ABSTRACT
SARS-CoV-2 non-structural protein 3 (Nsp3) contains a macrodomain that is essential for virus replication and is thus an attractive target for drug development. This macrodomain is thought to counteract the host interferon (IFN) response, an important antiviral signalling cascade, via the removal of ADP-ribose modifications catalysed by host poly(ADP-ribose) polymerases (PARPs). Here, we show that activation of the IFN response induces ADP-ribosylation of host proteins and that ectopic expression of the SARS-CoV-2 Nsp3 macrodomain reverses this modification in human cells. We further demonstrate that this can be used to screen for cell-active macrodomain inhibitors without the requirement for BSL-3 facilities. This IFN-induced ADP-ribosylation is dependent on the PARP9/DTX3L heterodimer, but surprisingly the expression of Nsp3 macrodomain or PARP9/DTX3L deletion do not impair STAT1 phosphorylation or the induction of IFN-responsive genes. Our results suggest that PARP9/DTX3L-dependent ADP-ribosylation is a downstream effector of the host IFN response and that the cellular function of the SARS-CoV-2 Nsp3 macrodomain is to hydrolyse this end product of IFN signalling, and not to suppress the IFN response itself.
ABSTRACT
Recent SARS-CoV-2 variants pose important concerns due to their higher transmissibility (1) and escape (2) from previous infections or vaccine-induced neutralizing antibodies (nAb). The receptor binding domain (RBD) of the Spike protein is a major nAb target (3), but data on its B cell epitopes are still lacking. Using a peptide microarray, we identified an immunodominant epitope (S415-429) recognized by 68% of sera from 71 convalescent Brazilians infected with the ancestral variant. In contrast with previous studies, we have identified a linear IgG and IgA antibody binding epitope within the RBD. IgG and IgA antibody levels for this epitope positively correlated with nAb titers, suggesting a potential target of antibody neutralizing activity. Interestingly, this immunodominant RBD region harbors the mutation hotspot site K417 present in P.1 (K417T) and B.1.351 (K417N) variants. In silico simulation analyses indicate impaired RBD binding to nAb in both variants and that a glycosylation in the B.1.351 417N could further hinder antibody binding as compared to the K417T mutation in P.1. This is in line with published data showing that nAb from either convalescents or anti-CoV-2 vaccinees are less effective towards B.1.351 than for P.1. Our data support the occurrence of immune pressure and selection involving this immunodominant epitope that may have critically contributed to the recent COVID-19 marked rise in Brazil and South Africa, and pinpoint a potential additional immune escape mechanism for SARS-CoV-2.
