SARS-CoV-2 spike protein and messenger ribonucleic acid found to translocate into the nucleus

In a recent study posted to the bioRxiv* preprint server, researchers reported severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) spike (S) protein and S messenger ribonucleic acid (mRNA) nuclear co-translocation as a novel feature of SARS-CoV-2 pathogenesis.

Study: Nuclear translocation of spike mRNA and protein is a novel pathogenic feature of SARS-CoV-2. Image Credit: Naeblys/Shutterstock
Study: Nuclear translocation of spike mRNA and protein is a novel pathogenic feature of SARS-CoV-2. Image Credit: Naeblys/Shutterstock

Background

The causative organism of coronavirus disease 2019 (COVID-19), SARS-CoV-2, has caused severe pathophysiological alterations among elder and vulnerable individuals and is reported to have greater transmissibility than severe acute respiratory syndrome coronavirus (SARS-CoV) or Middle East respiratory syndrome CoV (MERS-CoV).

SARS-CoV-2 S protein has been documented as a key factor for SARS-CoV-2 pathogenicity due to its broad tropism for the human angiotensin-converting enzyme 2 (hACE2) receptor.

Studies have investigated SARS-CoV-2 protein subcellular localization in vitro; however, comprehensive data on the S glycoprotein needs are limited and require further investigation.

About the study

In the present study, researchers explored S and S mRNA nuclear translocation as underlying mechanisms of SARS-CoV-2 pathogenicity.

Primary normal human bronchial epithelial (NHBE) cells were obtained from healthy non-smoker individuals and chronic obstructive pulmonary disease (COPD) patients to form a pseudostratified bronchial airway epithelium. The airway cells were infected with the SARS-CoV-2 USA/WA-CDC-WA1/2020 strain, SARS-CoV Urbani strain, and MERS-CoV for in vitro experiments.

The team investigated whether SARS-CoV-2 S translocated into the SARS-CoV-2-infected airway epithelial cell nucleus and colocalized with S mRNA. The cells were subjected to immunohistochemistry (IHC) analysis and were examined by confocal microscopy (CFM). Immunofluorescence analysis was performed for SARS-CoV-2 protein and S mRNA detection.

S sequences of both the viruses were aligned by the multiple sequence alignment (MSA) technique to determine whether the SARS-CoV-2 isolate has multiple novel sequence insertions (SIs) in S compared to the Urbani strain of SARS-CoV. Further, SARS-CoV-2 S was analyzed using in silico analysis to investigate whether S resembled or constituted protein motifs such as a nuclear localization signal (NLS) and to study the interactions between SARS-CoV-2 RNA and the S and nucleocapsid (N) proteins.

NLS estimation was performed for several pathogenic CoV S proteins. The team investigated whether the “RRAR” polybasic site could be an NLS motif and whether the NLS motif was functional concerning the polybasic RRAR site at the boundary between S subunits 1 (S1) and 2 (S2).

Results

Only SARS-CoV-2 S contained a functional NLS motif “PRRARSV” of pat7-type at the S1/S2 boundary due to the presence of the novel ‘NSPR’ SI and drove nuclear translocation of S protein (and S mRNA) in the airway epithelial cells infected by SARS-CoV-2. SARS-CoV-2 S mRNA was found to be nuclear (<10%) and abundant (~90%) in the cytoplasm, indicative of mRNA transition. In less than one percent of cases, complete translocation of S protein mRNA was observed.

Intracellular S and S protein mRNA distribution was observed, indicative of nuclear translocation, involving the outer surface and the inside of the nucleus. S and S protein mRNA colocalized and formed a protein-mRNA complex in the infected cells, 85% of which was detected outside the nucleus. The N proteins of MERS-CoV, SARS-CoV, and SARS-CoV-2 demonstrated nuclear translocation.

S translocated into the nucleus (25%) of the airway cells through the cytoplasmic endoplasmic reticulum (ER)-Golgi apparatus pathway, and 15% of the S protein was detected at the nuclear surface, indicative of a transitionary stage in protein translocation. Of note, the nuclear translocation of S mRNA located on the nucleus's surface was associated with and assisted by SARS-CoV-2 S. In contrast, S mRNA present in the cytoplasmic did not show such associations. NLS-facilitated translocations into the nucleus were observed for MERS-CoV, SARS-CoV, and SARS-CoV-2.

Conclusions

Overall, the study findings showed nuclear translocation and co-localization of the S glycoprotein and S mRNA, a novel mechanism of SARS-CoV-2 pathogenesis. Further, the S glycoprotein mRNA nuclear translocation was facilitated by the S protein due to the presence of the functional pat7 NLS “PRRARSV” motif in SARS-CoV-2 S.

The findings could aid the development of effective S-targeted agents to widen the therapeutic landscape of COVID-19.

The novel SARS-CoV-2 nuclear translocation indicates that S protein surface expression may be reduced, but its impact on host immune recognition needs to be determined. S and S mRNA colocalization indicate that the SARS-CoV-2 S may contain an RNA-binding motif and therefore, must be investigated further.

*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 behaviour, or treated as established information.

Journal reference:
Pooja Toshniwal Paharia

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Pooja Toshniwal Paharia

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

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