André P. Mäurer

Genome-wide RNAi screen identifies human host factors crucial for influenza virus replication

Influenza A virus, being responsible for seasonal epidemics and reoccurring pandemics, represents a worldwide threat to public health. High mutation rates facilitate the generation of viral escape mutants, rendering vaccines and drugs directed against virus-encoded targets potentially ineffective. In contrast, targeting host cell determinants temporarily dispensable for the host but crucial for virus replication could prevent viral escape. Here we report the discovery of 287 human host cell genes influencing influenza A virus replication in a genome-wide RNA interference (RNAi) screen. Using an independent assay we confirmed 168 hits (59%) inhibiting either the endemic H1N1 (119 hits) or the current pandemic swine-origin (121 hits) influenza A virus strains, with an overlap of 60%. Notably, a subset of these common hits was also essential for replication of a highly pathogenic avian H5N1 strain. In-depth analyses of several factors provided insights into their infection stage relevance. Notably, SON DNA binding protein (SON) was found to be important for normal trafficking of influenza virions to late endosomes early in infection. We also show that a small molecule inhibitor of CDC-like kinase 1 (CLK1) reduces influenza virus replication by more than two orders of magnitude, an effect connected with impaired splicing of the viral M2 messenger RNA. Furthermore, influenza-virus-infected p27-/- (cyclin-dependent kinase inhibitor 1B; Cdkn1b) mice accumulated significantly lower viral titres in the lung, providing in vivo evidence for the importance of this gene. Thus, our results highlight the potency of genome-wide RNAi screening for the dissection of virus–host interactions and the identification of drug targets for a broad range of influenza viruses.

Tarp regulates early Chlamydia-induced host cell survival through interactions with the human adaptor protein SHC1.

The Chlamydia-encoded protein Tarp is phosphorylated in the host cell cytoplasm and is a multivalent hub for antiapoptotic signaling molecules.

Reduced display of tumor necrosis factor receptor I at the host cell surface supports infection with Chlamydia trachomatis.

The obligate intracellular human pathogenic bacterium Chlamydia trachomatis has evolved multiple mechanisms to circumvent the host immune system. Infected cells exhibit a profound resistance to the induction of apoptosis and down-regulate the expression of major histocompatibility complex class I and class II molecules to evade the cytotoxic effect of effector immune cells. Here we demonstrate the down-regulation of tumor necrosis factor receptor 1 (TNFR1) on the surface of infected cells. Interestingly, other members of the TNFR family such as TNFR2 and CD95 (Fas/Apo-1) were not modulated during infection, suggesting a selective mechanism underlying surface reduction of TNFR1. The observed effect was not due to reduced expression since the overall amount of TNFR1 protein was increased in infected cells. TNFR1 accumulated at the chlamydial inclusion and was shed by the infected cell into the culture supernatant. Receptor shedding depended on the infection-induced activation of the MEK-ERK pathway and the metalloproteinase TACE (TNFα converting enzyme). Our results point to a new function of TNFR1 modulation by C. trachomatis in controlling inflammatory signals during infection.

A Loss-of-Function Screen Reveals Ras- and Raf-Independent MEK-ERK Signaling During Chlamydia trachomatis Infection

An siRNA-based screen of host factors that influence infection by Chlamydia reveals the decoupling of the canonical Ras-ERK signaling pathway. Decoupling a MAPK Pathway Chlamydia trachomatis (Ctr) is an obligate, intracellular bacterial pathogen that causes a number of sexually transmitted diseases and the infectious eye disease, trachoma. Ctr cycles between an extracellular, infectious state known as the elementary body and an intracellular, metabolically active and replicating state known as the reticulate body. Ctr reticulate bodies accumulate within the inclusion, a membrane-bound vacuole. Gurumurthy et al. performed an RNA interference (RNAi)–based screen of infected epithelial cells and identified 59 factors that regulated Ctr infectivity. Knockdown of two of these, K-Ras and Raf-1, resulted in the increased growth of Ctr. Infection by Ctr led to the phosphorylation and inactivation of Raf-1 and its recruitment to the inclusion rather than to the plasma membrane where it normally triggers the MEK-ERK pathway, which is important for cell survival. Despite the inactivation of Raf-1, ERK activation was normal in infected cells, ensuring survival of the cells and growth of the pathogen. Thus, Ctr differentially modulates components of the Ras-ERK pathway to its own advantage. Chlamydiae are obligate intracellular bacterial pathogens that have a major effect on human health. Because of their intimate association with their host, chlamydiae depend on various host cell functions for their survival. Here, we present an RNA-interference–based screen in human epithelial cells that identified 59 host factors that either positively or negatively influenced the replication of Chlamydia trachomatis (Ctr). Two factors, K-Ras and Raf-1, which are members of the canonical Ras–Raf–MEK (mitogen-activated or extracellular signal–regulated protein kinase kinase)–ERK (extracellular signal–regulated kinase) pathway, were identified as central components of signaling networks associated with hits from the screen. Depletion of Ras or Raf in HeLa cells increased pathogen growth. Mechanistic analyses revealed that ERK was activated independently of K-Ras and Raf-1. Infection with Ctr led to the Akt-dependent, increased phosphorylation (and inactivation) of Raf-1 at serine-259. Furthermore, phosphorylated Raf-1 relocalized from the cytoplasm to the intracellular bacterial inclusion in an Akt- and 14-3-3β–dependent manner. Together, these findings not only show that Chlamydia regulates components of an important host cell signaling pathway, but also provide mechanistic insights into how this is achieved.

HIF-1α is involved in mediating apoptosis resistance to Chlamydia trachomatis-infected cells.

Chlamydiae are obligate intracellular Gram‐negative bacteria that cause widespread diseases in humans. Due to the intimate association between bacterium and host, Chlamydia evolved various strategies to protect their host cell against death‐inducing stimuli, allowing the bacterium to complete its development cycle. An RNA interference (RNAi)‐based screen was used to identify host cell factors required for apoptosis resistance of human epithelial cells infected with Chlamydia trachomatis serovar L2. Among the 32 validated hits, the anti‐apoptotic Bcl‐2 family member Mcl‐1 was identified as a target. Protein network analyses implicated the transcription factor hypoxia‐induced factor 1 alpha (HIF‐1α) to be central to the regulation of many of the identified targets. Further mechanistic investigations showed that HIF‐1α was stabilized within the host cell cytoplasm during early infection time points, followed by its translocation to the nucleus and eventual transcriptional activation of Mcl‐1. siRNA‐mediated depletion of HIF‐1α led to a drastic decrease in Mcl‐1, rendering the cell sensitive to apoptosis induction. Taken together, our findings identify HIF‐1α as responsible for upregulation of Mcl‐1 and the maintenance of apoptosis resistance during Chlamydia infection.

RNA-interference based screen identifies new factors important for NF-kappaB activation and termination

The transcription factor NF-kappaB is a key mediator of the innate immune system. Although tremendous research efforts over the past decades have led to a more and more detailed understanding of NF-kappaB signaling, there are still missing pieces in the puzzle, especially upstream of the IKK complex and in the termination of the signaling. To identify more of the factors important for this signaling pathway we have conducted an RNA-interference based screen. For this purpose, we have developed an assay for high throughput analysis using a human epithelial cell line stably expressing a p65-GFP-fusion construct. The nuclear translocation of p65-GFP can be quantified by automated microscopic analysis. Three different stimuli were compared: the cytokines TNF-alpha and IL1-beta and the gastric pathogen Helicobacter pylori. We chose H. pylori as inducer because permanent infection with this bacterium can lead to chronic inflammation, ulceration and cancerogenesis and NF-kappaB is thought to be crucial in the promotion of this pathology. Furthermore, using different time points of the activation, we screened not only for factors important for activation, but also for termination of the signal. In terms of activation, the screen identified known factors like IKKalpha and IKKbeta as well as factors so far not linked to the NF-kappaB pathway. Interestingly, two factors were identified that are specific for NF-kappaB activation after H. pylori infection and not necessary for NF-kappaB activation by the cytokines TNFalpha or IL-1beta. Regarding termination, the screen identified among other factors an ubiquitin E3-Ligase so far not linked to the pathway. Upon down-regulation of this E3-Ligase, p65-GFP resides longer in the nucleus. This correlates with a strong degradation of IkappaBalpha. The screen was conducted with a library of siRNAs against 646 kinases and associated proteins, and is currently expanded to a genome wide scale.