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. 2024 Feb 1;17(2):dmm050297.
doi: 10.1242/dmm.050297. Epub 2024 Feb 28.

A Zika virus protein expression screen in Drosophila to investigate targeted host pathways during development

Nichole Link  1   2   3   4 , J Michael Harnish  3   4 , Brooke Hull  3   4   5 , Shelley Gibson  3   4 , Miranda Dietze  1 , Uchechukwu E Mgbike  1 , Silvia Medina-Balcazar  3   4 , Priya S Shah  6 , Shinya Yamamoto  3   4   5   7   8
Affiliations

A Zika virus protein expression screen in Drosophila to investigate targeted host pathways during development

Nichole Link et al. Dis Model Mech. .

Abstract

In the past decade, Zika virus (ZIKV) emerged as a global public health concern. Although adult infections are typically mild, maternal infection can lead to adverse fetal outcomes. Understanding how ZIKV proteins disrupt development can provide insights into the molecular mechanisms of disease caused by this virus, which includes microcephaly. In this study, we generated a toolkit to ectopically express ZIKV proteins in vivo in Drosophila melanogaster in a tissue-specific manner using the GAL4/UAS system. We used this toolkit to identify phenotypes and potential host pathways targeted by the virus. Our work identified that expression of most ZIKV proteins caused scorable phenotypes, such as overall lethality, gross morphological defects, reduced brain size and neuronal function defects. We further used this system to identify strain-dependent phenotypes that may have contributed to the increased pathogenesis associated with the outbreak of ZIKV in the Americas in 2015. Our work demonstrates the use of Drosophila as an efficient in vivo model to rapidly decipher how pathogens cause disease and lays the groundwork for further molecular study of ZIKV pathogenesis in flies.

Keywords: Drosophila; Degeneration; Microcephaly; Virus-host targets; Zika virus.

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Conflict of interest statement

Competing interests The authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
ZIKV proteins cause scorable phenotypes upon overexpression in Drosophila. (A) Graphical diagram of the Zika virus (ZIKV) polyprotein, which contains three structural and seven non-structural proteins at the endoplasmic reticulum (ER) membrane. (B) Diagram of the crossing scheme using which we crossed a fly containing a ZIKV protein under the control of a UAS element to another fly containing a GAL4 driver. The resulting fly expressed the ZIKV protein and was scored for phenotypes. (C) Table showing the phenotypes of the F1 generation resulting from a cross reared at 25°C. Dark blue represents lethality. Light blue represents semi-lethality, where less than 75% of expected Mendelian ratios are observed. Yellow indicates a morphological defect; the specific tissues affected are noted by the illustration (brain, thorax bristle and eye). The lethal stage for some crosses is indicated in Table S2.
Fig. 2.
Fig. 2.
Expression of ZIKV proteins in the notum causes bristle and split thorax phenotypes. (A) Control notum showing no morphological defects. (B) Expression of NS2A in the dorsocentral notum caused a supernumerary bristle phenotype. (C) Notum from animals with NS2B::NS3 expression (pnr-GAL4, UAS-NS2B::NS3) showed bristle loss. (D) A rare escaper notum from animals with NS4A expression demonstrates a split thorax phenotype and bristle defects. For each case, the penetrance of phenotype was 100%. All crosses were carried out at 29°C. Images are representative of at least 100 animals per genotype. Scale bar: 0.1 mm.
Fig. 3.
Fig. 3.
2K peptide alters NS4A and NS4B phenotypes. (A,B) Graphical diagram of the ZIKV NS4A and NS4A::2K peptides (A) and the 2K::NS4B and NS4B peptides (B) at the ER membrane. Both NS4A and NS4B can be found with the 2K peptide linker region. (C) Table showing the phenotypes of the F1 generation resulting from a cross reared with indicated drivers at 25°C or 29°C. Dark blue represents lethality. Light blue represents semi-lethality, where less than 75% of expected Mendelian ratios are observed. Yellow indicates a morphological defect; specific tissues affected are noted by the illustration (brain, thorax bristle and eye). Purple indicates an electrophysiological phenotype. The ‘vs’ column denotes whether one variant is more severe (> or <) or both have equal severity (=). The lethal stage for some crosses is indicated in Table S2. (D-H) Eye phenotypes as a result of GMR-GAL4 expression of control lacZ (D), NS4A (E), NS4A::2K (F), NS4B (G) and 2K::NS4B (H). Note that in general, the 2K peptide decreased the effect of NS4A but enhanced the phenotypes caused by NS4B. In each case, the penetrance of phenotype was 100%. Images are representative of at least 100 animals per genotype.
Fig. 4.
Fig. 4.
Multiple ZIKV proteins cause microcephaly upon overexpression in Drosophila. Expression of ZIKV proteins using either insc-GAL4 at 29°C (A-D,H) or Act-GAL4 at 25°C (E-G,I) caused microcephaly phenotypes. (A-G) Bright field images of brains from the indicated lines are shown. Scale bars: 100 µm. (H,I) Quantification of brain volume. Individual brain lobe volume measurements are plotted and the mean is represented by the line. Populations with smaller brain volumes and P<0.05 are open circles, whereas populations with P>0.05 are in closed circles. One-way ANOVA with multiple comparisons posttest compared to control (mCD8::GFP) was used to assess significance. Lethal crosses are indicated with a skull symbol and the number of animals for each condition are shown as N. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.
Fig. 5.
Fig. 5.
Subcellular localization of ZIKV proteins differs when expressed in neuronal stem cells. (A) Neural stem cells from wild-type animals stained with DAPI (blue) and Calnexin 99A (white) to highlight ER structure. (B) Animals expressing mCD8::GFP as a control with DAPI (blue), Dpn (magenta) and mCG8::GFP (green). (C-M) Neural stem cells from third instar larvae with insc-GAL4 ZIKV protein expression stained for the C-terminal HA tag (green) to mark ZIKV proteins, Dpn (magenta) to indicate neuroblasts and DAPI to highlight DNA. Each panel represents a single stem cell in interphase. Scale bars: 5 µm. Note that the 2K peptide altered protein localization of NS4A and NS4B, whereas NS5 was localized in the nucleus. Images are representative of at least 20 animals per genotype.
Fig. 6.
Fig. 6.
Expression of some ZIKV proteins causes electrophysiological defects in the fly visual system. (A-C) Representative electroretinograms (ERGs) from control (luciferase) (A) and prM::E (B) and NS4A (C) expressing animals 30 days after eclosion in a 12 h/12 h light/dark cycle. (D) Quantification of depolarization amplitude showed reduced depolarization amplitude in animals with neuronal expression of prM::E. (E,F) Quantification of on/off transient amplitudes showed loss of on- and off-transients with NS4A expression. (G-L) ERG depolarization amplitude (G,J) and on (H,K) and off (I,L) transient quantification of NS4A-expressing animals at 5 days after eclosion (G-I) and NS4A- and NS4A::2K-expressing animals at 30 days after eclosion (J-L). No defect was documented in 5-day-old animals (G-I), indicating that NS4A causes degenerative ERG defects over time. Comparisons between NS4A alone or with NS4A::2K (J-L) showed that only NS4A alone caused neuronal phenotypes at 30 days after eclosion. (M,N) Transmission electron microscopy of the retina of control animals expressing luciferase (M) or NS4A (N) with Rh1-GAL4 in photoreceptors. Note that NS4A induced loss of photoreceptors, likely corresponding to the decrease in ERG amplitude over time. Images are representative of three animals per genotype. Scale bars: 4 µm. In D-L, data show the mean±s.e.m. One-way ANOVA with multiple comparisons posttest compared to control (luciferase) was used to assess significance. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.
Fig. 7.
Fig. 7.
ZIKV protein expression phenotypes for prM::E and NS1 are different based on the viral strain. (A) Phenotypes when prM::E and NS1 from either the Puerto Rican or Cambodian strains of ZIKV were expressed by various GAL4 drivers. The ‘vs’ column denotes whether one variant is more severe (> or <) or both have equal severity (=). ND, not determined. (B,C) Representative ERG traces of control, prM::Epr- and prM::Ecam-expressing animals at 5 days (B) or 30 days (C) after eclosion. (D,E) Depolarization amplitudes of 5-day-old (D) or 30-day-old (E) animals expressing luciferase (control), prM::Epr or prM:Ecam show developmental ERG defects that are stable with age. The prM::Ecam phenotype is more severe than the prM::Epr phenotype. (F,G) On transient (F) and off transient (G) amplitudes were also reduced with expression of prM::Epr or prM:Ecam shown at 5 days after eclosion. Data show the mean±s.e.m. One-way ANOVA with multiple comparisons posttest compared to control (luciferase) was used to assess significance. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.
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