Interactions between SIRT5 and SARS-CoV-2 Nsp14 protein
In a recent study published in the PLOS Pathogens journal, researchers investigated the interactions between sirtuin 5 (SIRT5) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nonstructural protein (Nsp14) protein.
The highly conserved enzyme called SARS-CoV-2 Nsp14 is essential for viral replication. The non-structural protein Nsp14 has exoribonuclease and N7-methyltransferase activity and forms a stable complex with Nsp10. Human SIRT5 has been found in protein-interactome investigations as a potential Nsp14 binding partner.
The nicotinamide adenine dinucleotide (NAD) -dependent protein deacylase SIRT5 has been found to strip succinyl and malonyl groups from lysine residues, which is essential for cellular metabolism.
About the study
In the present study, researchers assessed the characteristics of SARS-CoV-2 Nsp14 and SIRT5 interaction and SIRT5's function in SARS-CoV-2 infection.
The team first validated and defined the nature of Nsp14-SIRT5 interaction. The Nsp14-strep mammalian expression vector was employed for affinity purification. After being transfected into HEK-293T cells with either Nsp14-strep or a green fluorescent protein (GFP) control for 48 hours, tagged proteins were extracted.
The alterations in the thermal stability of Nsp14 and SIRT5 in viable cells were then measured using the cellular thermal shift assay (CETSA). The shift in thermal stability was measured by western blotting after Nsp14 and SIRT5 expression plasmids were transfected into HEK-293T cells, either separately or together.
The study also determined whether Nsp10 and SIRT5 were components of separate complexes or whether they interacted as well. Using clustered regularly interspaced short palindromic repeats (CRISPR) interference in HEK-293T cells, the team created a SIRT5 knockdown cell line (SIRT5-KD) to remove endogenous expression of SIRT5. A single guide ribonucleic acid (RNA) was chosen after testing several others throughout the investigation. In SIRT5-KD cells, expression plasmids corresponding to SIRT5, Nsp14-strep, and Nsp10 with a Flag tag (Nsp10-Flag) were transfected singly or collectively for 48 hours. Removing a potential succinyl, malonyl, or glutaryl group from Nsp14 was the main hypothesis for how SIRT5 could alter the Nsp14 protein.
The study showed that western blots confirmed previous mass spectrometry findings by demonstrating that SIRT5 was particularly co-purifying with Nsp14. Nsp14 and SIRT5 co-localized into the same cellular compartments, with a predominance of cytoplasmic and perinuclear localization, as further demonstrated by immunofluorescence observed in human alveolar basal epithelial A549 cells transfected with Nsp14 expression plasmid.
When the two proteins were co-transfected, researchers noticed a significant improvement in the stability of both proteins. When transfected alone, Nsp14 was poorly expressed and scarcely visible. However, SIRT5 enabled a robust signal. Altogether, these preliminary findings demonstrated that SIRT5 and Nsp14 interacted in human cells and that SIRT5 significantly stabilized Nsp14 expression.
Strep affinity purification (Strep-AP) confirmed SIRT5 and Nsp10 interactions with Nsp14. However, using Flag-IP to remove Nsp10 revealed that only Nsp14 co-purified with Nsp10. This demonstrated that Nsp10 and SIRT5 did not interact. The team also hypothesized that Nsp10 and SIRT5 were in direct competition for Nsp14 binding because the SIRT5 signal after Strep-AP appeared to be diminished in the presence of Nsp10. Because SIRT5 and Nsp10 could competitively bind to Nsp14, it was predicted that Nsp14/SIRT5 and Nsp14/Nsp10 would form different complexes.
The team also observed that genes such as Y102 and T105 interacted with the extended acidic chains of succinyl or malonyl groups, Q140 and I142 were involved in NAD binding, and H158 was catalytically required to remove a proton from NAD. H158, Q140, and I142 are conserved in all sirtuins; however, SIRT5-specific Y102 and T105 mediated the selectivity to longer-chain acidic groups.
SIRT5 binding was completely lost in the H158Y and Q140A mutants and was only partially recovered in the Y102F mutation. This finding demonstrated that the interaction with Nsp14 required an intact SIRT5 catalytic domain. The stability of the overexpressed catalytic mutants was unaffected by the treatment of HEK293T cells with the proteasome inhibitor MG-132, which indicated that the various proteins could fold correctly.
The team observed that SIRT1 and Nsp14 interacted, but no interactions were observed with SIRT2, SIRT3, SIRT6, or SIRT7. The SIRT1 signal seemed to be weaker than the SIRT5 signal. Notably, similar to the SIRT5 findings, the mutation of the SIRT1 catalytic domain or treatment of cells with Ex-527, a SIRT1 inhibitor, blocked the interaction. This indicated that SIRT1 catalytic activity was essential for Nsp14 interaction. On the other hand, treatment of cells with a specific SIRT1 activator like SRT1720 or a non-specific activator like Resveratrol did not result in any evaluable impact on Nsp14 binding. While both these activators showed cellular toxicity at high concentrations, the reduction in binding noted after affinity purification was associated with decreased levels of SIRT5 and Nsp14 in input lanes.
The team also observed that SIRT5 was not present in SIRT5-KO cells. RT-qPCR measured viral RNA after three days in SARS-CoV-2 wildtype (WT) and SIRT5-KO cells following infection with SARS-CoV-2 Wuhan strain at an MOI of 0.1 and 1. Notably, a two-to-three-fold reduction was also found in SARS-CoV-2 mRNA in SIRT5-KO cells at both MOIs. These findings suggested that SIRT5 was essential for the replication and/or spread of SARS-CoV-2.
Overall, the study findings demonstrated that SIRT5 interacted with the viral protein Nsp14 and that this interaction occurred without the involvement of Nsp10.
- Walter M, Chen IP, Vallejo-Gracia A, Kim IJ, Bielska O, et al. (2022). SIRT5 is a proviral factor that interacts with SARS-CoV-2 Nsp14 protein. PLOS Pathogens. doi: https://doi.org/10.1371/journal.ppat.1010811 https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1010811
Posted in: Medical Science News | Medical Research News | Disease/Infection News
Tags: Adenine, Assay, Cell, Cell Line, Coronavirus, Coronavirus Disease COVID-19, CRISPR, Enzyme, Fluorescent Protein, Genes, Interactome, Lysine, Mass Spectrometry, Metabolism, Mutation, Palindromic Repeats, Plasmid, Proteasome Inhibitor, Protein, Respiratory, Resveratrol, Ribonucleic Acid, RNA, SARS, SARS-CoV-2, Severe Acute Respiratory, Severe Acute Respiratory Syndrome, Spectrometry, Structural Protein, Syndrome
Bhavana Kunkalikar is a medical writer based in Goa, India. Her academic background is in Pharmaceutical sciences and she holds a Bachelor's degree in Pharmacy. Her educational background allowed her to foster an interest in anatomical and physiological sciences. Her college project work based on ‘The manifestations and causes of sickle cell anemia’ formed the stepping stone to a life-long fascination with human pathophysiology.
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