Scientists image a single HIV particle being born

A mapmaker and a mathematician may seem like an unlikely duo, but together they worked out a way to measure longitude – and kept millions of sailors from getting lost at sea. Now, another unlikely duo, a virologist and a biophysicist at Rockefeller University, is making history of their own. By using a specialized microscope that only illuminates the cell’s surface, they have become the first to see, in real time and in plain view, hundreds of thousands of molecules coming together in a living cell to form a single particle of the virus that has, in less than 25 years, claimed more than 25 million lives: HIV.

This work, published in the May 25 advanced online issue of Nature, may not only prove useful in developing treatments for the millions around the globe still living with the lethal virus but the technique created to image its assembly may also change the way scientists think about and approach their own research.

“The use of this technique is almost unlimited,” says Nolwenn Jouvenet, a postdoc who spearheaded this project under the direction of HIV expert Paul Bieniasz and cellular biophysicist Sandy Simon, who has been developing the imaging technique since 1992. “Now that we can actually see a virus being born, it gives us the opportunity to answer previously unanswered questions, not only in virology but in biology in general.”

Unlike a classical microscope, which shines light through a whole cell, the technique called total internal reflection microscopy only illuminates the cell’s surface where HIV assembles. “The result is that you can see, in exquisite detail, only events at the cell surface. You never even illuminate anything inside of the cell so you can focus on what you are interested in seeing the moment it is happening,” says Simon, professor and head of the Laboratory of Cellular Biophysics.

When a beam of light passes through a piece of glass to a cell’s surface, the energy from the light propagates upward, illuminating the entire cell. But when that beam is brought to a steeper angle, the light’s energy reflects off the cell’s surface, illuminating only the events going on at its most outer membrane. By zeroing in at the cell’s surface, the team became the first to document the time it takes for each HIV particle, or virion, to assemble: five to six minutes. “At first, we had no idea whether it would take milliseconds or hours,” says Jouvenet. “We just didn’t know.”

“This is the first time anyone has seen a virus particle being born,” says Bieniasz, who is an associate professor and head of the Laboratory of Retrovirology at Rockefeller and a scientist at the Aaron Diamond AIDS Research Center. “Not just HIV,” he clarifies, “any virus.”

To prove that what they were watching was virus particles assembling at the surface (rather than an already assembled virion coming into their field of view from inside the cell), the group tagged a major viral protein, called the Gag protein, with molecules that fluoresce, but whose color would change as they packed closer together. Although many different components gather to form a single virion, the Gag protein is the only one necessary for assembly. It attaches to the inner face of the cell’s outer membrane and when enough Gag molecules flood an area, they coalesce in a way that spontaneously forms a sphere.

Simon, Bieniasz and Jouvenet found that the Gag molecules are recruited from the inside of the cell and travel to the cell’s surface. When enough Gag molecules get close and start bumping into each other, the cell’s outer membrane starts to bulge outward into a budding virion and then pinches off to form an individual, infectious particle. At this point, the researchers showed that the virion is a lone entity, no longer exchanging resources with the cell. By using tricks from optics and physiology, they were able to watch the steps of viral assembly, budding, and even scission off the cell surface. With such a view they can start to describe the entire lifeline in the birth of the virus.

“I think that you can begin to understand events on a different level if you actually watch them happen instead of inferring that they might occur using other techniques,” says Bieniasz. “This technique and this collaboration made that possible.”

Source: Rockefeller University

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Citation: Scientists image a single HIV particle being born (2008, May 25) retrieved 23 February 2019 from
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May 26, 2008
So the formation of viral particles was observed in detail.

What tells us that

1) those are HIV viral particles?


2) that those particles are "infective"?

May 26, 2008
It tells us a couple of things: The virus was formed at the outer cell surface. Confirming a hypothesis. It doesn't tell us that the new cell is infectious. We already know that. But it does show us how the virion is formed, and thus introduces us to some intervention possibilities.

May 27, 2008
I have a feeling that it may not be scientifically justified to assume that what is observed are HIV particles budding, or that these particles are infective.

What if the budding of such particles is a common occurrence of normal cell metabolism - has anyone checked?

"It doesn't tell us that the new cell is infectious. We already know that."

And how, pray tell, do we know?

May 27, 2008
This article does not mention what kind of cell culture was utilized in the experimental imaging of the so-called budding phenomenon. I am confident the original article, published in Nature, would have had to report such facts. However, any commentary based upon the reporting in this review article is largely a waste of time. I recommend that those who are interested in debating the merits of the original article, first read the original article. In a vaccum, Sepp's question is a fair one: by what means was the infected specimen obtained? How was that culture treated? How was it determined that this specimen was, indeed, infected with a virus? Once "viral budding" was observed, did the researchers then attempt to cull said virions and infect a healthy animal (Koch's Postulates)? Noting that there is no approved standard for the direct determination of HIV infection, exactly how did the researchers decide that they were, in fact, working with an HIV-infected specimen? Again, only a close reading of the original article will answer these questions. More repartee on this review article is pointless. Good luck analyzing the original data!

May 30, 2008
Here is a comment of a French scientist that explains the difficulty of preparing any kind of solution that actually shows the presence of HIV viral particles:

Blood samples are prepared for "viral load" by separating circulating leucocytes, and extracting their cellular nuclei. It is on such chromatin rich samples preparations that PCR technology is applied to identify and amplify nucleic acid sequences analogous to retroviral genome, the results being interpreted as measuring a hypothetical "viral load".

It is well known that an impressive proportion of the human genome is analogous to retroviral sequences. It is, therefore, not surprising at all that retroviral sequences are found in such samples, and could easily be amplified by PCR. But these findings
have NOTHING to do with the hypothetical presence, and even less the quantification, of retroviral particles in the blood sample from the studied patient!

This method simply bypasses what should be the obvious initial step, ie the separation of RETROVIRAL PARTICLES of HIV !!!

If, instead of using extracts from leucocyte nuclei, the patient blood plasma was first treated with one of the classic methods for the isolation of retroviral particles (like sucrose gradient centrifugation), and if the nucleic acid of these isolated particles was next analysed by PCR technology, then I would think that the PCR results might have something to do with the presence of an hypothetical retrovirus.

But this is NEVER done! I have been debating this for many years! I remember very well, in Pretoria, in May 2000, during the first Mbeki's conference, spending a long evening with Luc Montagnier and explaining to him that the PCR viral load technology was plain nonsense because it bypasses the essential initial step of retroviral particle isolation. Montagnier agreed that particle isolation should indeed be the first step in the procedure, and he even invited me to his lab at Pasteur in Paris to do the experiment with him! I should have jumped !!

This is perhaps the most striking example of a huge problem in specimen preparation, a problem that completely destroys all significance of so-called viral load measurements!!

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