The YXXΦ motif within the severe acute respiratory syndrome coronavirus (SARS-CoV) 3a protein is crucial for its intracellular transport

Abstract

BackgroundThe SARS coronavirus (SARS-CoV) 3a protein functions as an ion channel, induces apoptosis and is important for viral pathogenesis. It is expressed on the cell surface and contains a tyrosine-based sorting motif and a di-acidic motif, which may be crucial for its intracellular trafficking. However the role of these motifs is not fully understood in the case of 3a protein.MethodsThe subcellular distribution of the 3a protein was studied by immunofluorescence staining of cells transfected with wild type and mutant constructs along with markers for different intracellular compartments. Semi-quantitative RT-PCR was performed to estimate the mRNA where as western blotting was carried out to detect protein levels of wild type and mutant 3a proteins. In vitro transcription- translation was performed to estimate cell free protein synthesis.ResultsWhile the wild type 3a protein is efficiently transported to the plasma membrane, the protein with mutations in the tyrosine and valine residues within the YXXV motif (ΔYXXΦ) accumulated in the Golgi compartment. However the 3a protein with mutations within the EXD di-acidic motif (ΔEXD) showed an intracellular distribution similar to the wild type protein. Increased retention of the ΔYXXΦ protein in the Golgi compartment also increased its association with lipid droplets. The ΔYXXΦ protein also expressed at significantly lower levels compared to the wild type 3a protein, which was reversed with Brefeldin A and Aprotinin.ConclusionsThe data suggest that the YXXΦ motif of the SARS-CoV 3a protein is necessary for Golgi to plasma membrane transport, in the absence of which the protein is targeted to lysosomal degradation compartment via lipid droplets.

Porcine epidemic diarrhea virus ORF3 gene prolongs S-phase, facilitates formation of vesicles and promotes the proliferation of attenuated PEDV

“…The ORF3a protein of SARS-CoV reportedly forms an ion channel and may modulate virus release [14]. Also, the SARS-CoV 3a protein is necessary for Golgi to plasma membrane transport [15]. Considering the similar genome structure between SARS-CoV and PEDV, a previous study adapted same methods used for the SARS-CoV 3a protein to demonstrate that the PEDV ORF3 protein also functioned as an ion channel and regulates virus production [16].…”

Functional Immune Deficiency Syndrome via Intestinal Infection in COVID-19

“…127 ORF3a is an integral transmembrane protein, localized mostly in the Golgi complex, cytoplasm, and cell surface. [128][129][130] Deletion of several accessory proteins including ORF3a do not dramatically decrease viral replication, suggesting they are not essential for viral growth. 131 However, the most significant decrease in viral growth was observed when ORF3a was deleted.…”

“…134 The tyrosine motif Tyr-Asn-Ser-Val (160 -163) was found essential for a proper ORF3a export to the cell surface. 129 Mutations at Tyr-Asn-Ser-Val motif, increase ORF3a retention at Golgi complex and lysosome degradation. 129 Further, the motifs Tyr-Asn-Ser-Val, di-acidic (Glu-Gly-Asp, 172 -173) and cysteine rich motifs, have been associated with apoptotic function of ORF3a.…”

“…129 Mutations at Tyr-Asn-Ser-Val motif, increase ORF3a retention at Golgi complex and lysosome degradation. 129 Further, the motifs Tyr-Asn-Ser-Val, di-acidic (Glu-Gly-Asp, 172 -173) and cysteine rich motifs, have been associated with apoptotic function of ORF3a. 139 Mutations at 176 -274 residues pointed this region to be important to induce cell cycle arrest at G1 stage.…”

A Review of Functional Motifs Utilized by Viruses

“…The 3a protein encoded by severe acute respiratory syndrome-associated coronavirus (SARS-CoV) functions as an ion channel protein [59]. It harbors the Yxxϕ motif, which is necessary for endocytosis, intracellular trafficking, and surface transport of SARS-CoV.…”