SARS-CoV-2 Spike Proteins And mRNAs Can Translocate Into The Nucleus Of Host Cells Unlike Any Other Coronaviruses

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A new study involving researchers from the U.S.NIH, US. NIAID, University of North Dakota School of Medicine & Health and University of Nebraska Medical Center have alarmingly found that unlike any other known coronaviruses, the SARS-CoV-2 spike proteins and S mRNAs are able to translocate into the nucleus of human host cells and contribute to pathogenesis.

The study findings were published on a preprint server and are currently being peer reviewed.
https://www.biorxiv.org/content/10.1101/2022.09.27.509633v1
 

In the context of SARS-CoV, one of the controversies regarding the natural origin of SARS-CoV-2 is that its S gene has multiple novel sequence insertions. Zhang C. et al. analyzed the report by Pradhan et al. (withdrawn) [41] on the presence of four unique novel sequences in the SARS-CoV-2 S gene and showed that these four sequence insertions were not related to the receptor-binding domain (RBD) [21].

A recent study identified S gene novel sequence insertions among several key genomic features that differentiate SARS-CoV-2 from other beta-coronaviruses, particularly SARS-CoV and MERS-CoV [14]. The source and characterization of these sequence insertions have yet to be determined; however, the closest BLAST hit of these sequences is bat coronavirus RaTG13 [42].

Similar to a previous report [21], we found four multiple sequence insertions in the SARS-CoV-2 S protein: IS1 “GTNGKTR”, IS2 “YYHK”, “HRSY”, and IS4 “NSPR” (Fig. 1).

Here, we showed that the fourth novel sequence insertion in the S gene was an NLS and resulted in nuclear translocation of the S protein, which not only complemented previous in silico findings [14] but also identified a novel pathogenic genomic feature of the S gene. Interestingly, the fourth significant insertion has received attention due to the description of a polybasic site “RRAR”, which may contribute to increased serin protease-driven entry of SARS-CoV-2 [19] and is implicated in broader tropism and/or enhanced viral transmissibility compared to SARS-CoV [20].

However, we found that the IS4 “NSPR” created a pat7 NLS “PRRARSV” in the S protein, which was unique to SARS-CoV-2. We first reported that the S protein translocated into the nucleus in the SARS-CoV-2-infected airway epithelium, which is an appropriate lung model for studying respiratory virus infection in vitro [2, 43].

Our results confirmed that the SARS-CoV-2 S protein was a unique addition to the list of viral proteins that possess NLSs and consequently translocate into the nucleus of infected cells [17, 30, 31, 44].

Among coronaviruses, SARS-CoV-2 S protein is the first type-1 transmembrane glycoprotein that translocates into the nucleus. Vesicular stomatitis virus (VSV), which is a negative sense RNA virus, has a glycoprotein that translocates to the nucleus as well. A study by the University of Illinois at Urbana Champaign showed exactly how the glycoprotein on VSV was able to travel to the nucleus of hamster kidney cells [45].

NLS-driven S protein nuclear translocation is a novel pathogenic feature of SARS-CoV-2 infection compared to other pathogenic coronaviruses. However, the pathogenic contribution of the S protein’s NLS motif to virus-induced pathophysiology is yet to be determined. Our results suggested that the S protein translocated into the nucleus due to the NLS, which also raised two important points.

First, we investigated whether the proposed polybasic site “RRAR” could itself be an NLS motif. The answer was that the proposed polybasic site was not an NLS motif because an NLS is a well characterized and predefined amino acid sequence motif [15-17]. Additionally, the amino acid sequence of the probable sequence insertion “NSPR” [21] was also not an NLS but was part of the P7 “PRRARSV” NLS. Thus, the inserted sequence creates the NLS in the S protein of SARS-CoV-2 and may make SARS-CoV-2 unique among human pathogenic coronaviruses.

The second important point was whether the NLS motif was functional in the context of the described polybasic site at the S1/S2 boundary. All type-1 transmembrane glycoproteins are processed through the ER-Golgi pathway before signal peptide-driven cellular surface localization. The proposed polybasic site was functional (availability to proteases) when the S protein was on the virion for host cell entry.

A fully posttranslationally processed S protein surface translocation could also provide a polybasic site to be processed by furin cleavage. However, there is no information on the availability or usability of the S protein’s polybasic site by furin proteases in the cytoplasm before virus assembly. Thus, the NLS is functional in SARS-CoV-2-infected cells, and the polybasic site only functions during the viral entry step.

The NLS is obviously functional in infected cells, and no furin cleavage at the polybasic site is necessary other than for viral entry. Our results confirmed that the S protein NLS motif was functional in SARS-CoV-2-infected cells. Although mutating the polybasic site (which also mutated the NLS) may impact viral S protein function in vitro, the result will not confirm or deny that one is more important than the other between the polybasic site and the NLS. While our result does provide direct evidence for the presence of the NLS motif and nuclear translocation of the S protein, our results do not confirm nor deny that the NSPR sequence has a natural origin.

Instead, our results showed that the inserted sequence NSPR was a functional NLS motif, which increased the intracellular distribution of the S protein, including novel nuclear translocation. The novel nuclear translocation of the SARS-CoV-2 S protein suggests that: 1. the nuclear translocation of the S protein reduces its surface expression, but whether it contributes to evading host immune recognition remains to be determined; and 2. the colocalization of the S protein with S mRNA suggests that the S protein has an RNA binding motif, which remains to be determined. One of the important ways of confirming a functional NLS motif is to use site-directed mutational analysis.

Plasmid-driven transient expression of S protein in the human lung airway A549 cell line and primary normal human branchial epithelial cells showed robust S expression but was toxic to the cells. Therefore, the success of site-directed mutational analysis of the S protein in a transient expression system is doubtful and the characterization of NLS by a mutational analysis is yet to be determined. Thus, our novel findings emphasize further research on the NLS motif of the SARS-CoV-2 S protein.

One of the most important findings in our study was the simultaneous detection of the different spatial distributions of S protein and S mRNA at the single-molecule level in a single infected cell. We confirmed that S mRNA translocated into the nucleus by image analysis of the colocalization of S mRNA with nuclear staining.

The SARS-CoV-2 N protein has already been shown to bind to RNA [46]. There was no information available confirming whether the S protein could bind to S mRNA for nuclear translocation. Our results revealed that S mRNA nuclear translocation was mediated by the S protein because S mRNA nuclear translocation was always associated with the S protein.

For example, S mRNA colocalized with the S protein inside and outside the surface of the nucleus. Although the primer-probe was designed to target S mRNA, the SARS-CoV-2 positive-strand RNA genome (whole or partial) can be targeted by the same probe due to the sequence similarity between S mRNA and the whole or partial genome. Thus, our results lack sufficient detail contributing to the discussion of the controversial scientific topic of whether there is any possibility of SARS-CoV-2 genome integration into the host DNA [47, 48].

Additionally, one of the significant differences in the S protein sequences of SARS-CoV and SARS-CoV-2 is the pat7 NLS motif. Whether S protein expression by the current vaccine platforms causes suboptimal expression of S protein on the cell surface due to the NLS remains to be determined [49].

In conclusion, the SARS-CoV-2 S protein has a functional pat7 NLS “PRRARSV”, that results in one out of four S proteins translocating into the nucleus in infected cells. S Protein appears to shuttle S mRNA (possibly the genome) into the nucleus as well. Thus, the NLS of the S protein may contribute to the evasion of the host immune response and is a novel pathogenic feature of SARS-CoV-2.

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