Functional interrogation of cellular Lp(a) uptake by genome-scale CRISPR screening.

An elevated level of lipoprotein(a), or Lp(a), in the bloodstream has been causally linked to the development of atherosclerotic cardiovascular disease and calcific aortic valve stenosis. Steady state levels of circulating lipoproteins are modulated by their rate of clearance, but the identity of the Lp(a) uptake receptor(s) has been controversial. In this study, we performed a genome-scale CRISPR screen to functionally interrogate all potential Lp(a) uptake regulators in HuH7 cells. Strikingly, the top positive and negative regulators of Lp(a) uptake in our screen were LDLR and MYLIP, encoding the LDL receptor and its ubiquitin ligase IDOL, respectively. We also found a significant correlation for other genes with established roles in LDLR regulation. No other gene products, including those previously proposed as Lp(a) receptors, exhibited a significant effect on Lp(a) uptake in our screen. We validated the functional influence of LDLR expression on HuH7 Lp(a) uptake, confirmed in vitro binding between the LDLR extracellular domain and purified Lp(a), and detected an association between loss-of-function LDLR variants and increased circulating Lp(a) levels in the UK Biobank cohort. Together, our findings support a central role for the LDL receptor in mediating Lp(a) uptake by hepatocytes.

Type I interferon signaling induces a delayed antiproliferative response in Calu-3 cells during SARS-CoV-2 infection.

Disease progression during SARS-CoV-2 infection is tightly linked to the fate of lung epithelial cells, with severe cases of COVID-19 characterized by direct injury of the alveolar epithelium and an impairment in its regeneration from progenitor cells. The molecular pathways that govern respiratory epithelial cell death and proliferation during SARS-CoV-2 infection, however, remain poorly understood. We now report a high-throughput CRISPR screen for host genetic modifiers of the survival and proliferation of SARS-CoV-2-infected Calu-3 respiratory epithelial cells. The top 4 genes identified in our screen encode components of the same type I interferon signaling complex - IFNAR1, IFNAR2, JAK1, and TYK2. The 5th gene, ACE2, was an expected control encoding the SARS-CoV-2 viral receptor. Surprisingly, despite the antiviral properties of IFN-I signaling, its disruption in our screen was associated with an increase in Calu-3 cell fitness. We validated this effect and found that IFN-I signaling did not sensitize SARS-CoV-2-infected cultures to cell death but rather inhibited the proliferation of surviving cells after the early peak of viral replication and cytopathic effect. We also found that IFN-I signaling alone, in the absence of viral infection, was sufficient to induce this delayed antiproliferative response. Together, these findings highlight a cell autonomous antiproliferative response by respiratory epithelial cells to persistent IFN-I signaling during SARS-CoV-2 infection. This response may contribute to the deficient alveolar regeneration that has been associated with COVID-19 lung injury and represents a promising area for host-targeted therapeutic development.

Genetic diversity of porcine enteric caliciviruses in pigs raised in Rio de Janeiro State, Brazil.

Porcine enteric caliciviruses (PEC) belong to the genera Norovirus and Sapovirus within the family Caliciviridae. They are enteric pathogens and are considered potential zoonotic agents. In this study, the circulation of PEC was evaluated by RT-PCR of stool samples and intestinal contents of pigs raised in Rio de Janeiro State, Brazil. Both porcine norovirus (PoNoV) and porcine sapovirus (PoSaV) were detected. The PoNoV strains were classified as genogroup II, genotypes 11, 18 and 19. The PoSaV strains were classified as genogroups III and VII, though some strains could not be classified into any established genogroup, potentially representing a new one. PEC were detected mainly in animals without clinical signs of gastroenteritis.

Recent advances in understanding noroviruses.

Noroviruses are the leading cause of acute gastroenteritis around the world. An individual living in the United States is estimated to develop norovirus infection five times in his or her lifetime. Despite this, there is currently no antiviral or vaccine to combat the infection, in large part because of the historical lack of cell culture and small animal models. However, the last few years of norovirus research were marked by a number of ground-breaking advances that have overcome technical barriers and uncovered novel aspects of norovirus biology. Foremost among them was the development of two different in vitro culture systems for human noroviruses. Underappreciated was the notion that noroviruses infect cells of the immune system as well as epithelial cells within the gastrointestinal tract and that human norovirus infection of enterocytes requires or is promoted by the presence of bile acids. Furthermore, two proteinaceous receptors are now recognized for murine norovirus, marking the first discovery of a functional receptor for any norovirus. Recent work further points to a role for certain bacteria, including those found in the gut microbiome, as potential modulators of norovirus infection in the host, emphasizing the importance of interactions with organisms from other kingdoms of life for viral pathogenesis. Lastly, we will highlight the adaptation of drop-based microfluidics to norovirus research, as this technology has the potential to reveal novel insights into virus evolution. This review aims to summarize these new findings while also including possible future directions.