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. 2005 Nov 18;337(2):720-9.
doi: 10.1016/j.bbrc.2005.09.098. Epub 2005 Sep 26.

In vivo functional characterization of the SARS-Coronavirus 3a protein in Drosophila

S L Alan Wong  1 , Yiwei Chen, Chak Ming Chan, C S Michael Chan, Paul K S Chan, Y L Chui, Kwok Pui Fung, Mary M Y Waye, Stephen K W Tsui, H Y Edwin Chan
Affiliations

In vivo functional characterization of the SARS-Coronavirus 3a protein in Drosophila

S L Alan Wong et al. Biochem Biophys Res Commun. .

Abstract

The Severe Acute Respiratory Syndrome-Coronavirus (SARS-CoV) 3a locus encodes a 274 a.a. novel protein, and its expression has been confirmed in SARS patients. To study functional roles of 3a, we established a transgenic fly model for the SARS-CoV 3a gene. Misexpression of 3a in Drosophila caused a dominant rough eye phenotype. Using a specific monoclonal antibody, we demonstrated that the 3a protein displayed a punctate cytoplasmic localization in Drosophila as in SARS-CoV-infected cells. We provide genetic evidence to support that 3a is functionally related to clathrin-mediated endocytosis. We further found that 3a misexpression induces apoptosis, which could be modulated by cellular cytochrome c levels and caspase activity. From a forward genetic screen, 78 dominant 3a modifying loci were recovered and the identity of these modifiers revealed that the severity of the 3a-induced rough eye phenotype depends on multiple cellular processes including gene transcriptional regulation.

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Figures

Fig. 1
Fig. 1
Misexpression of the SARS-CoV 3a gene in Drosophila. (A,B) Misexpression of the SARS-CoV 3a gene caused external eye disruption in adult Drosophila. Misexpression of the control EGFP transgene (A) showed normal external eye morphology, whereas misexpression of the SARS-CoV EGFP-3a transgene (B) caused a rough eye phenotype as characterized by loss of regularity of the external eye structure. (C–G) Subcellular localization of the EGFP-3a fusion protein in Drosophila. Misexpression of the EGFP control protein showed homogeneous intracellular green fluorescence signals in both third instar eye imaginal disc (C) and salivary gland (E) cells. A distinct punctate cytoplasmic expression pattern of the EGFP-3a fusion protein was observed in both eye imaginal disc (D) and salivary gland cells (F). (G) Location of salivary gland cell nuclei of (F). Propidium iodide (PI) was used to stain cell nuclei in eye imaginal disc cells (C,D).
Fig. 2
Fig. 2
Genes involved in endocytosis are related to the EGFP-3a-induced rough eye phenotype. Unlike the gmr-GAL4 control (A), EGFP-3a misexpression resulted in a rough eye phenotype (B). A mutant allele of the endocytic gene Eps15 (Eps15EP2513) dominantly suppressed the EGFP-3a-induced rough eye phenotype (C). Nedd4 monoubiquitinates Eps15 and such modification is essential for Eps15 function. A P-element insert line in the Nedd4 locus (Nedd4EY00500) showed dominant suppression of the EGFP-3a phenotype (D).
Fig. 3
Fig. 3
Apoptotic pathway is related to the EGFP-3a-induced rough eye phenotype. (A–F) Overexpression of anti-apoptotic genes suppresses the EGFP-3a-induced rough eye phenotype. gmr-GAL4 alone (A) and misexpression of EGFP-lacZ fusion protein by gmr-GAL4 (B) showed no external eye deformation. Overexpression of the pro-apoptotic gene reaper caused a rough eye phenotype and reduction in eye size (C). The EGFP-3a-induced rough eye phenotype (D) was suppressed when coexpressed with either P35 (E) or DIAP1 (F). (G–M) Coexpression of anti-apoptotic genes reduces the number of apoptotic cells in third instar larval eye discs. Comparable numbers of acridine orange-positive apoptotic cells were observed in gmr-GAL4 control eye discs (G) and discs misexpressed with a GFP-lacZ fusion protein (H). Expression of both reaper (I) and EGFP-3a (J) showed increased number of acridine orange-positive cells in eye discs. The EGFP-3a-induced apoptosis was suppressed by coexpression of baculoviral anti-apoptotic genes P35 (K), DIAP1 (L); and cytochrome c dc3 (M). Arrows indicate the location of the morphological furrows.
Fig. 4
Fig. 4
Cellular cytochrome c levels correlate with EGFP-3a-induced rough eye phenotype in Drosophila. Misexpression of EGFP-3a, but not EGFP (A), caused a rough eye phenotype (B). A chromosomal deletion Df(2L)Exel6039 of the cytochrome c gene region enhanced the EGFP-3a phenotype (C). In contrast, a P-element insert line, dc3EP2305, in the dc3 locus suppressed (D) the EGFP-3a-induced rough eye phenotype. (E) Genomic structure of cytochrome c genes in Drosophila shows that a P-element insert line, EP2305, is located in the 5′ untranslated region of the dc3 locus. (F) Western blot analysis showed a significant reduction of cytochrome c protein levels in the deletion line, Df(2L)Exel6039. (G) RT-PCR analysis showed overexpression of the dc3 gene in the P-element insert line, dc3EP2305. (H) Immunofluorescence of cytochrome c protein in salivary gland cells. Overexpressed cytochrome c protein, via dc3EP2305, was only detected by antibodies that recognize the denatured, but not native, cytochrome c.
Fig. 5
Fig. 5
EGFP-3a interacts with gene transcription regulatory machineries. Misexpression of the SARS-CoV 3a gene disrupted the normal external eye structure (A; gmr-GAL4) and caused a rough eye phenotype in Drosophila (B). The EGFP-3a-induced rough eye phenotype was dominantly suppressed by a chromosome deletion Df(3R)Exel8157 (C) which uncovers the 87D8-10 region, and by a null allele of C-terminal-binding protein (CtBP87De-10) (D). The EGFP-3a-induced rough eye phenotype was suppressed by the Ubc9 mutant lesswright (lwr02858) which is an E2 enzyme that SUMOylates CtBP (E). A P-element allele of Suppressor of hairless (Su(H)EY07695), inserted in the 5′ untranslated region of Su(H), dominantly suppressed EGFP-3a-induced rough eye phenotype (F).
Fig. 6
Fig. 6
extra macrochaetae dominantly modifies EGFP-3a-induced rough eye phenotype. Misexpression of EGFP-3a caused a rough eye phenotype (A). A chromosomal deletion Df(3L)Exel6086 which uncovers the extra macrochaetae (emc) genomic region 61C9 (B), and an UAS-emc line (C) showed dominant suppression of the EGFP-3a phenotype. (D) RT-PCR analysis showed misexpression of EGFP-3a caused down-regulation of emc gene transcription.
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