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

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.

SUBMITTER: Wong SL

PROVIDER: S-EPMC7117541 | biostudies-literature | 2005 Nov

REPOSITORIES: biostudies-literature

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

Wong S L Alan SL Chen Yiwei Y Chan Chak Ming CM Chan C S Michael CS Chan Paul K S PK Chui Y L YL Fung Kwok Pui KP Waye Mary M Y MM Tsui Stephen K W SK Chan H Y Edwin HY

Biochemical and biophysical research communications 20050926 2

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 pr ...[more]

PMID: 16212942

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Project description:BackgroundA recent publication reported that a tyrosine-dependent sorting signal, present in cytoplasmic tail of the spike protein of most coronaviruses, mediates the intracellular retention of the spike protein. This motif is missing from the spike protein of the severe acute respiratory syndrome-coronavirus (SARS-CoV), resulting in high level of surface expression of the spike protein when it is expressed on its own in vitro.Presentation of the hypothesisIt has been shown that the severe acute respiratory syndrome-coronavirus genome contains open reading frames that encode for proteins with no homologue in other coronaviruses. One of them is the 3a protein, which is expressed during infection in vitro and in vivo. The 3a protein, which contains a tyrosine-dependent sorting signal in its cytoplasmic domain, is expressed on the cell surface and can undergo internalization. In addition, 3a can bind to the spike protein and through this interaction, it may be able to cause the spike protein to become internalized, resulting in a decrease in its surface expression.Testing the hypothesisThe effects of 3a on the internalization of cell surface spike protein can be examined biochemically and the significance of the interplay between these two viral proteins during viral infection can be studied using reverse genetics methodology.Implication of the hypothesisIf this hypothesis is proven, it will indicate that the severe acute respiratory syndrome-coronavirus modulates the surface expression of the spike protein via a different mechanism from other coronaviruses. The interaction between 3a and S, which are expressed from separate subgenomic RNA, would be important for controlling the trafficking properties of S. The cell surface expression of S in infected cells significantly impacts viral assembly, viral spread and viral pathogenesis. Modulation by this unique pathway could confer certain advantages during the replication of the severe acute respiratory syndrome-coronavirus.

Project description:The entry of the SARS coronavirus (SCV) into cells is initiated by binding of its spike envelope glycoprotein (S) to a receptor, ACE2. We and others identified the receptor-binding domain (RBD) by using S fragments of various lengths but all including the amino acid residue 318 and two other potential glycosylation sites. To further characterize the role of glycosylation and identify residues important for its function as an interacting partner of ACE2, we have cloned, expressed and characterized various soluble fragments of S containing RBD, and mutated all potential glycosylation sites and 32 other residues. The shortest of these fragments still able to bind the receptor ACE2 did not include residue 318 (which is a potential glycosylation site), but started at residue 319, and has only two potential glycosylation sites (residues 330 and 357). Mutation of each of these sites to either alanine or glutamine, as well as mutation of residue 318 to alanine in longer fragments resulted in the same decrease of molecular weight (by approximately 3 kDa) suggesting that all glycosylation sites are functional. Simultaneous mutation of all glycosylation sites resulted in lack of expression suggesting that at least one glycosylation site (any of the three) is required for expression. Glycosylation did not affect binding to ACE2. Alanine scanning mutagenesis of the fragment S319-518 resulted in the identification of ten residues (K390, R426, D429, T431, I455, N473, F483, Q492, Y494, R495) that significantly reduced binding to ACE2, and one residue (D393) that appears to increase binding. Mutation of residue T431 reduced binding by about 2-fold, and mutation of the other eight residues--by more than 10-fold. Analysis of these data and the mapping of these mutations on the recently determined crystal structure of a fragment containing the RBD complexed to ACE2 (Li, F, Li, W, Farzan, M, and Harrison, S. C., submitted) suggested the existence of two hot spots on the S RBD surface, R426 and N473, which are likely to contribute significant portion of the binding energy. The finding that most of the mutations (23 out of 34 including glycosylation sites) do not affect the RBD binding function indicates possible mechanisms for evasion of immune responses.

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

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

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