Researchers from the University of Campinas (Unicamp)-Brazil and the University of São Paulo (USP)-Brazil has validated evidence that could have various startling medical and health implications for all those that have been exposed to the SARS-CoV-2 coronavirus.
The study findings were published in the peer reviewed journal: Frontiers in Cellular and Infection Microbiology. https://www.frontiersin.org/articles/10.3389/fcimb.2022.849017/full
The research team explored the previously reported SARS-CoV-2 structural membrane protein (M) interaction with human Proliferating Cell Nuclear Antigen (PCNA).
https://pubmed.ncbi.nlm.nih.gov/32353859/
https://pubmed.ncbi.nlm.nih.gov/33845483/
The SARS-CoV-2 M protein is responsible for maintaining virion shape, and PCNA is a marker of DNA damage which is essential for DNA replication and repair.
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After nearly two years since the start of the COVID-19 pandemic, understanding the underlying mechanisms of SARS-CoV-2 infection and the search for treatment are still in high demand. Large-scale analysis of SARS-CoV-2 human infection interactome indicated several protein-protein interactions that require further validation (Bouhaddou et al., 2020; Gordon et al., 2020; Li et al., 2021; Stukalov et al., 2021).
In this work, we aimed to characterize the interactions of viral proteins and PCNA. According to Gordon et al. (2020), the score of SAINT probability of PCNA for E protein was zero (a low score of interaction probability), and for M was 1.0 (a high score of interaction probability), while PCNA did not appear as an interactor for N protein. Through immunoprecipitation and PLA assays (Figure 1), we validated the M-PCNA interaction.
PLA assay indicates that M-PCNA interaction occurs in the cytoplasm (Figure 1C), and to better characterize it, we performed a co-localization assay by immunofluorescence and subcellular fractioning. Our data indicate that in M expression (Figure 2) or upon SARS-CoV-2 infection (Figures 5A-C), PCNA translocates from nucleus to cytoplasm.
In agreement with PLA data, upon M expression there is also a partial co-localization among M and PCNA in the cytoplasm documented by confocal microscopy (Figure 3 and Supplementary Figure S3). This can be hypothesized as a need for the virus to have PCNA partially translocated from the nucleus to the cytoplasm, and that M is responsible, at least in part, for this translocation in the context of the viral infection.
PCNA acts as a co-factor for DNA polymerases in normal conditions, being essential for DNA replication (O’Donnell et al., 2013; Siddiqui et al., 2013). PCNA also participates in DNA repair, on the metabolism of DNA, and chromatin by recruiting various enzymes, and not only by acting as a scaffold and localizing these factors, but also activating their enzymatic activities (Choe and Moldovan, 2017).
Aside from this, post-translational modifications in PCNA alter its function in different ways (Hoege et al., 2002; Moldovan et al., 2007; Choe and Moldovan, 2017). The interactome described by Stukalov et al. (2021) demonstrated that higher ubiquitination occurs in specific regions of PCNA in SARS-CoV-2 infection, indicating a regulatory mechanism of PCNA by the virus infection (Stukalov et al., 2021).
In cells transfected with FLAG-M (Figure 4) or infected with SARS-CoV-2, we observed an increase of PCNA and γH2AX (Figures 5D–H), and both proteins are associated with DNA repair. We also found a high expression of PCNA and γH2AX in a COVID-19 patient (Figure S4). Li et al. (2021) already reported a high PCNA expression in moderate COVID-19 patients compared to the control group by proteomics analysis (Li et al., 2021). It has been reported that virus infection in human cells can cause DNA damage, inhibiting the association of the DNA polymerase to the DNA stalling and collapsing the replication forks, which results in DNA double-strand breaks (DSB) (Kannouche et al., 2004; Luftig, 2014).
This damage activates a stress response, mediated by checkpoint kinases, which help stabilize and restart the replication forks, thus preventing the generation of DNA damage and genomic instability (Zeman and Cimprich, 2014). In this case, one of the strategies of the cells is to trigger the PCNA ubiquitination. When PCNA is polyubiquitinated, it searches for damaged DNA to assemble the replication complex, with a less specific DNA polymerase (Boehm et al., 2016). This mechanism is known as translesion synthesis (TLS) and is activated to bypass damaged DNA (Kannouche et al., 2004).
Thus, the increase of PCNA could be a strategy for the viral infection to maintain cell viability. Also, after DNA damage, several proteins are activated to manage the DNA lesion. One of them is γH2AX, the phosphorylated form of H2AX, a specific marker of DNA damage that is responsible for recruiting repair proteins to deal with stalled forks (Dickey et al., 2009; Choe et al., 2016), and also increases the expression of p53 and phosphorylation of p53, leading to cell growth inhibition (Dillehay et al., 2014; Dillehay et al., 2015).
PCNA is also found in the cytoplasm of cancer cells. The accumulation of PCNA in the cytoplasm of cancer cells evidenced interactions between PCNA and the proteins in the cytoplasm (Byung and Lee, 2006). PCNA can bind to enzymes of the glycolysis pathway and regulate energy production in the mitochondria; maintain cytoskeleton integrity; and participate in other signaling pathways (Neuman et al., 2011; Bouhaddou et al., 2020). Li et al. (2021) described that SARS-CoV-2 M protein has its activity connected to ATP biosynthesis and metabolic processes (Li et al., 2021). Considering these data, the PCNA translocation from the nucleus to cytoplasm reported here may also be associated with the regulation of metabolism in infected cells to maintain SARS-CoV-2 replication.
Another hypothesis is that cytosolic PCNA, in association with the M protein, could bind to proteins that inhibit the immune response. Several studies pointed out that cytoplasmic PCNA is found in mature neutrophils associated with procaspases, thus preventing neutrophils from apoptosis (Witko-Sarsat et al., 2010).
The accumulation of PCNA in the cytoplasm of mature neutrophils is due to the activity of the chromosome region maintenance 1 (CRM1)-dependent nuclear-to-cytoplasmic relocalization during granulocytic (Bouayad et al., 2012). Cytoplasmic PCNA is also associated with Caspase-9 in the SH-SY5Y neuroblastoma cell line, and S-nitrosylation of PCNA at the residues C81 and C162 decreases this interaction, leading to caspase-9 activation (Yin et al., 2015).
Verdinexor is a selective inhibitor of nuclear export (SINE), a molecular drug that binds to CRM-1 and blocks the transport of proteins from the nucleus to the cytoplasm, including PCNA (Bouayad et al., 2012; Widman et al., 2018). The CRM-1 inhibitors have demonstrated activity against over 20 different DNA and RNA viruses, including influenza and respiratory syncytial virus (Perwitasari et al., 2014; Widman et al., 2018; Jorquera et al., 2019).
In addition, Selinexor, another SINE, reduced SARS-CoV-2 infection in vitro and protected the respiratory system in an in vivo model (Kashyap et al., 2021). We found that Verdinexor 0.1 µM was a safe dose and enough to reduced viral replication by 15% (Figure 6), corroborating the previous SINE study. Nonetheless, SINEs are drugs that could act in the translocation of different proteins, not only in PCNA. We also treated cells with different doses of PCNA inhibitor (PCANI1).
This inhibitor stabilizes the PCNA trimer and prevents the PCNA translocation from the nucleus to the cytoplasm and reallocation inside the nucleus (Lu and Dong, 2019). This inhibits the PCNA action on the replication forks and indirectly leads tumor cells to a higher sensitivity to anticancer DNA damaging drugs (Klein et al., 2020; Wyler et al., 2020). Our results show that PCNA I1 0.5 µM reduced viral replication by 20% (Figure 6), no difference was seen when the cells were treated with PCNA I1 0.1 µM. Our results indicate a potential use of PCNA and nuclear translocation inhibitors as treatments for COVID-19.
Conclusions
