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Background and AimsHigh expression of phosphatidylinositol 4-kinase III alpha (PI4KIIIα) correlates with poor survival rates in patients with hepatocellular carcinoma (HCC). In addition, Hepatitis C virus (HCV) infections activate PI4KIIIα and contribute to HCC progression. We aimed at mechanistically understanding the impact of PI4KIIIα on the progression of liver cancer and the potential contribution of HCV in this process.MethodsSeveral hepatic cell culture and mouse models were used to study functional importance of PI4KIIIα on liver pathogenesis. Antibody arrays, gene silencing and PI4KIIIα specific inhibitor were applied to identify the involved signaling pathways. The contribution of HCV was examined by using HCV infection or overexpression of its nonstructural protein.ResultsHigh PI4KIIIα expression and/or activity induced cytoskeletal rearrangements via increased-phosphorylation of paxillin and cofilin. This led to morphological alterations and higher migratory and invasive properties of liver cancer cells. We further identified the liver specific lipid kinase phosphatidylinositol 3-kinase C2 domain-containing subunit gamma (PIK3C2γ) working downstream of PI4KIIIα in regulation of the cytoskeleton. PIK3C2γ generates plasma membrane (PM) phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2]- enriched, invadopodia-like structures which regulate cytoskeletal reorganization by promoting Akt2 phosphorylation.ConclusionsPI4KIIIα regulates cytoskeleton organization via PIK3C2γ/Akt2/paxillin-cofilin to favor migration and invasion of liver cancer cells. These findings provide mechanistic insight into the contribution of PI4KIIIα and HCV to progression of liver cancer and identify promising targets for therapeutic intervention.IMPACT AND IMPLICATIONSUnderstanding mechanistically how high PI4KIIIα expression are associated with poor clinical outcomes of liver cancer is important to develop pharmaceutical interventions. Our study sheds light on the importance of the two lipid kinases PI4KIIIα and PIK3C2γ as well as the contribution of HCV on liver cancer progression, unraveling the signaling pathway governing this process. This preclinical study contributes to better understanding the complex connection of phospholipids, cytoskeleton and liver cancer and suggests strategies to improve therapeutic outcomes by targeting important signaling molecules.Graphical abstract

Glycan–protein interactions are highly specific yet transient, rendering glycans ideal recognition signals in a variety of biological processes. In human norovirus (HuNoV) infection, histo-blood group antigens (HBGAs) play an essential but poorly understood role. For murine norovirus infection (MNV), sialylated glycolipids or glycoproteins appear to be important. It has also been suggested that HuNoV capsid proteins bind to sialylated ganglioside head groups. Here, we study the binding of HBGAs and sialoglycans to HuNoV and MNV capsid proteins using NMR experiments. Surprisingly, the experiments show that none of the norovirus P-domains bind to sialoglycans. Notably, MNV P-domains do not bind to any of the glycans studied, and MNV-1 infection of cells deficient in surface sialoglycans shows no significant difference compared to cells expressing respective glycans. These findings redefine glycan recognition by noroviruses, challenging present models of infection.

Three recent papers highlight the impact of an entry receptor on murine norovirus pathogenesis. CD300lf was shown to be the first proteinaceous entry receptor for a norovirus, specialized intestinal epithelial cells constitute the reservoir for persistence, and a new link between the receptor and a lipid biosynthetic enzyme was revealed.

Background Chikungunya virus (CHIKV) is a re-emerging arthropod-borne (arbo)virus that causes chikungunya fever in humans and is predominantly transmitted by Aedes aegypti mosquitoes. The CHIKV replication machinery consists of four non-structural proteins (nsP1-4) that additionally require the presence of a number of host proteins for replication of the viral RNA. NsP3 is essential for CHIKV replication and has a conserved macro, central and C-terminal hypervariable domain (HVD). The HVD is intrinsically disordered and interacts with various host proteins via conserved short peptide motifs: A proline-rich (P-rich) motif that has affinity for SH3-domain containing proteins and duplicate FGDF motifs with affinity for G3BP and its mosquito homologue Rasputin. The importance of these motifs for infection of mammalian cells has previously been implicated. However, their role during CHIKV infection of mosquito cells and transmission by mosquitoes remains unclear. Methodology / Principal findings Here, we show that in-frame deletion of the P-rich motif is lethal for CHIKV replication in both mosquito and mammalian cells. However, while mutagenesis of the P-rich motif negatively affects replication both in mammalian and mosquito cells, it did not compromise the infection and transmission of CHIKV by Ae. aegypti mosquitoes. Mutagenesis of both FGDF motifs together completely inactivated CHIKV replication in both mammalian and mosquito cells. Importantly, mutation of a single FGDF motif attenuated CHIKV replication in mammalian cells, while replication in mosquito cells was similar to wild type. Surprisingly, CHIKV mutants containing only a single FGDF motif were efficiently transmitted by Ae. aegypti. Conclusions / Significance The P-rich motif in CHIKV nsP3 is dispensable for transmission by mosquitoes. A single FGDF motif is sufficient for infection and dissemination in mosquitoes, but duplicate FGDF motifs are required for the efficient infection from the mosquito saliva to a vertebrate host. These results contribute to understanding the dynamics of the alphavirus transmission cycle and may help the development of arboviral intervention strategies., Author summary Chikungunya virus (CHIKV) is a re-emerging arthropod-borne virus that is transmitted predominantly by Aedes aegypti mosquitoes. In 2016 alone CHIKV caused over 100.000 infections in South-America, exemplifying the impact of CHIKV disease. Previous research has suggested that the CHIKV non-structural protein 3 (nsP3) may determine the infection of mosquitoes. NsP3 is known to interact with several host proteins through a conserved proline (P)-rich and duplicate FGDF motifs that are present in its C-terminal domain. Here we investigated the importance of these conserved motifs for the infection and replication of CHIKV in both Aedes mosquito cells and mammalian cells. Furthermore, we assessed the role of these motifs for the transmission by Ae. aegypti mosquitoes via infectious bloodmeal experiments. We show that mutation of the P-rich motif negatively affects the replication of CHIKV in both mammalian and mosquito cells. In contrast, mutating the P-rich motif did not affect the transmission by Ae. aegypti. Mutation of both FGDF motifs together completely inactivated CHIKV in mammalian and mosquito cells, while mutation of a single FGDF motif negatively affected replication only in mammalian cells. Importantly, CHIKV containing only a single FGDF motif was still efficiently transmitted by Ae. aegypti mosquitoes. These results contribute to understanding the key interactions between alphaviruses and their mosquito vector.

Aspergillus niger is a filamentous fungus that is extensively used in industrial fermentations for protein expression and the production of organic acids. Inherent biosynthetic capabilities, such as the capacity to secrete these biomolecules in high amounts, make A. niger an attractive production host. Although A. niger is renowned for this ability, the knowledge of the molecular components that underlie its production capacity, intercellular trafficking processes and secretion mechanisms is far from complete. Here, we introduce a standardized set of tools, consisting of an N-terminal GFP-actin fusion and codon optimized eforRed chromoprotein. Expression of the GFP-actin construct facilitates visualization of the actin filaments of the cytoskeleton, whereas expression of the chromoprotein construct results in a clearly distinguishable red phenotype. These experimentally validated constructs constitute the first set of standardized A. niger biomarkers, which can be used to study morphology, intercellular trafficking, and secretion phenomena.