Single virus tracing sets the stage for new infection-fighting drugs and gene-therapy strategies, Science authors say

November 29, 2001

This release has been updated as of Nov. 27.

This release is available in German by clicking here.

MUNICH, GERMANY--The single virus, tagged with one fluorescent dye molecule, bumps against a living cell once, twice--up to five times--making contact for less than a second on each approach before the cell suddenly engulfs it. Only a fraction of all single viruses make it past the external cell membrane, new research suggests. But, once inside, the successful invading virus has a good chance of penetrating its target, the nucleus, within a few minutes.

Surprisingly, researchers report in the 30 November 2001 issue of the journal Science, key infection steps occur much faster than previously believed. Moreover, tubular structures might serve as pipelines for viruses, which perhaps ride piggyback on "motor proteins" to reach the nuclear area, where genetic expression occurs, promoting infection.

Such insights--illustrated in real-time movies created by scientists of the Ludwig-Maximilians-Universität München--may set the stage for better anti-viral drugs, or for delivering gene-therapy medicines directly to the cell nucleus. "For the first time, we have been able to observe a single virus, labeled with only one dye molecule, on its infectious entry pathway into a living cell," explains researcher Christoph Bräuchle, senior author on the Science paper, published with his University colleagues, Georg Seisenberger, Martin U. Ried, Thomas Endreß, Hildegard Büning and Michael Hallek. "Labeling with only one dye molecule minimizes the distortion of virus-cell interactions. In addition, the single virus tracing technique shows us that infection happens much more rapidly than we previously assumed-within minutes, rather than hours."

To study different stages of infection pathways, so far researchers typically have used a conventional technique called fluorescence microscopy, often in conjunction with sample-staining methods. For example, the protective shell of protein that surrounds the genetic material of a virus, known as the capsid, can be labeled with fluorescent dye molecules. Labeled capsids can be visualized using light to excite the dyes, which then release fluorescent light as they de-energize.

Unfortunately, Bräuchle and Hallek say, this conventional approach requires a high concentration of viruses, or a high degree of labeling, or both. The former makes it difficult to see individual components within the crowded sample, whereas large quantities of dye may interfere with cell-virus interactions, possibly distorting natural, physiological events, he added.

The teams of Bräuchle and Hallek used a more sensitive fluorescent microscopy system, capable of measuring the signal from a lone fluorophore, to investigate the movements of Adeno-associated viruses (AAVs)--harmless versions of parvoviruses that are non-pathogenic for human beings. Individual viruses were labeled with a single Cy5 dye molecule, then brought in contact with HeLa cells (a commonly used line of living human cells, so named after the late Henrietta Lacks, a cancer patient whose cells now provide a model system for scientists).

Within a total of 74 cells at different stages of infection, the research team analyzed 1,009 trajectories of single, dye-labeled viral particles. Again and again, they watched the virus slow down as it approached the cell surface. This deceleration was followed by a series of brief "touching events," with the viral particle making contact with the cell surface, as if ringing a doorbell four or five times. "It is not clear yet whether these touching events represent binding-and-release processes at a receptor, or simply unspecific adsorption of the virus to the cell surface," the researchers noted. Clearly, however, the virus was trying to gain entry to the cell.

In 13 percent of all approaches having membrane contact (43 of 339 trajectories) the virus penetrated the cellular surface. Within a fraction of a second, the virus was then sucked into the cytoplasm through endocytosis, an engulfment process that begins when the cellular membrane forms a cellular receiving compartment called an endosome. Bräuchle's group was even able to distinguish between viruses contained within 50-nanometer endosomes and faster-moving, free viruses, thus confirming that endosomes do indeed play a key role in transporting single viruses into cells following penetration.

In 15 minutes, half of the 74 cells were infected, with at least one dye-labeled virus detected in the nuclear area. "This indicates a much faster infection compared to the two-hour infection time" previously measured using conventional methods, the Science paper points out.

"Viruses are clever particles, capable of using the cell's own transportation system, that consists of motor proteins which run along microtubules as tracks. Riding on the back of the motor proteins, they are transported within the cytoplasm," says Bräuchle.

Even in the nuclear area, where such motor proteins have not been observed before, viral particles seem to follow such tracks: "To our great surprise, in 34 of the trajectories within the nuclear area, the viral particles underwent directed motion along well-defined pathways," the German research team reported. "Some pathways were used several times consecutively, by different viral particles." They speculate that the tracks for the motor proteins have extended into the existing, well-known tubular structures, or "pipelines," which are formed inside the nuclear area during infection events (resulting from an invagination of the nuclear envelope). In this way, the directed motion of viruses inside the nuclear area may be described as piggyback transportation of motor proteins on the tracks inside such pipelines.

"In our measurement, the whole entry process takes place in a time range from some seconds to a few minutes," Bräuchle said. "From our observation of the virus infection pathway into a living cell, with a high degree of clarity and detail, we can obtain the 'movie script' of the infection."

In the future, Bräuchle notes, such detailed, step-by-step information on viral pathways may help pharmaceutical makers find new ways to block infections. Understanding their pathways will also improve delivery vectors for gene therapy, Hallek adds.
-end-
The authors, Georg Seisenberger, Thomas Endreß and Christoph Bräuchle, are at the Department of Chemistry of the Ludwig-Maximilians-Universität München. Christoph Bräuchle is also a member of its Center of NanoScience (CeNS).

Authors Martin U. Ried, Hildegard Büning and Michael Hallek are from the Gene Center of the Ludwig-Maximilians-Universität München. Michael Hallek is also at the University Hospital Grosshadern.

MEDIA NOTE: A press conference for journalists only--jointly planned by Ludwig-Maximilians-Universität Munchen and the journal, Science--will take place at 14:00 Hours in the City of Munich, Geschwister-Scholl-Platz 1, Senatssaal (subway station "Universität"). Journalists are asked to pre-register by contacting Cornelia Glees-zur Bonsen of Ludwig-Maximilians-Universität Munchen, 49-89-2180-3423, cornelia.glees@verwaltung.uni-muenchen.de; or Tobias Ernst of HCC DeFacto, 49-89-3866-7460, t.ernst@hccdefacto.de

Reporters who wish to receive an advance copy of the embargoed Science paper, please contact Ginger Pinholster at +202-326-6421, gpinhols@aaas.org, or Lisa Onaga, +202-326-7088, lonaga@aaas.org

CONTACTS: Cornelia Glees-zur Bonsen, Ludwig-Maximilians-Universität, 49-89-2180-3423, cornelia.glees@verwaltung.uni-muenchen.de; Ernst Tobias, HCC DeFacto, 49-89-3866-7460, t.ernst@hccdefacto.de; Ginger Pinholster,

American Association for the Advancement of Science

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