Multiple layers of innate immune response antagonism of SARS-CoV-2
Authors: Zhou Fuchun , Periasamy Sivakumar , Jackson Nathaniel D. , Cheng Wan Sze , Soto Acosta Ruben , Ilinykh Philipp A. , Ye Chengjin , Chauhan Shailendra , Nudelman German , Zaslavsky Elena , Widen Steven G. , Martinez-Sobrido Luis , Sealfon Stuart C. , Bukreyev Alexander 
Published: January 2024

ABSTRACTSeveral SARS-CoV-2 proteins have been shown to counteract the host innate immune response, mostly usingin vitroprotein expression, which may not fully reflect their role in the context of viral infection. In addition, while each viral protein was characterized in a different experimental system, their relative contribution in immunosuppression remains unclear. Here we used a SARS-CoV-2 bacterial artificial chromosome withen passantmutagenesis to recover a panel of twelve infectious recombinant SARS-CoV-2 viruses, each with mutations in either NSP1, NSP2, NSP3, NSP6, NSP12, NSP13, NSP14, NSP15, NSP16, ORF3a, ORF6 or ORF8. We used the interferon-stimulated response element (ISRE)-driven luciferase assay in 293T-ACE2/TMPRSS2 cells to test the panel, demonstrating that mutations in many proteins, especially in NSP1 and NSP15, increased the type I interferon response relative to the parental wild-type virus. RNA-seq analysis of mutant-virus infected Calu-3 cells showed that the mutations in NSP1 or NSP15 lead to higher expression of multiple genes involved in innate immune response, cytokine-mediated signaling and regulation of lymphocyte proliferation. Furthermore, mutations in either NSP1 or NSP15 resulted in a greater maturation of human monocyte-derived dendritic cellsin vitro. Infection of K18 hACE2 transgenic mice with either NSP1 or NSP15 mutated viruses demonstrated attentuated respiratory tract replication. Analysis of lung immune cells from infected mice by single-cell RNA-seq identified 15 populations of major myeloid and lymphoid cells with changes in the pattern of their activation associated with viral infection. The effects of mutations in NSP1 or NSP15 on these responses are consistent with differences in the immunosuppressive mechanisms utilized by the two proteins. Overall, these data demonstrate different and redundant mechanisms of innate immune antagonism by SARS-CoV-2 and suppression of activation of antigen presenting cells and T and B lymphocytes mediated by multiple viral proteins.AUTHOR SUMMARYThe mechanisms by which SARS-CoV-2 and its proteins modulate host immunity, specifically the interferon response, are still not clear. We generated twelve infectious SARS-CoV-2 viruses with mutations in individual proteins and demonstrated that many of them have interferon-antagonizing activity and immunosuppressive effects in human cells and in the K18 hACE mouse model of infection. We idemtified distinct and redundant mechanisms of immunosuppression of SARS-CoV-2 mediated by multiple individual viral proteins, with 9 out of the 12 tested proteins showing some immunosuppressive effect in at least one experimental system. The demonstrated immunosuppressive effects extend from the innate response to immune cells to pathologic changesin vivo. Importantly, this work shows, for the first time, a comparison of the effects of multiple viral proteins in the context of authentic viral infection, rather than in a surrogate system, and shows the relative contribution of each viral protein under identical experimental conditions. Overall, our data indicates that SARS-CoV-2 antagonizes multiple immune mechanisms, particularly type I interferon signaling, activation of innate immune cells and T and B lymphocyte functions with the greatest effects due to NSP1 and NSP15.