SARS-CoV-2 hijacks cholesterol trafficking to fuel infection and evade immune responses

Researchers uncover how SARS-CoV-2 rewires cholesterol metabolism, highlighting a novel target to combat COVID-19's metabolic and cardiovascular effects.

Study: Manipulation of Host Cholesterol by SARS-CoV-2. Image Credit: Kateryna Kon / Shutterstock

*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

In a recent research paper posted to the bioRxiv preprint* server, researchers in the United States investigated the potential effect of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on cholesterol metabolism, focusing on the role of the viral protein open reading frame 3a (ORF3a).

They found that SARS-CoV-2 causes cholesterol sequestration in lysosomes via the ORF3a protein, which disrupts protein trafficking and reduces the levels of bis(monoacylglycero)phosphate (BMP) in the cell, enhancing viral survival.

Background

Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, disrupts lipid metabolism, particularly cholesterol homeostasis, which can persist during and after infection. This is linked to disease severity and long-term complications like dyslipidemia and cardiovascular diseases.

Cholesterol is crucial for cellular function and is primarily transported through lysosomes, where proteins like Niemann-Pick C1 and C2 (NPC1 and NPC2) facilitate its release. SARS-CoV-2 may exploit plasma membrane cholesterol to enhance infectivity.

Disruptions in the lysosomal cholesterol pathway can cause cholesterol buildup, impairing cellular functions, and viruses like Ebola are known to hijack this mechanism. Notably, BMP plays a dual role: it aids in cholesterol transport and contributes to viral infection by promoting viral fusion with lysosomal membranes.

In the present study, researchers investigated the potential impact of SARS-CoV-2 infection on cholesterol transport in cells, focusing on the role of the viral protein ORF3a.

About the Study

A variety of experimental techniques were employed, including culturing A549, HeLa, and Vero E6 cells, followed by SARS-CoV-2 infection at different multiplicities of infection. SARS-CoV-2 ORF3a-VPS39 interaction was studied using mutations at key residues (notably W193 and Y184, which were identified as critical for this interaction). Immunofluorescence, filipin staining, and confocal microscopy were used to assess cholesterol localization and vesicular dynamics, while high-content imaging quantified cell-specific responses.

Cholesterol levels were measured using gas chromatography-mass spectrometry (GC-MS), and lipid species were analyzed through shotgun lipidomics. For further protein analysis, western blotting was performed to detect secreted NPC2 and cathepsin D, along with cell lysates. Data were analyzed using ImageJ and Prism 9, and statistical significance was determined by t-tests or analysis of variance.

Results and Discussion

SARS-CoV-2 infection was found to increase filipin-positive puncta in lysosomes of A549-hACE2 and Vero E6 cells, indicating altered cholesterol distribution, especially in lysosomes, without affecting total cholesterol levels. Among the 28 viral proteins tested, ORF3a showed the strongest increase in filipin puncta, suggesting significant lysosomal cholesterol sequestration.

Notably, SARS-CoV-2 ORF3a localized to lysosomes and caused them to swell, whereas SARS-CoV ORF3a did not induce such effects, highlighting a distinct pathogenic strategy unique to SARS-CoV-2.

ORF3a was found to interact with VPS39, a key component of the HOPS complex involved in cholesterol egress from lysosomes. Key residues W193 and Y184 were shown to form a hydrophobic binding interface critical for this interaction, distinguishing SARS-CoV-2 ORF3a from its SARS-CoV counterpart. Mutations at W193 and Y184 disrupted this interaction, while S171 and H182 had no significant effect.

SARS-CoV-2 ORF3a expression was shown to cause cholesterol accumulation in lysosomes, which was reduced by the W193A mutation. It also led to the mislocalization of NPC2 and increased its secretion, indicating disrupted NPC2 trafficking, likely due to interference with TGN-to-endosome transport. Additionally, BMP levels were significantly reduced in infected cells, which likely exacerbates lysosomal cholesterol sequestration.

In SARS-CoV-2-infected Vero E6 cells, BMP levels were found to decrease at 12 hours post-infection, coinciding with increased cholesterol at 18 hours. In HeLa-Flp-In cells, SARS-CoV-2 ORF3a was found to reduce BMP levels by 20%, with partial rescue in the W193A mutant. Lipidomics confirmed this reduction, correlating BMP loss with cholesterol accumulation and suggesting BMP reduction may contribute to cholesterol sequestration.

SARS-CoV-2 may reduce plasma membrane cholesterol to limit secondary infections, as shown by decreased SARS-CoV-2 infection in NPC1 inhibitor-treated cells. This supports the hypothesis that the virus manipulates cholesterol distribution to optimize replication conditions. Interestingly, the virus also appears to reduce its own infectivity within a single cell, suggesting a self-regulating mechanism to prevent viral overload and ensure broader host-level spread.

Scheme of the pathways participating in lysosomal cholesterol egress. In healthy cells, lysosomes process endocytosed low-density lipoproteins (LDLs) to release free cholesterol, which is exported via the cholesterol transporters Niemann-Pick C 2 (NPC2) and NPC1. NPC2 also binds with the phospholipid bis(monoacylglycero)phosphate (BMP) to export cholesterol independently of NPC1. Newly synthesized NPC2 associates with the sorting receptor mannose-6-phosphate receptors, such as CI-MPR, in the trans-Golgi network (TGN) and is delivered to late endosomes. Here, CI-MPR is retrieved and recycled back to the TGN with help from the HOPS complex. In SARS-CoV-2-infected cells, ORF3a interacts with the HOPS subunit VPS39, disrupting CI-MPR recycling. This leads to NPC2 trafficking defects, i.e., mislocalization and increased secretion. Additionally, the ORF3a-VPS39 interaction reduces BMP levels by an undefined mechanism, together resulting in cholesterol sequestration within lysosomes.

Conclusion

In conclusion, a novel mechanism by which SARS-CoV-2 disrupts host cell lipid metabolism, specifically through cholesterol sequestration in lysosomes, has been elucidated. By uncovering the specific interaction between the viral protein ORF3a and host protein VPS39, the study highlights a critical role of lysosomal cholesterol trafficking disruption in SARS-CoV-2 pathogenesis.

This discovery opens potential therapeutic avenues to target lipid dysregulation in COVID-19, which could help mitigate both the disease's immediate and long-term metabolic consequences, including dyslipidemia and cardiovascular complications.

*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.