Scientists in the USA – from the Johns Hopkins University and the University of Maryland – have recently uncovered the mechanism by which severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) is released from the infected cells. They have demonstrated that the open reading frame 3a (ORF3a) protein of SARS CoV-1 induces the viral release process (egress) by disrupting Golgi morphology, reducing cargo trafficking, and neutralizing Golgi pH. The study is currently available on the bioRxiv* preprint server.
*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
Background
Human coronaviruses are enveloped RNA viruses that assemble by budding into the endoplasmic reticulum (ER) – Golgi intermediate compartment. Although the mechanism of viral egress is not fully known, there is evidence indicating that proteins present on the viral envelope such as the spike, membrane, and envelope proteins play essential roles in the egress process. For instance, the envelope protein of coronaviruses is a viroporin that acts as an ion channel and facilitates viral egress.
In cells infected with infectious bronchitis virus (IBV), it has been observed that the envelope protein triggers the viral egress process by fragmenting and neutralizing the Golgi complex and reducing cargo trafficking. In addition, the envelope protein has been found to protect the viral spike protein from aberrant proteolysis.
Unlike IBV, which is a gamma coronavirus, human coronaviruses belong to the alpha and beta genera. All lethal human coronaviruses, such as SARS-CoV-1, SARS-CoV-2, and Middle East respiratory syndrome coronavirus (MERS-CoV), belong to the beta genus. Previous studies on coronavirus egress have shown that, unlike IBV, the envelope protein of SARS-CoV-1 is not involved in Golgi neutralization and fragmentation to facilitate viral egress. In the current study, the scientists have investigated the mechanism of SARS-CoV-1 egress.
Study design
The scientists specifically examined the role of viral accessory protein ORF3a in the egress process, because transient overexpression of this protein has been found to disrupt the Golgi morphology. Moreover, similar to the envelope protein, ORF3a possesses ion channel activity which is known to associate with viral egress.
To examine cargo trafficking and measure Golgi luminal pH, they conducted a series of experiments using African green monkey kidney cells transiently overexpressing SARS-CoV-1 ORF3a, IBV envelope protein (positive control), or IBV membrane protein (negative control).
Important observations
The analysis of Golgi morphology revealed that ORF3a is transported through the Golgi to the plasma membrane, whereas both IBV membrane and envelope proteins are located in the Golgi region. Dispersion of Golgi complex proteins was observed in cells overexpressing ORF3a or envelope protein. However, the dispersion caused by ORF3a was less extensive compared to that by IBV envelope protein. The scientists hypothesized that since the envelope protein is tightly placed in the Golgi region, its impact on dispersion is more widespread.
To validate this hypothesis, they produced a mutant form of ORF3a, which is expected to remain in the Golgi region. However, they failed to validate their hypothesis because the mutant ORF3a was retained in the ER instead of the Golgi region.
To investigate cargo trafficking, they examined carbohydrate processing and surface expression of the vesicular stomatitis virus glycoprotein (VSV-G), which is a membrane protein processed in the Golgi during its transportation to the plasma membrane. The analysis revealed that both carbohydrate processing and surface expression of VSV-G was reduced in cells expressing SARS-CoV-1 ORF3a or IBV envelope protein. However, the reduction in cargo trafficking was highest in cells expressing IBV envelope protein, an observation similar to the Golgi dispersion effect. The scientists believe that a reduction in virus release due to reduced cargo trafficking may be an acceptable compromise to ensure the retention of viral infectivity.
By analyzing the fluorescence emission spectra of a Golgi-targeted luminal pHlorin molecule by flow cytometry, the scientists observed that the pH of the Golgi lumen increased significantly in cells expressing ORF3a. However, the intensity of pH change was lower than that observed in cells expressing IBV envelope protein. Taken together, these observations suggest that the dispersion of Golgi and reduction in cargo trafficking are due to increased Golgi luminal pH.
With further analysis, the scientists confirmed that, unlike IBV envelope protein, the ion channel activity of SARS-CoV-1 ORF3a protein is required for the Golgi dispersion and cargo trafficking reduction.
In a separate set of experiments, the scientists investigated the effects of SARS-CoV-2 proteins on Golgi morphological alterations. Their analysis revealed that the dispersion of Golgi complex proteins in cells expressing SARS-CoV-2 ORF3a is similar to that observed in cells expressing SARS-CoV-1 ORF3a. Because the amino acid residues associated with the ion channel activity of ORF3a are conserved between SARS-CoV-1 and SARS-CoV-2, the scientists believe that both viruses share a similar mechanism for Golgi morphology disruption.
*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
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