Single‐cell analysis of population context advances RNAi screening at multiple levels

Snijder, Berend; Sacher, Raphael; Rämö, Pauli; Liberali, Prisca; Mench, Karin; Wolfrum, Nina; Burleigh, Laura; Scott, Cameron C.; Verheije, Monique H.; Mercer, Jason; Moese, Stefan; Heger, Thomas; Theusner, Kristina; Jurgeit, Andreas; Lamparter, David; Balistreri, Giuseppe; Schelhaas, Mario; Haan, Cornelis A. M. de; Marjomäki, Varpu; Hyypiä, Timo; Rottier, Peter J. M.; Sodeik, Beate; Marsh, Mark; Grüenberg, Jean; Amara, Ali; Greber, Urs F.; Helenius, Ari; Pelkmans, Lucas

doi:10.1038/msb.2012.9

Cited by 158 publications

(143 citation statements)

References 53 publications

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“…Therefore, viral entry pathways can be described only for a particular cell line, and general concepts can be obtained only after testing several host factors (75). This notion is also supported by a small RNAi screen targeting 50 protein kinases that has shown that HSV-1 gene expression relies on different kinases in the HeLaKyoto or HeLaMZ cell lines (36).…”

Section: Discussionmentioning

confidence: 90%

Herpes Simplex Virus Internalization into Epithelial Cells Requires Na ⁺ /H ⁺ Exchangers and p21-Activated Kinases but neither Clathrin- nor Caveolin-Mediated Endocytosis

Devadas

Koithan

Diestel

et al. 2014

J Virol

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Herpes simplex virus 1 (HSV-1) is an alphaherpesvirus that has been reported to infect some epithelial cell types by fusion at the plasma membrane but others by endocytosis. To determine the molecular mechanisms of productive HSV-1 cell entry, we perturbed key endocytosis host factors using specific inhibitors, RNA interference (RNAi), or overexpression of dominant negative proteins and investigated their effects on HSV-1 infection in the permissive epithelial cell lines Vero, HeLa, HEp-2, and PtK 2 . HSV-1 internalization required neither endosomal acidification nor clathrin-or caveolin-mediated endocytosis. In contrast, HSV-1 gene expression and internalization were significantly reduced after treatment with 5-(N-ethyl-N-isopropyl)amiloride (EIPA). EIPA blocks the activity of Na ؉ /H ؉ exchangers, which are plasma membrane proteins implicated in all forms of macropinocytosis. HSV-1 internalization furthermore required the function of p21-activated kinases that contribute to macropinosome formation. However, in contrast to some forms of macropinocytosis, HSV-1 did not enlist the activities of protein kinase C (PKC), tyrosine kinases, C-terminal binding protein 1, or dynamin to activate its internalization. These data suggest that HSV-1 depends on Na ؉ /H ؉ exchangers and p21-activated kinases either for macropinocytosis or for local actin rearrangements required for fusion at the plasma membrane or subsequent passage through the actin cortex underneath the plasma membrane. IMPORTANCE After initial replication in epithelial cells, herpes simplex viruses (HSVs) establish latent infections in neurons innervating these regions.Upon primary infection and reactivation from latency, HSVs cause many human skin and neurological diseases, particularly in immunocompromised hosts, despite the availability of effective antiviral drugs. Many viruses use macropinocytosis for virus internalization, and many host factors mediating this entry route have been identified, although the specific perturbation profiles vary for different host and viral cargo. In addition to an established entry pathway via acidic endosomes, we show here that HSV-1 internalization depended on sodium-proton exchangers at the plasma membrane and p21-activated kinases. These results suggest that HSV-1 requires a reorganization of the cortical actin cytoskeleton, either for productive cell entry via pHindependent fusion from macropinosomes or for fusion at the plasma membrane, and subsequent cytosolic passage to microtubules that mediate capsid transport to the nucleus for genome uncoating and replication.

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Section: Discussionmentioning

confidence: 90%

Herpes Simplex Virus Internalization into Epithelial Cells Requires Na ⁺ /H ⁺ Exchangers and p21-Activated Kinases but neither Clathrin- nor Caveolin-Mediated Endocytosis

Devadas

Koithan

Diestel

et al. 2014

J Virol

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“…HeLa cells were from the ATCC. HeLa Kyoto cells were a kind gift from L. Pelkmans (Institute of Molecular Life Sciences, Zurich, Switzerland) (34). HaCaT cells that originated from N. Fusenig (DKFZ, Heidelberg, Germany) (35) were a kind gift from the Schiller lab (NIH, National Cancer Institute, Bethesda, MD, USA).…”

Section: Methodsmentioning

confidence: 99%

Kallikrein-8 Proteolytically Processes Human Papillomaviruses in the Extracellular Space To Facilitate Entry into Host Cells

Cerqueira

Ventayol

Vogeley

et al. 2015

J Virol

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104

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The entry of human papillomaviruses into host cells is a complex process. It involves conformational changes at the cell surface, receptor switching, internalization by a novel endocytic mechanism, uncoating in endosomes, trafficking of a subviral complex to the Golgi complex, and nuclear entry during mitosis. Here, we addressed how the stabilizing contacts in the capsid of human papillomavirus 16 (HPV16) may be reversed to allow uncoating of the viral genome. Using biochemical and cell-biological analyses, we determined that the major capsid protein L1 underwent proteolytic cleavage during entry. In addition to a dispensable cathepsin-mediated proteolysis that occurred likely after removal of capsomers from the subviral complex in endosomes, at least two further proteolytic cleavages of L1 were observed, one of which was independent of the low-pH environment of endosomes. This cleavage occurred extracellularly. Further analysis showed that the responsible protease was the secreted trypsin-like serine protease kallikrein-8 (KLK8) involved in epidermal homeostasis and wound healing. Required for infection, the cleavage was facilitated by prior interaction of viral particles with heparan sulfate proteoglycans. KLK8-mediated cleavage was crucial for further conformational changes exposing an important epitope of the minor capsid protein L2. Occurring independently of cyclophilins and of furin that mediate L2 exposure, KLK8-mediated cleavage of L1 likely facilitated access to L2, located in the capsid lumen, and potentially uncoating. Since HPV6 and HPV18 also required KLK8 for entry, we propose that the KLK8-dependent entry step is conserved. IMPORTANCEOur analysis of the proteolytic processing of incoming HPV16, an etiological agent of cervical cancer, demonstrated that the capsid is cleaved extracellularly by a serine protease active during wound healing and that this cleavage was crucial for infection. The cleavage of L1 is one of at least four structural alterations that prime the virus extracellularly for receptor switching, internalization, and possibly uncoating. This step was also important for HPV6 and HPV18, which may suggest that it is conserved among the papillomaviruses. This study advances the understanding of how HPV16 initially infects cells, strengthens the notion that wounding facilitates infection of epidermal tissue, and may help the development of antiviral measures. Human papillomaviruses (HPVs) comprise a large family of small, nonenveloped DNA viruses with transforming potential. HPVs selectively infect basal keratinocytes of stratified skin and mucosal epithelia and persist, mostly without clinical symptoms, in virtually every part of the human skin. The biological costs of HPV persistence range from benign papilloma and genital warts over preneoplastic lesions to anogenital or oropharyngeal cancers (1). In fact, infection by the so-called "high-risk" HPV causes about 5% of all human cancers (2). Of these, cervical cancers are the most prevalent. However, HPV-associated oropharyngea...

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“…We find that seed effects are also present in published largescale RNAi datasets that have been corrected for indirect effects occurring through changes in a single cell's microenvironment (27,28) ("population context") (Fig. S7).…”

Section: Discussionmentioning

confidence: 74%

Specific inhibition of diverse pathogens in human cells by synthetic microRNA-like oligonucleotides inferred from RNAi screens

Franceschini

Meier

Casanova

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Systematic genetic perturbation screening in human cells remains technically challenging. Typically, large libraries of chemically synthesized siRNA oligonucleotides are used, each designed to degrade a specific cellular mRNA via the RNA interference (RNAi) mechanism. Here, we report on data from three genome-wide siRNA screens, conducted to uncover host factors required for infection of human cells by two bacterial and one viral pathogen. We find that the majority of phenotypic effects of siRNAs are unrelated to the intended "on-target" mechanism, defined by full complementarity of the 21-nt siRNA sequence to a target mRNA. Instead, phenotypes are largely dictated by "off-target" effects resulting from partial complementarity of siRNAs to multiple mRNAs via the "seed" region (i.e., nucleotides 2-8), reminiscent of the way specificity is determined for endogenous microRNAs. Quantitative analysis enabled the prediction of seeds that strongly and specifically block infection, independent of the intended ontarget effect. This prediction was confirmed experimentally by designing oligos that do not have any on-target sequence match at all, yet can strongly reproduce the predicted phenotypes. Our results suggest that published RNAi screens have primarily, and unintentionally, screened the sequence space of microRNA seeds instead of the intended on-target space of protein-coding genes. This helps to explain why previously published RNAi screens have exhibited relatively little overlap. Our analysis suggests a possible way of identifying "seed reagents" for controlling phenotypes of interest and establishes a general strategy for extracting valuable untapped information from past and future RNAi screens.high-throughput RNAi screening | antimicrobials

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Single‐cell analysis of population context advances RNAi screening at multiple levels

Abstract: A large set of high-content RNAi screens investigating mammalian virus infection and multiple cellular activities is analysed to reveal the impact of population context on phenotypic variability and to identify indirect RNAi effects.

Cited by 158 publications

References 53 publications

Herpes Simplex Virus Internalization into Epithelial Cells Requires Na ⁺ /H ⁺ Exchangers and p21-Activated Kinases but neither Clathrin- nor Caveolin-Mediated Endocytosis

Herpes Simplex Virus Internalization into Epithelial Cells Requires Na ⁺ /H ⁺ Exchangers and p21-Activated Kinases but neither Clathrin- nor Caveolin-Mediated Endocytosis

Kallikrein-8 Proteolytically Processes Human Papillomaviruses in the Extracellular Space To Facilitate Entry into Host Cells

Specific inhibition of diverse pathogens in human cells by synthetic microRNA-like oligonucleotides inferred from RNAi screens

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Single‐cell analysis of population context advances RNAi screening at multiple levels

Abstract: A large set of high-content RNAi screens investigating mammalian virus infection and multiple cellular activities is analysed to reveal the impact of population context on phenotypic variability and to identify indirect RNAi effects.

Cited by 158 publications

References 53 publications

Herpes Simplex Virus Internalization into Epithelial Cells Requires Na + /H + Exchangers and p21-Activated Kinases but neither Clathrin- nor Caveolin-Mediated Endocytosis

Herpes Simplex Virus Internalization into Epithelial Cells Requires Na + /H + Exchangers and p21-Activated Kinases but neither Clathrin- nor Caveolin-Mediated Endocytosis

Kallikrein-8 Proteolytically Processes Human Papillomaviruses in the Extracellular Space To Facilitate Entry into Host Cells

Specific inhibition of diverse pathogens in human cells by synthetic microRNA-like oligonucleotides inferred from RNAi screens

Contact Info

Product

Resources

About

Herpes Simplex Virus Internalization into Epithelial Cells Requires Na ⁺ /H ⁺ Exchangers and p21-Activated Kinases but neither Clathrin- nor Caveolin-Mediated Endocytosis

Herpes Simplex Virus Internalization into Epithelial Cells Requires Na ⁺ /H ⁺ Exchangers and p21-Activated Kinases but neither Clathrin- nor Caveolin-Mediated Endocytosis