Ari Helenius

ETH Zurich | ETH Zürich · Department of Biology

The cell entry mechanism of SARS-CoV-2, the causative agent of the COVID-19 pandemic, is not fully understood. Most animal viruses hijack cellular endocytic pathways as an entry route into the cell. Here, we show that in cells that do not express serine proteases such as TMPRSS2, genetic depletion of all dynamin isoforms blocked the uptake and stro...

The cell entry mechanism of SARS-CoV-2, the causative agent of the COVID-19 pandemic, is not fully understood. Most animal viruses hijack cellular endocytic pathways as an entry route into the cell. Here, we show that in cells that do not express serine proteases such as TMPRSS2, genetic depletion of all dynamin isoforms blocked the uptake and stro...

Viruses are infectious agents that multiply by entering live cells and using the biosynthetic machinery and other services of the host cells to produce new virus particles. Because they themselves have no metabolism, motility, or other complex functions associated with cell life, they can be extremely small and their structure can be simple. Howeve...

Another host factor for SARS-CoV-2 Virus-host interactions determine cellular entry and spreading in tissues. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the earlier SARS-CoV use angiotensin-converting enzyme 2 (ACE2) as a receptor; however, their tissue tropism differs, raising the possibility that additional host factors are...

My coworkers and I have used animal viruses and their interaction with host cells to investigate cellular processes difficult to study by other means. This approach has allowed us to branch out in many directions, including membrane protein characterization, endocytosis, secretion, protein folding, quality control, and glycobiology. At the same tim...

SARS-CoV-2 is the causative agent of COVID-19, a coronavirus disease that has infected more than 6.6 million people and caused over 390,000 deaths worldwide. The Spike (S) protein of the virus forms projections on the virion surface responsible for host cell attachment and penetration. This viral glycoprotein is synthesized as a precursor in infect...

Influenza A virus is a pathogen of great medical impact. To develop novel antiviral strategies, it is essential to understand the molecular aspects of virus–host cell interactions in detail. During entry, the viral ribonucleoproteins (vRNPs) that carry the RNA genome must be released from the incoming particle before they can enter the nucleus for...

Viruses are infectious agents that multiply by entering live cells and using the biosynthetic machinery and other services of the host cells to produce new virus particles. Because they themselves have no metabolism, motility, or other complex functions associated with cell life, they can be extremely small and their structure can be simple. Howeve...

Significance The surface of mammalian cells contains abundant plasma membrane invaginations termed “caveolae.” Caveolae are important for various cellular functions, e.g. signaling, membrane regulation, and vesicular trafficking. Assembly and stability of caveolae depends on a protein coat formed by complexes of cavin and caveolin proteins whose st...

Transport of newly synthesized proteins from the endoplasmic reticulum (ER) to the Golgi complex is highly selective. As a general rule, such transport is limited to soluble and membrane-associated secretory proteins that have reached properly folded and assembled conformations. To secure the efficiency, fidelity, and control of this crucial transp...

Phosphoinositide-3-kinases have been shown to be involved in influenza virus pathogenesis. They are targeted directly by virus proteins and are essential for efficient viral replication in infected lung epithelial cells. However, to date the role of PI3K signaling in influenza infection in vivo has not been thoroughly addressed. Here we show that o...

p110γ is not required for T cell development in the periphery. Naive WT and p110γ-KD mice were analysed using flow cytometry. Shown is a quantification of T cells in lung (A) and blood (B)(mean ± SEM) (n = 5). (C-D) Shown is the activation state of T cells in naive animals in lung and blood. Activated T cells were defined as CD44+CD62L- cells (mean...

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early...

siRNA screen identifies BTB domain containing proteins relevant for IAV infection. A549 cells were depleted of individual BTB domain containing proteins by using up to 4 different siRNAs in a 96-well plate setting. After 72 hr of transfection cells were infected with IAV. Infected cells were visualized by immunofluorescence staining of the viral pr...

The present application provides a method for treating an influenza virus infection in a subject characterised in that a therapeutically effective amount of a modulator of the ubiquitin-binding property of HDAC6 is administered to said subject. The present application also provides an antibody binding to HADC6 and decreasing or blocking its ubiquit...

Human Cytomegalovirus (HCMV) is an important and widespread pathogen in the human population. While infection by this β-herpesvirus in endothelial, epithelial, and dendritic cells depends on endocytosis, its entry into fibroblasts is thought to occur by direct fusion of the viral envelope with the plasma membrane. To characterize individual steps d...

Figure S6: VACV MV infection does not require MT dynamics. HeLa cells were pre‐treated with the indicated compounds at 10 µm for 1 h prior to infection. Cells were then infected with WR E EGFP L mCherry virus (MOI = 2). At 12 h p.i., cells were harvested and analyzed by flow cytometry for both EGFP (black bars; early gene expression) and mCherry (g...

Movie S1. Dynamic colocalization of VACV MVs with Rab5a. HeLa cells expressing EGFP‐Rab5a were infected with WR mCherry‐A4 MVs. Cells were imaged 15 min p.i. at 0.5 Hz. Note coordinated dynamic movement of VACV MVs and Rab5a. Regions of interest are boxed in white.

Movie S3. Dynamic colocalization of VACV MVs with Rab7a‐positive late macropinosomes. HeLa cells expressing EGFP‐Rab7a were infected with WR mCherry‐A4 MVs. Cells were imaged 30 min p.i. at 0.5 Hz. Note the large size of the macropinosome in Movie S3 which appears to contain multiple MVs. Regions of interest are boxed in white.

Movie S7. Dynamic colocalization of VACV MVs with EGFP‐FYVEEEA1. HeLa cells expressing EGFP‐FYVEEEA1 were infected with WR mCherry‐A4 MVs and cells were imaged 15 min p.i. at 0.5 Hz. Note the acquisition of EGFP‐FYVEEEA1 on MVs and their subsequent movement within EGFP‐FYVEEEA1‐positive compartments. Regions of interest are boxed in white.

Figure S1: Automated analysis of VACV internalization and endosome colocalization. A) Detection of endocytic vesicles (endosomes) and viral particles using the imaris software. Digital images of endocytic vesicles, total virus particles (VACV tot) and external virus particles (VACV ext) generated using the imaris spot detection are displayed. B) Au...

Table S1: The ‘usual suspects’ siRNA library. Listed are the three independent siRNAs used for depletion of 162 human genes involved in endocytosis and membrane trafficking. Information includes Entrez gene id (column A), NCBI gene symbol (column B), gene description (column C) and siRNA target sequence (column E).

Table S3: Trafficking hits required for EV infection. Listed are the 10 MV hits (see Table S2) whose depletion resulted in a 40% or greater decrease in EV infection. For each siRNA, the Entrez gene Id (column A), NCBI gene symbol (column B), average relative infection (column H) and average number of nuclei (column J) are displayed.

Movie S4. Dynamic colocalization of VACV MVs with Rab7a‐positive late macropinosomes. HeLa cells expressing EGFP‐Rab7a were infected with WR mCherry‐A4 MVs, and cells imaged 30 min. p.i. at 0.5 Hz. Note the fusion of VACV MV containing late macropinosomes with large Rab7a positive vacuoles.

Movie S6. Dynamic colocalization of VACV MVs with LAMP1. HeLa cells expressing LAMP1‐EGFP were infected with WR mCherry‐A4 MVs, and cells imaged 30 min. p.i. at 0.5 Hz. Note the fusion of VACV MV containing LAMP1‐positive vacuoles.

Figure S2: Colocalization of VACV with endogenous Rab5, Rab7 and LAMP1. A–C) HeLa cells were bound with VACV WR mCherry‐A4 MVs at an MOI of 2 at 4°C. Cells were washed and shifted to 37°C for the indicated time points. Non‐permeabilized cells were then subjected to immunostaining with α‐L1R to distinguish external (blue) versus internalized (red) v...

Figure S3: RNAi screen workflow, image analysis and cell number correction. A) The ‘usual suspects’ siRNA library consists of two 384‐well plates. Three copies of the library (six plates in total) were used in each experiment. The siRNAs were introduced into HeLa ATCC cells by reverse transfection. At 72 h post‐transfection, cells were infected wit...

Figure S4: Colocalization of VACV MVs with SNX3. Cells transfected with EGFP‐SNX3 were infected with WR mCherry‐A4 MVs at an MOI of 2. At the indicated time points, cells were fixed and non‐permeabilized cells were subjected to immunostaining with α‐L1R to distinguish bound virions (purple). White arrows represent colocalization events and represen...

Figure S5: VACV MV infection relies on Rab34 function. HeLa cells were transfected with WT, C/A or D/N versions of EGFP‐Rab34. At 18 h p.i., cells were infected with WR E/L mRFP MVs. Cells were harvested for flow cytometry, and 10 000 transfected cells were scored for infection. Results are displayed as the percent infection relative to infection o...

Table S2: VACV MV trafficking hits. The thirty‐three cell factors whose depletion caused at least a 40% decrease in MV infection are listed. For each siRNA used, the Entrez Gene Id (column A), NCBI gene symbol (column B), average relative infection (column H) and average number of nuclei (column J) are displayed.

Movie S2. Dynamic colocalization of VACV MVs with Rab5a. HeLa cells expressing EGFP‐Rab5a were infected with WR mCherry‐A4 MVs, and cells imaged 15 min. p.i. at 0.5 Hz. Note the fusion of VACV MV containing early macropinosomes with large Rab5a positive vacuoles.

Movie S5. Dynamic colocalization of VACV MVs with LAMP1. HeLa cells expressing LAMP1‐EGFP were infected with WR mCherry‐A4 MVs and cells were imaged 30 min p.i. at 0.5 Hz. Note the large size and highly dynamic movement of the LAMP1‐positive compartments. A VACV MV fusing from a LAMP1‐positive late macropinosome can be seen in Movie S5 (center box)...

The prototypic poxvirus, vaccinia virus (VACV), occurs in two infectious forms, mature virions (MVs) and extracellular virions (EVs). Both enter HeLa cells by inducing macropinocytic uptake. Using confocal microscopy, live-cell imaging, targeted RNAi screening, and perturbants of endosome maturation, we analyzed the properties and maturation pathwa...

Large-scale RNAi screening has become an important technology for identifying genes involved in biological processes of interest. However, the quality of large-scale RNAi screening is often deteriorated by off-targets effects. In order to find statistically significant effector genes for pathogen entry, we systematically analyzed entry pathways in...

During cell entry, capsids of incoming influenza A viruses (IAVs) must be uncoated before viral ribonucleoproteins (vRNPs) can enter the nucleus for replication. After hemagglutinin-mediated membrane fusion in late endocytic vacuoles, the vRNPs and the matrix proteins dissociate from each other and disperse within the cytosol. Here, we found that f...

In addition to classically defined immune mechanisms, cell-intrinsic processes can restrict virus infection and have shaped virus evolution. The details of this virus-host interaction are still emerging. Following a genome-wide siRNA screen for host factors affecting replication of Semliki Forest virus (SFV), a positive-strand RNA (+RNA) virus, we...

Unlabelled: Influenza A virus (IAV) uses the low pH in late endocytic vacuoles as a cue for penetration by membrane fusion. Here, we analyzed the prefusion reactions that prepare the core for uncoating after it has been delivered to the cytosol. We found that this priming process occurs in two steps that are mediated by the envelope-embedded M2 io...

Of the many pathogens that infect humans and animals, a large number use cells of the host organism as protected sites for replication. To reach the relevant intracellular compartments, they take advantage of the endocytosis machinery and exploit the network of endocytic organelles for penetration into the cytosol or as sites of replication. In thi...

Unlabelled: The Bunyaviridae constitute a large family of enveloped animal viruses, many of which are important emerging pathogens. How bunyaviruses enter and infect mammalian cells remains largely uncharacterized. We used two genome-wide silencing screens with distinct small interfering RNA (siRNA) libraries to investigate host proteins required...

Author Summary Certain human papillomaviruses (HPV) are the etiological cause of cervical cancers and other epithelial tumors. Recent advances in the development of anti-HPV vaccines and their increasing deployment provide hope for a significant decrease of these cancers in the future. However, many details of the transmission of HPV between infect...

Significance Pathogens can enter into human cells using a variety of specific mechanisms, often hitchhiking on naturally existing transport pathways. To uncover parts of the host machinery that are required for entry, scientists conduct infection screens in cultured cells. In these screens, human genes are systematically inactivated by short RNA ol...

Poxvirus genome uncoating is a two-step process. First, cytoplasmic viral cores are activated and early viral genes are expressed. Next, cores are disassembled and the genomes released. This second step depends on an early viral factor(s) that has eluded identification for over 40 years. We used a large-scale, high-throughput RNAi screen directed a...

Incoming Simian Virus 40 particles bind to their cellular receptor, the glycolipid GM1, in the plasma membrane and thereby induce membrane deformation beneath the virion leading to endocytosis and infection. Efficient membrane deformation depends on receptor lipid structure and the organization of binding sites on the internalizing particle. To det...

The arenavirus Lassa (LASV) causes a severe hemorrhagic fever with high mortality in humans. Antigen-presenting cells, in particular dendritic cells (DC), are early and preferred targets of LASV and their productive infection contributes to virus-induced immunosuppression observed in fatal disease. Here we characterized the role of the C-type lecti...

Host cell entry of vaccinia virus, the prototypic poxvirus, involves a membrane fusion event delivering the viral core and two proteinaceous lateral bodies (LBs) into the cytosol. Uncoating of viral cores is poorly characterized, and the composition and function of LBs remains enigmatic. We found that cytosolic cores rapidly dissociated from LBs an...

Influenza A virus (IAV) represents a worldwide threat to public health by causing severe morbidity and mortality every year. Due to high mutation rate, new strains of IAV emerge frequently. These IAVs are often drug-resistant and require vaccine reformulation. A promising approach to circumvent this problem is to target host cell determinants cruci...

Western blot showing the protein amount of ATP6V1B2 in the cells treated with AllStars and ATP6V1B2 siRNAs. β-actin actin was used as loading control. (TIF)

Validation of the EE, EA, EU, and EI assays with relevant positive controls. (TIF)

Image analysis steps of each assay. (TIF)

Summary of the virus amounts and the detection time-points of the EB, EE, EA, EF, EU, EI, and infection assays. (TIF)

Sequences of siRNAs targeting ATP6V1B2, ATP6AP2, ATP6V1A, CUL3, and CSE1L genes. (TIF)

Binding of IAV on the cell membrane (EB assay) of AllStars negative and ATP6V1B2 siRNA-treated cells. (TIF)

Human respiratory syncytial virus is a human pathogen that causes severe infection of the respiratory tract. Current information about the structure of the virus and its interaction with host cells is limited. We carried out an electron cryotomographic characterization of cell culture-grown human respiratory syncytial virus to determine the archite...

RSV cell binding to cells treated with inhibitors. HeLa cells were pretreated with solvent (MOCK), genistein (Gen), CAS879127-07- 8, iressa, wortmannin (Wort), LY294002, PI-103, staurosporine (Stau), rottlerin (Rott), calphostin C (CalphC), blebbistatin (Bleb), ML7 at indicated concentrations and individual inhibitor were continuously present at ea...

RSV enters A549 cells by macropinocytosis. (A). RSV (moi ∼0.5) was bound to A549 cells at 4°C followed by 30 min at 37°C. Cells were by IIF with anti-F-AF488 (green), anti-N-AF594 (red), and phalloidin- AF647 (pseudocolored white) for confocal microscopy as, and Z-stack image series acquired. The orthogonal views of image Z-stacks (pseudo-colored w...

SRM assays used to study F0 (UniProt accession number P03420, FUS_HRSVA). (DOCX)

Dose dependent influence of inhibitors on the RSV infection. HeLa cells were pretreated with solvent (MOCK), or (A) chlorpromazine (Chlp), dynasore, dyngo-4a, dynol-34-2, bafilomycin A (BafA), ammonium chloride (NH4Cl), monensin (Mon) (B) cytochalasin D (CytoD), latrunculin A (LatA), jasplakinolide (Jas), nocodazole (Noc), taxol (Tax), NCS23766, pi...

RSV infection of cells expressing Rab5 and Rab7. HeLa cells were transiently transfected with a GFP-Rab5 WT, Rab5 Q79L (C/A), Rab5 S34N (D/N), Rab7 WT, Rab7 Q67L (C/A), Rab7 T22N (D/N) expressing constructs. After 12 h of transient expression cells were infected with rrRSV expressing m-RFF for additional 18 h. After fixation cells were imaged with...

RSV induces transient blebbing of HeLa cells. HeLa cells were inoculated with a purified RSV (moi 30) and immediately imaged with Olympus CellR microscope with DIC settings with the 20× objective, 1 frame per 10 sec speed at 37C. (AVI)

Respiratory Syncytial Virus (RSV) is a highly pathogenic member of the Paramyxoviridae that causes severe respiratory tract infections. Reports in the literature have indicated that to infect cells the incoming viruses either fuse their envelope directly with the plasma membrane or exploit clathrin-mediated endocytosis. To study the entry process i...

The fluid mosaic model for biological membranes was formulated 40 years ago. Ten years later endosomes were discovered as important prelysosomal organelles. At the outset of my research career, I was fortunate to witness both these turning points in biochemistry and cell biology from close up, and to participate in some of the studies. In this shor...

A two-step, automated, high-throughput RNAi silencing screen was used to identify host cell factors required during vaccinia virus infection. Validation and analysis of clustered hits revealed previously unknown processes during virus entry, including a mechanism for genome uncoating. Viral core proteins were found to be already ubiquitinated durin...

Cholera toxin enters cells via an unusual pathway that involves trafficking through endosomes to the endoplasmic reticulum (ER). Whether the toxin induces its own pathway or travels along a physiological retrograde route is not known. To study its trafficking, we labeled cholera toxin B (CTB) or endogenous plasma membrane proteins with a small chem...

This article has been corrected since Online Publication. Extra information giving access to an external website has been removed. Isogenic cells in culture show strong variability, which arises from dynamic adaptations to the microenvironment of individual cells. Here we study the influence of the cell population context, which determines a single...

Supplementary Figures S1–32, Supplementary Tables S1–4