The Legionella pneumophila effector DenR hijacks the host NRas proto-oncoprotein to downregulate MAPK signaling.

Small GTPases of the Ras subfamily are best known for their role as proto-oncoproteins, while their function during microbial infection has remained elusive. Here, we show that Legionella pneumophila hijacks the small GTPase NRas to the Legionella-containing vacuole (LCV) surface. A CRISPR interference screen identifies a single L. pneumophila effector, DenR (Lpg1909), required for this process. Recruitment is specific for NRas, while its homologs KRas and HRas are excluded from LCVs. The C-terminal hypervariable tail of NRas is sufficient for recruitment, and interference with either NRas farnesylation or S-acylation sites abrogates recruitment. Intriguingly, we detect markers of active NRas signaling on the LCV, suggesting it acts as a signaling platform. Subsequent phosphoproteomics analyses show that DenR rewires the host NRas signaling landscape, including dampening of the canonical mitogen-activated protein kinase pathway. These results provide evidence for L. pneumophila targeting NRas and suggest a link between NRas GTPase signaling and microbial infection.

The intracellular inhibition of HCV replication represents a novel mechanism of action by the innate immune Lactoferrin protein.

Hepatitis C virus (HCV) is a major public-health problem with 130-170 million individuals chronically infected worldwide. In order to halt the epidemic, therapy against HCV will need to be both effective and widely available. Studies focusing on safe and affordable natural product active against HCV have revealed the antiviral activity of the human Lactoferrin (hLF) protein which binds and neutralizes the circulating virion. In the current study, investigation of hLF activity on the HCV subgenomic replicon system, which is independent from viral entry and shedding, revealed a distinct antireplicative activity of hLF against HCV. Hepatocellular uptake of hLF was confirmed and correlated with qualitative HCV staining reduction. Quantitative dose-response inhibition assays confirmed an hLF-mediated and dose-dependent HCV replication reduction reaching up to 60%. The in cellulo anti-HCV activity of hLF was additive to both Ribavirin and Interferon-α-2b. Further investigation of hLF activity against the essential viral proteins involved in HCV genome replication revealed an inhibitory activity against the HCV ATPase/Helicase NS3 protein but not against the HCV RNA-dependent RNA polymerase (NS5B protein). NS3 inhibition was mediated by a direct and specific interaction between hLF and an allosteric binding site on NS3. Taken together, our findings reveal a new antiviral mechanism of action by which hLF inhibits intracellular HCV replication.