Evolution of enhanced innate immune evasion by SARS-CoV-2

Emergence of SARS-CoV-2 variants of concern (VOCs) suggests viral adaptation to enhance human-to-human transmission1,2. Although much effort has focused on characterisation of spike changes in VOCs, mutations outside spike likely contribute to adaptation. Here we used unbiased abundance proteomics, phosphoproteomics, RNAseq and viral replication assays to show that isolates of the Alpha (B.1.1.7) variant3 more effectively suppress innate immune responses in airway epithelial cells, compared to first wave isolates. We found that Alpha has dramatically increased subgenomic RNA and protein levels of N, Orf9b and Orf6, all known innate immune antagonists. Expression of Orf9b alone suppressed the innate immune response through interaction with TOM70, a mitochondrial protein required for RNA sensing adaptor MAVS activation. Moreover, the activity of Orf9b and its association with TOM70 was regulated by phosphorylation. We propose that more effective innate immune suppression, through enhanced expression of specific viral antagonist proteins, increases the likelihood of successful Alpha transmission, and may increase in vivo replication and duration of infection4. The importance of mutations outside Spike in adaptation of SARS-CoV-2 to humans is underscored by the observation that similar mutations exist in the Delta and Omicron N/Orf9b regulatory regions

Impact of SARS-CoV-2 ORF6 and its variant polymorphisms on host responses and viral pathogenesis

: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encodes several proteins that inhibit host interferon responses. Among these, ORF6 antagonizes interferon signaling by disrupting nucleocytoplasmic trafficking through interactions with the nuclear pore complex components Nup98-Rae1. However, the roles and contributions of ORF6 during physiological infection remain unexplored. We assessed the role of ORF6 during infection using recombinant viruses carrying a deletion or loss-of-function (LoF) mutation in ORF6. ORF6 plays key roles in interferon antagonism and viral pathogenesis by interfering with nuclear import and specifically the translocation of IRF and STAT transcription factors. Additionally, ORF6 inhibits cellular mRNA export, resulting in the remodeling of the host cell proteome, and regulates viral protein expression. Interestingly, the ORF6:D61L mutation that emerged in the Omicron BA.2 and BA.4 variants exhibits reduced interactions with Nup98-Rae1 and consequently impairs immune evasion. Our findings highlight the role of ORF6 in antagonizing innate immunity and emphasize the importance of studying the immune evasion strategies of SARS-CoV-2

Influence of NaCl and pH on intracellular enzymes that influence Cheddar cheese ripening

The influence of NaCl and reduced pH was determined for aminopeptidase, lipase / esterase and methanethiol-producing capability in selected lactic acid bacteria and brevibacteria in simulated cheese-like conditions. The observations on simulated cheese-like conditions were confirmed in 60% reduced-fat Cheddar cheese. The activity of each enzyme decreased with NaCl addition and when the pH was reduced to approximate Cheddar cheese conditions (5% NaCl, pH 5.2). Residual intracellular aminopeptidase activity was dominated by general aminopeptidase activity (aminopeptidase N and / or aminopeptidase C) in laboratory, simulated cheese-like conditions, and 60% reduced-fat Cheddar cheese curd. During cheese aging, total lipase / esterase activity peaked at 120 d then decreased, even though starter culture populations remained high. Methanethiol-producing capability occurred under cheese-like conditions in whole cells, but not in cell-free extracts. Met and Met-containing peptides induced methanethiol-producing capability for 2-3 generations and could be re-induced later in the growth cycle of Brevibacterium linens BL2. Aminopeptidase and lipase / esterase activity in reduced fat cheese were not correlated to an increase in Cheddar-type flavor, but a culture\u27s methanethiol-producing capability was associated with higher cheese consumer preference scores. Results suggest that use of cheese-like conditions may aid in selecting cultures to increase desirable flavors for low-fat cheese manufacture. Additionally, data suggest that whole cells are important for proper flavor development in 60% reduced-fat Cheddar cheese