 or attP40 landing sites20 on Chr-2, or at the attP18 landing site21 on Chr-X (GenetiVision Corporation, Houston, TX). Experimental crosses (Fig. 1) were performed between age-matched RNAi-sensor (female) and test (male) flies, followed by progeny collection at 4 days-post-eclosion for further analyses.

Eye pigment assays

Eye pigment extractions were performed as described36 with few modifications. Heads of 5 virgin female adults at 4 days-post-eclosion were homogenized in acidified methanol (0.1% HCl), followed by gentle rocking for 24 h at 4 °C. After pigment extraction, samples were incubated at 50 °C for 5 min, followed by high-speed centrifugation. Optical density of each clarified supernatant sample was read as absorbance at 480 nm.

Western blotting

Total proteins were extracted by homogenizing 5 heads of virgin female flies at 4 days-post-eclosion in ice-cold protein extraction buffer [250 mM HEPES–KOH, 100 mM KCl, 10 mM EDTA, 0.1% Triton X, 5% Glycerol with freshly added 5 mM DTT and Protease Inhibitor Cocktail (Roche, Indianapolis, IN, USA)]. The supernatant was resolved by SDS-PAGE analysis, followed by dry-transfer onto a PVDF membrane as described40 using iBlot 3 System (Invitrogen). Two identically loaded protein blots (Fig. S3 and S4) were incubated in 5% (w/v) Blotting-Grade Blocker (Bio-Rad, Hercules, CA, USA), followed by FHV-B2-specific polyclonal monospecific rabbit antiserum (Pacific Immunology, San Diego, CA), or β-actin-specific polyclonal rabbit antibodies (Thermo Scientific, Waltham, MA, USA) at 1:10,000. Goat anti-rabbit HRP conjugate (Thermo Scientific) was used as a secondary antibody at 1:25,000. Immunoblots were developed using Immobilin Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA), followed by visualization of signals with iBright CL1500 Imaging System (Thermo Scientific).