Newswise — Viruses have at least a billion years experience in entering host cells whereas cell biologists have had little more than sixty years to figure out how viruses do it. Spurred on by the disease implications of viruses from influenza to HIV, researchers have pieced together the viral entry story, at least its broad outlines. Most animal viruses, they say, use a Trojan horse strategy, binding themselves to the surface of the cell and waiting to be carried inside by a process called endocytosis. For the prototypic endocytic pathway, a lysosome is waiting at the end to degrade this seemingly passive trespasser. But the virus strikes first, sensing the lowering pH of the route to the lysosome and instead maneuvering into the cytoplasm where it often heads for the nucleus to hijack the cell's genetic replication machinery.
That's the story that Alicia Smith, Ari Helenius and their colleagues at the Swiss Federal Institute of Technology in Zurich expected to record when they applied advanced light microscopy techniques to film several different viruses types as they navigated the viral entry process step-by-step in living cells. Using real-time fluorescence microscopy (epifluorescence, confocal, and total internal reflection fluorescence) in combination with biochemical and biophysical assays, Helenius and company added fluorescently labeled virus particles to cells and watched as they undertook the endocytic journey. Two types of the glowing viruses took unexpected infection routes.
Both involve cholesterol-dependent, virus-triggered pathways that involve pH-neutral, non-endosomal intermediate compartments. The first was revealed by the simian virus 40 (SV40) which made its entry through caveolae (small caves) in the plasma membrane made up of discrete lipid collections called "lipid rafts" that pinched off into the cell interior. The virus was then delivered to a novel organelle that the researchers named a caveosome, a sealed membrane organelle that directed SV40 to the endoplasmic reticulum where it launched its viral attack.
(See the videos at http://www.ascb.org/publicpolicy/pressbooks/pressbook04.html) A SV40 relative, the murine polyomavirus, revealed a second non-endosomal viral entry pathway. The polyoma virus seems to bind to carbohydrate-containing cell surface lipids. This generates a signal inside the cell that leads to formation of small, uncoated, cholesterol-containing membrane vesicles at the plasma membrane. The vesicle detaches with the virus inside, starting the invader on a pathway through a series of organelles culminating in the endoplasmic reticulum where the virus escapes into the cytoplasm.
The unexpected diversity of viral entry routes raises a number of important questions says Helenius. How many viral entry pathways are there? How can they be inhibited to prevent infection? What normal cellular functions do these pathways provide? Clearly there are a few more viral and cellular secrets hidden behind the billion years of experience.
Alternate endocytic pathways revealed by virus entry into animal cells, A. E. Smith,1 H. Ewers,1 I. Sbalzarini,2 P. Koumoutsakos,2 A. Helenius1 ; 1 Institute of Biochemistry, Swiss Federal Institute of Technology, Zurich, Switzerland, 2 Institute of Computational Science, Swiss Federal Institute of Technology, Zurich, Switzerland.
At the meeting: Session 231, Endocytosis I, Poster Presentation 1085, Halls D/E. Author presents: Sunday, Dec. 6, Noon — 1:30 PM.
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