How SARS-CoV-2 hijacks Fragile X proteins to fuel infection: New clues in COVID-19 and genetic disorders

*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.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 study posted to the bioRxiv preprint* server, an international team of researchers investigated interactions between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) non-structural protein 3 (NSP3) and fragile X mental retardation proteins (FMRPs) and explored their contribution to coronavirus disease 2019 (COVID-19) pathophysiology.

Stress granules, large protein-ribonucleic acid (RNA) assemblies, are crucial for viruses to replicate in host cells. The granules, composed of ribonucleic acid-binding protein molecules such as Ras-GTPase-activating SH3 domain-binding-proteins 1 and 2 (G3BP1/2) and ubiquitin-associated protein 2 (UBAP2L), are essential for antiviral signaling. Fragile X syndrome, a genetic form of mental retardation, is linked to deregulated expression of these proteins. However, the exact molecular basis of Fragile X syndrome remains unknown, and the interactions between viruses and host cells are crucial for viral replication.

About the study

The present study investigated the SARS-CoV-2 non-structural protein 3-FMRPs interaction and its role in COVID-19.

Using SARS-CoV-2 non-structural protein 3, the researchers explored the connections between SARS-CoV-2 and cellular host components. They performed mass spectrometry to compare YFP-tagged NSP3 and control purification. Furthermore, YFP-tagged fragments of NSP3 were produced in Henrietta Lacks (HeLa) cells, followed by immunological purification and Western blot analysis to assess fragile X mental retardation protein 1 (FXR1) binding. They altered blocks of 10 amino acids to Ala in the inherently disordered areas of NSP3 1-181 to find the FMRP binding site.

In all, nine mutants were created, two of which covered the inherently disordered N-terminus and seven of which followed the Ubl1 domain. African green monkey (VeroE6) and human lung adenocarcinoma (Calu3) cells were used to study the effect of the NSP3-FMRP interaction on SARS-CoV-2 infection. Wild-type (WT) viruses demonstrated a 0.50 to 1.30 log drop in SARS-CoV-2 titer after interferon-stimulated genes (ISG) activation in comparison to untreated. NSP3 mutants, on the other hand, were two to three times more sensitive than WT.

To study the contribution of the SARS-CoV-2 non-structural protein 3-FMRP interactions in the in vivo settings, the researchers infected hamsters with the SARS-CoV-2 wild-type strain and non-structural protein 3 mutant, observing weight reduction and illness over seven days. Furthermore, the researchers used AlphaFold multimer to construct a structural model of the complex and evaluated whether NSP3 binding rewires the FMRP interactome.

The team mapped the binding site in UBAP2L to FMRPs and constructed a peptide array encompassing the UBAP2L area with 20mer peptides moved by two amino acids at a time to investigate whether the interactome data indicated that UBAP2L and NSP3 compete for binding to a comparable interface on FMRPs. FXR1 localization was studied in VeroE6 cells to assess the capacity of NSP3 to counteract host cell antiviral systems via an influence on stress granule composition and assembly.

Results

The team discovered a new connection between SARS-CoV-2 non-structural protein 3 and FMR1 and FXR1-2. SARS-CoV-2 NSP3 mutants that prevented FMRP binding slowed proliferation in vitro and delayed illness onset in animals. The I304N mutation in a fragile X syndrome patient impaired the binding of a unique peptide motif in NSP3 to the two main KH domains of FMRPs. Through direct competition with a peptide motif in UBAP2L, NSP3 binding to FMRPs inhibited their interaction with the stress granule component UBAP2L, preventing FMRP integration into stress granules.

The RNA-binding protein FMR1 and the closely related FXR1 and FXR2 proteins were the most notable cellular host factors co-purifying with NSP3. The results characterized the motif in NSP3 binding to FMRPs and showed that it was conserved across the Sarbecovirus family. The viral mutants were weakened in proliferation after 24 hours of infection compared to the SARS-CoV-2 wild-type strain in VeroE6 cells. The diminished capability of non-structural protein 3 mutants in interferon-deficient VeroE6 cells indicated that IFN-stimulated genes (ISGs) may not be responsible for attenuation.

In comparison to WT control animals, hamsters infected with the mut1 as well as mut2 mutants of NPS3 exhibited no significant difference in weight loss or illness. Similarly, histopathology showed that NSP3 mutant infected hamsters had significantly less cellular infiltration and damage than controls. The SARS-CoV-2 non-structural protein 3 sequence containing 20 amino acids was necessary and adequate to interact with FMRPs. A 23mer peptide of NSP3, derived from alphaviruses, showed fragile X mental retardation protein 1 and FXR1-122 binding, indicating that the viruses hijack fragile X mental retardation proteins through a different method. The team identified three NSP3 residues that were critical for FMRP binding, i.e., F145A, G140, and Y138.

The non-structural protein 3 peptide interacted with the FMRP KH2 domain's GxxG motif in the same way as RNA and deoxyribonucleic acid have been demonstrated to bind to KH domains. In the presence of the NSP3 WT peptide, the team found a dramatic shift of constituents of stress granules from FXR1. UBAP2L exhibited a significant decrease in simultaneous purification, which is consistent with an earlier two-hybrid screen that revealed probable direct interactions between FXR1 215 to 360 and UBAP2L. The TOP3B-TDRD3-FMRP complex was found to be displaced, which corresponded to the inability of the fragile X mental retardation protein 1 I304N genetic mutant to bind with NSP3.

The mutant did not bind to stress granule constituents and UBAP2L in cells. The FMRP interaction mechanism was conserved between UBAP2 and UBAP2L, and the NSP3 peptide and UBAP2L peptide competed for isothermal titration calorimetry (ITC) binding to FXR1 260-315. This inhibited the UBAP2L-FMRP interaction, which is required for FMRP attachment with stress granules, and effectively antagonized antiviral defense mechanisms during the early stages of infection.

Overall, the study findings provided unique insights into ways in which SARS-CoV-2 hijacks proteins from host cells for effective infection, as well as molecular explanations for the potential underlying genetic deficiencies in fragile X syndrome.

*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.

Written by

Pooja Toshniwal Paharia

Dr. based clinical-radiological diagnosis and management of oral lesions and conditions and associated maxillofacial disorders.