Systematic functional analysis of SARS-CoV-2 proteins uncovers viral innate immune antagonists and remaining vulnerabilities.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evades most innate immune responses but may still be vulnerable to some. Here, we systematically analyze the impact of SARS-CoV-2 proteins on interferon (IFN) responses and autophagy. We show that SARS-CoV-2 proteins synergize to counteract anti-viral immune responses. For example, Nsp14 targets the type I IFN receptor for lysosomal degradation, ORF3a prevents fusion of autophagosomes and lysosomes, and ORF7a interferes with autophagosome acidification. Most activities are evolutionarily conserved. However, SARS-CoV-2 Nsp15 antagonizes IFN signaling less efficiently than the orthologs of closely related RaTG13-CoV and SARS-CoV-1. Overall, SARS-CoV-2 proteins counteract autophagy and type I IFN more efficiently than type II or III IFN signaling, and infection experiments confirm potent inhibition by IFN-γ and -λ1. Our results define the repertoire and selected mechanisms of SARS-CoV-2 innate immune antagonists but also reveal vulnerability to type II and III IFN that may help to develop safe and effective anti-viral approaches.

Comparison of different cytotoxicity assays for in vitro evaluation of mesoporous silica nanoparticles.

Colorimetric or luminogenic cytotoxicity assays are typically applied for in vitro cytotoxicity evaluations due to their easy handling and low cost. However, the results may be strongly assay-dependent. Furthermore, when applied to nanoparticle toxicity screening, nanoparticle-specific interferences can occur. Therefore, it is important to evaluate the assays for different classes of nanoparticles. Mesoporous silica nanoparticles (MSNs) have emerged as a promising platform for both diagnostic and therapeutic applications but a comparison between the commonly employed colorimetric formazan-dependent MTT and WST-1 and luminescent ATP-dependent cytotoxicity assays is still missing. In this work, we evaluated the applicability of four different in vitro cell viability assays for the cytotoxicity analysis of three differently functionalized mesoporous silica nanoparticles towards TZM-bl indicator cells. The results derived from the colorimetric measurements of cell-viability were compared with results obtained by cell count experiments, flow cytometry, and optical microscopy. The correlation between the viability assay results and the viable cell count was observed to be both assay and particle dependent. The MTT assay generally overestimated the cytotoxicity of the mesoporous silica particles, while the WST-1 assay sometimes clearly underestimated their cytotoxicity and even suggested a viability exceeding 100%. Of the two ATP-based assays, the CellTiterGlo assay gave the best correlation with cell count data, although some particle-dependent effects were observed. In conclusion, ATP-based assays seem most suitable for in vitro cytotoxicity evaluation of MSNs.

Utilization of replication-competent XMRV reporter-viruses reveals severe viral restriction in primary human cells.

The gammaretrovirus termed xenotropic murine leukemia virus-related virus (XMRV) was described to be isolated from prostate cancer tissue biopsies and from blood of patients suffering from chronic fatigue syndrome. However, many studies failed to detect XMRV and to verify these disease associations. Data suggesting the contamination of specimens in particular by PCR-based methods and recent reports demonstrating XMRV generation via recombination of two murine leukemia virus precursors raised serious doubts about XMRV being a genuine human pathogen. To elucidate cell tropism of XMRV, we generated replication competent XMRV reporter viruses encoding a green fluorescent protein or a secretable luciferase as tools to analyze virus infection of human cell lines or primary human cells. Transfection of proviral DNAs into LNCaP prostate cancer cells resulted in readily detectably reporter gene expression and production of progeny virus. Inoculation of known XMRV susceptible target cells revealed that these virions were infectious and expressed the reporter gene, allowing for a fast and highly sensitive quantification of XMRV infection. Both reporter viruses were capable of establishing a spreading infection in LNCaP and Raji B cells and could be easily passaged. However, after inoculation of primary human blood cells such as CD4 T cells, macrophages or dendritic cells, infection rates were very low, and a spreading infection was never established. In line with these results we found that supernatants derived from these XMRV infected primary cell types did not contain infectious virus. Thus, although XMRV efficiently replicated in some human cell lines, all tested primary cells were largely refractory to XMRV infection and did not support viral spread. Our results provide further evidence that XMRV is not a human pathogen.