TURNING POINTS Virus entry: What has pH got to do with it? Ari Helenius After several years of working on membrane For almost two years, the virus entry path- Over the following months, we validated proteins using the Semliki Forest virus (SFV) as way and mechanism of host penetration con- all the predictions of our pH hypothesis. For a model, I decided in 1976 to focus my research tinued to elude us. However, in the spring example, we could demonstrate a membrane on the mechanisms by which animal viruses of 1978, I had the chance to participate in a fusion reaction in vitro by simply mixing such as SFV enter and infect their host cells. Dahlem Conference in Berlin called ‘Transport liposomes and SFV, and briefly dropping the pH I had just moved from Finland with the lab of of Macromolecules in Cellular Systems’. It was to 6 or below. Moreover, when we added acidic Kai Simons to the newly founded European an opportune time for a meeting on this topic: medium to cells, we could ‘fool’ surface-bound Molecular Biology Laboratory (EMBL) in pathways of vesicle transport were being dis- viruses into fusing with the plasma membrane; Heidelberg, Germany. I was eager to tackle a covered, receptor-mediated trafficking pro- the entry block caused by weak bases was challenging problem — and one that did not cesses were beginning to be described, and the bypassed and the cells became infected. require work with detergents. important role of clathrin-coated vesicles was Joined by Judy White, Mark Marsh and Relying almost exclusively on electron finally properly appreciated. All the leaders in Karl Matlin as postdoctoral fellows in the lab, microscopy, previous work on animal virus the membrane cell biology field were present. we extended the acid-activation concept to entry had left the field divided between pro- During a coffee break, I asked William Sly, a other viruses. For the influenza A virus, the ponents of direct virus penetration through pioneer in the trafficking of mannose-6-phos- threshold was a pH-unit lower, and exposure the plasma membrane of the host cell and phate receptors, whether he knew what weak to low pH induced a dramatic, irreversible those who favoured penetration into the bases such as ammonium chloride and chlo- conformational change in the haemagglutinin cytosol from intracellular vacuoles after endo- roquine do to cells. He directed me to a paper glycoprotein. We realized, moreover, that the cytosis. Together with Jürgen Kartenbeck, a by Brian Poole showing that these agents raise acidic organelles where viruses were activated colleague in the German Cancer Research the pH in lysosomes. were not lysosomes, but pre-lysosomal vacu- Center (where we were temporarily housed), When Erik Fries and I were discussing the oles. We called these vacuoles ‘endosomes’, a and Erik Fries, a Swedish post-doctoral fel- conference in the laboratory the following name that soon became the general term used low, we studied what happened when SFV was week, he made a remark that changed every- for these complicated and dynamic compart- added to cells in tissue culture. Using electron thing. He simply asked, “Could pH have some- ments. microscopy, we visualized virus particles thing to do with it?” Suddenly, all the pieces of Today, we know that most viruses (whether binding to the cell surface, becoming inter- the puzzle fell into a coherent picture. We real- enveloped or not) use endocytic entry mecha- nalized by coated vesicles and accumulating ized that the acidic pH in lysosomes and other nisms. For the majority, a drop in pH serves as in intracellular vacuoles. Biochemical assays endocytic vacuoles was not only required to a trigger for host penetration. Some bacterial with radioactive virus particles demonstrated optimize the degradative action of acid hydro- toxins also rely on low pH as a cue. Moreover, that endocytosis was rapid and efficient. We lases, but could also serve as a ‘cue’ for viruses it is well known that differences in the pH confirmed that infection was prevented by to activate their penetration mechanism. between compartments regulate directional weak bases such as ammonium chloride and Exposure to low pH ‘told’ the viruses that they transport of cargo in the endocytic and secre- chloroquine, and showed that this involved had entered a cell and reached the endocytic tory pathways. Endosomes have attained a vis- inhibition of virus entry. However, these com- pathway, and that it was time to activate the ibility and recognition that those of us working pounds did not inhibit the endocytic uptake penetration machinery. We speculated that low on them in the early 1980s could hardly have of SFV. To our disappointment, we had no pH induced a change in the spike glycoproteins dreamt of. I have had other turning points evidence of fusion between host and virus present on the virus envelope that allowed along my scientific path, but none of them with membranes. fusion of the viral envelope from the lumenal quite as many significant consequences as the side with the limiting membrane of the vacuole. one involving endosomal entry of SFV and the As a result, the viral capsid was released into role of pH. Ari Helenius is at the Institute of Biochemistry, ETH Zurich, HPM E6.3, 8093 Zurich, Switzerland. the cytosol without itself having to cross the COMPETING FINANCIAL INTERESTS e-mail: [email protected] hydrophobic barriers formed by a membrane. The author declares no competing financial interests. NATURE CELL BIOLOGY VOLUME 15 | NUMBER 2 | FEBRUARY 2013 125 © 2013 Macmillan Publishers Limited. All rights reserved.