Host cells reprogram lipid droplet synthesis through YY1 to resist PRRSV infection.

Metabolism in host cells can be modulated after viral infection, favoring viral survival or clearance. Here, we report that lipid droplet (LD) synthesis in host cells can be modulated by yin yang 1 (YY1) after porcine reproductive and respiratory syndrome virus (PRRSV) infection, resulting in active antiviral activity. As a ubiquitously distributed transcription factor, there was increased expression of YY1 upon PRRSV infection both in vitro and in vivo. YY1 silencing promoted the replication of PRRSV, whereas YY1 overexpression inhibited PRRSV replication. PRRSV infection led to a marked increase in LDs, while YY1 knockout inhibited LD synthesis, and YY1 overexpression enhanced LD accumulation, indicating that YY1 reprograms PRRSV infection-induced intracellular LD synthesis. We also showed that the viral components do not colocalize with LDs during PRRSV infection, and the effect of exogenously induced LD synthesis on PRRSV replication is nearly lethal. Moreover, we demonstrated that YY1 affects the synthesis of LDs by regulating the expression of lipid metabolism genes. YY1 negatively regulates the expression of fatty acid synthase (FASN) to weaken the fatty acid synthesis pathway and positively regulates the expression of peroxisome proliferator-activated receptor gamma (PPARγ) to promote the synthesis of LDs, thus inhibiting PRRSV replication. These novel findings indicate that YY1 plays a crucial role in regulating PRRSV replication by reprogramming LD synthesis. Therefore, our study provides a novel mechanism of host resistance to PRRSV and suggests potential new antiviral strategies against PRRSV infection.IMPORTANCEPorcine reproductive and respiratory virus (PRRSV) has caused incalculable economic damage to the global pig industry since it was first discovered in the 1980s. However, conventional vaccines do not provide satisfactory protection. It is well known that viruses are parasitic pathogens, and the completion of their replication life cycle is highly dependent on host cells. A better understanding of host resistance to PRRSV infection is essential for developing safe and effective strategies to control PRRSV. Here, we report a crucial host antiviral molecule, yin yang 1 (YY1), which is induced to be expressed upon PRRSV infection and subsequently inhibits virus replication by reprogramming lipid droplet (LD) synthesis through transcriptional regulation. Our work provides a novel antiviral mechanism against PRRSV infection and suggests that targeting YY1 could be a new strategy for controlling PRRSV.

An optimized protocol for the extraction and quantification of cytosolic DNA in mammalian cells.

Leakage of mitochondrial or nuclear DNA into the cytosol can occur following viral infections, radiation damage, and some cancers. Here, we present an optimized protocol for isolating and quantifying cytosolic DNA from mammalian cells. We describe steps for collecting cytosolic fractions from cells, extracting DNA using columns, and quantifying extracted DNA using qPCR. This straightforward protocol can be completed in as little as 5 hours, and allows for the identification of the source of DNA. For complete details on the use and execution of this protocol, please refer to Jahun et al.1.

Porcine Sapovirus Protease Controls the Innate Immune Response and Targets TBK1.

Human sapoviruses (HuSaVs) and noroviruses are considered the leading cause of acute gastroenteritis worldwide. While extensive research has focused on noroviruses, our understanding of sapoviruses (SaVs) and their interactions with the host's immune response remains limited. HuSaVs have been challenging to propagate in vitro, making the porcine sapovirus (PSaV) Cowden strain a valuable model for studying SaV pathogenesis. In this study we show, for the first time, that PSaV Cowden strain has mechanisms to evade the host's innate immune response. The virus 3C-like protease (NS6) inhibits type I IFN production by targeting TBK1. Catalytically active NS6, both during ectopic expression and during PSaV infection, targets TBK1 which is then led for rapid degradation by the proteasome. Moreover, deletion of TBK1 from porcine cells led to an increase in PSaV titres, emphasizing its role in regulating PSaV infection. Additionally, we successfully established PSaV infection in IPEC-J2 cells, an enterocytic cell line originating from the jejunum of a neonatal piglet. Overall, this study provides novel insights into PSaV evasion strategies, opening the way for future investigations into SaV-host interactions, and enabling the use of a new cell line model for PSaV research.

Microfluidic antibody profiling after repeated SARS-CoV-2 vaccination links antibody affinity and concentration to impaired immunity and variant escape in patients on anti-CD20 therapy.

Background: Patients with autoimmune/inflammatory conditions on anti-CD20 therapies, such as rituximab, have suboptimal humoral responses to vaccination and are vulnerable to poorer clinical outcomes following SARS-CoV-2 infection. We aimed to examine how the fundamental parameters of antibody responses, namely, affinity and concentration, shape the quality of humoral immunity after vaccination in these patients. Methods: We performed in-depth antibody characterisation in sera collected 4 to 6 weeks after each of three vaccine doses to wild-type (WT) SARS-CoV-2 in rituximab-treated primary vasculitis patients (n = 14) using Luminex and pseudovirus neutralisation assays, whereas we used a novel microfluidic-based immunoassay to quantify polyclonal antibody affinity and concentration against both WT and Omicron (B.1.1.529) variants. We performed comparative antibody profiling at equivalent timepoints in healthy individuals after three antigenic exposures to WT SARS-CoV-2 (one infection and two vaccinations; n = 15) and in convalescent patients after WT SARS-CoV-2 infection (n = 30). Results: Rituximab-treated patients had lower antibody levels and neutralisation titres against both WT and Omicron SARS-CoV-2 variants compared to healthy individuals. Neutralisation capacity was weaker against Omicron versus WT both in rituximab-treated patients and in healthy individuals. In the rituximab cohort, this was driven by lower antibody affinity against Omicron versus WT [median (range) KD: 21.6 (9.7-38.8) nM vs. 4.6 (2.3-44.8) nM, p = 0.0004]. By contrast, healthy individuals with hybrid immunity produced a broader antibody response, a subset of which recognised Omicron with higher affinity than antibodies in rituximab-treated patients [median (range) KD: 1.05 (0.45-1.84) nM vs. 20.25 (13.2-38.8) nM, p = 0.0002], underpinning the stronger serum neutralisation capacity against Omicron in the former group. Rituximab-treated patients had similar anti-WT antibody levels and neutralisation titres to unvaccinated convalescent individuals, despite two more exposures to SARS-CoV-2 antigen. Temporal profiling of the antibody response showed evidence of affinity maturation in healthy convalescent patients after a single SARS-CoV-2 infection, which was not observed in rituximab-treated patients, despite repeated vaccination. Discussion: Our results enrich previous observations of impaired humoral immune responses to SARS-CoV-2 in rituximab-treated patients and highlight the significance of quantitative assessment of serum antibody affinity and concentration in monitoring anti-viral immunity, viral escape, and the evolution of the humoral response.