Spanish researchers from Universidade de Santiago de Compostela, Instituto de Salud Carlos III and the Hospital Clínico Universitario de Santiago (SERGAS) are also proposing that SARS-CoV-2 are also oncogenic viruses as they have found yet another mechanism by which SARS-CoV-2 infections can also contribute to the rise of cancers.
PLP1/2 of the alphacoronavirus NL63 as well as the PLpros of SARS-CoV and MERS-CoV from the genus betacoronavirus physically interact with and stabilize RCHY1, and lead to p53 degradation. Because PLpros and PLPs are relatively well conserved among coronaviruses, it is highly likely that all of the coronaviruses share this strategy to antagonize the antiviral host factor p53. SUD alone strongly binds to and stabilizes RCHY1, leading to stimulation of p53 ubiquitination and degradation.
The SUD–PLpro fusion interacts with RCHY1 more intensively and causes stronger endogenous p53 degradation than SARS-CoV PLpro alone. Hence, SUD functions as an enhancer for SARS-CoV Nsp3 to counteract p53, and this might contribute to the high virulence of SARS-CoV.
The mechanism of how SUD and PLpro/PLPs stimulate the accumulation of RCHY1 remains unclear. It is known that phosphorylation by CAMKII or CDK9 leads to polyubiquitination and subsequent degradation of RCHY1 (14, 24), but SUD does not impair the phosphorylation activity of CAMK2D toward RCHY1.
Because papain-like proteases of SARS-CoV, MERS-CoV, and NL63 possess deubiquitinating activity (39, 46, 47) and can stabilize RCHY1, we originally hypothesized that the latter may be a possible substrate for these enzymes. However, an in vitro deubiquitination assay has shown that SARS-CoV PLpro does not deubiquitinate RCHY1. Thus, SUD and PLpro should stabilize RCHY1 through other mechanisms, e.g., by counteracting the binding of RCHY1 to CDK9 or interfering with the homodimerization of RCHY1 for self-ubiquitination.
Because both SUD and PLpro physically interact with RCHY1, their mechanisms of RCHY1 stabilization might also be different from one another.
The protein level of RCHY1 is dramatically increased in the presence of SUD. As a result, RCHY1-mediated ubiquitination of p53 is enhanced and p53 degradation is stimulated. However, enhanced p53 degradation should not be the only consequence of RCHY1 accumulation. Besides p53, the substrate targets of the E3 ubiquitin ligase RCHY1 include transcription factors p63, p73, and c-Myc; checkpoint kinase Chk2; DNA polymerase polH; histone deacetylase HDAC1; and CDK inhibitor p27Kip1 (9, 10, 48–52).
It can be hypothesized that during HCoV infection, the protein levels of various genes might also be down-regulated. SUD also physically interacts with CAMK2D, although it does not disturb phosphorylation of RCHY1 by CAMK2D. However, RCHY1 is neither the only substrate nor the only interacting partner of CAMK2D. The association of SUD and CAMK2D might alter the affinity of CAMK2D for other proteins, e.g., for its candidate binding partner IFN-γ receptor 2 (IFNGR2), which was discovered by Y2H screen (53).
Alternatively, the interaction between SUD and CAMK2D might interfere with phosphorylation of STAT1 by CAMK2D, which is required for maximum IFN-γ–stimulated gene (ISG) expression (54). In addition to stimulation of p53 degradation, SUD probably features even more complex interferences with host cellular signaling pathways.
To date, p53 was found to serve as an antiviral factor against diverse positive-sense single-stranded RNA (ssRNA) viruses such as hepatitis C virus (HCV) and poliovirus; negative-sense ssRNA viruses such as influenza A virus; and the retrovirus HIV-1 (29, 31, 55, 56). p53 antiviral activity on Sendai virus replication was reported in the context of deubiquitination of MDM2 by coronaviral PLpro and ubiquitination of p53 by MDM2 (30).
In this mainly biochemical study, the inhibitory effect of p53 was not tested for coronaviruses. The authors had hypothesized regulatory influence of HCoV-NL63 PLP2 on the stability of MDM2, due (i) to structural similarities of PLP2, PLpro, and the cellular deubiquitinase HAUSP, and (ii) to the preferential deubiquitination of MDM2 by HAUSP. Indeed, interaction of PLP2 and MDM2, deubiquitination of MDM2 by PLP2, and ubiquitination of p53 leading to proteasomal degradation was demonstrated.
Applying unbiased protein–protein interaction Y2H screens (34), we identified interaction of SUD with the cellular ubiquitin ligase RCHY1, which exerts similar ubiquitination activities on p53 as MDM2. We directly demonstrate, to our knowledge for the first time, that p53 inhibits replication of coronaviruses and provide evidence that SUD and PLpro/PLPs antagonize the host defense factor p53 via stabilizing the E3 ubiquitin ligase RCHY1. So far, we have no hints on a conceivable involvement of MDM2, though it may well be that CoVs have developed different ways to down-regulate p53.
In addition, the deISGylating activity, exhibited by PLpro domains, might further interfere with the ISGylation status of host proteins (57, 58). Decreased ISGylation of host proteins seems to positively mediate virus infection. It has been demonstrated that cellular ISGylation contributes to inhibition of replication of murine hepatitis virus strain 3 (59).
Thus, CoV Nsp3 proteins containing SUD PLpro/PLPs counteract the host defense in two distinct manners—one is p53 independent: the PLpro/PLPs within Nsp3 exert a direct deISGylation activity to interfere with cellular ISGylation; the other is p53 dependent: Nsp3 proteins lead to p53 degradation by stabilizing RCHY1. These results may open new possibilities to therapeutically target SARS infections in the future.