While public health experts were exploring strategies to prevent the spread of HIV and clinicians were attempting to develop drugs that might treat or perhaps even cure AIDS, laboratory scientists were studying the virus itself. From the first sequence of HIV, published in 1985 (see Wain-Hobson, this series), it was clear that the virus differed from other known retroviruses by including additional genes besides the usual gag, pol and env. What were the functions of these genes? Did they affect virus replication, pathogenesis or transmission? In his commentary, Dr. Alan Frankel recalls the almost serendipitous discovery he made in 1988 of an interesting property of the Tat protein. He observed that Tat had the unusual ability to cross both the plasma membrane and nuclear envelope, going directly to the nucleus. This discovery became important beyond the HIV field, as investigators in other fields exploited this phenomenon to develop new methods for protein delivery into cells.
Cellular Uptake of the Tat Protein from Human lmmunodeficiency Virus
Cell, Vol. 55, 1189-l 193. December 23, 1988, Copyright 1988 by Cell Press
Alan Frankel and Carl Pabo
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1987 < All years > 1989
Commentary by Dr. Alan Frankel
Alan Frankel, Professor
Department of Biochemistry and Biophysics, UCSF
One of the greatest pleasures of scientific research is experiencing an “aha moment.” I had such a moment in 1988, about a year after beginning work on the biochemistry of HIV-1 Tat. I was a postdoctoral fellow with Carl Pabo, a crystallographer at Johns Hopkins, and our goal was to determine the structure of Tat, a suspected zinc-binding protein. At the time, the function of Tat was poorly understood but was believed to be a DNA-binding transcription factor, not an RNA-binding factor as it is now known to be. My prior experience with DNA-binding zinc fingers allowed me to purify Tat expressed in bacteria but, without a known binding site, I needed some other way to measure its functional activity. I came across a technique in which a cell scraper was used to literally rip open the membranes of adherent tissue culture cells, allowing exogenous proteins to enter. So I added Tat to scraped HeLa cells containing an LTR reporter and, lo and behold, observed massive stimulation of the reporter gene. Then I performed an obvious control, adding Tat to non-scraped cells and – the “aha moment” – observed the same stimulation. Somehow, Tat could enter cells and make its way into the nucleus.
This bit of serendipity was followed by an agonizing week in which I could not reproduce the result. However, the initial data were so striking I simply could not believe they were wrong. Finally, I realized that in my subsequent experiments I had streamlined the protocol, washing the cells only once, not twice, before adding fresh Tat-containing medium. The residual trypsin remaining from the passage solution was enough to degrade the added Tat, and so, reverting to the original protocol, I was able to confirm that the “aha moment” was real.
I immediately thought the finding was interesting for two reasons: First, it suggested that extracellular Tat might play some role in HIV biology. For example, Tat released by killed infected cells might enter other latently infected cells to stimulate viral replication, or Tat might enter uninfected cells and alter expression of cellular genes. Second, the ability of a nuclear protein to so efficiently enter cells – it turns out many different cell types – might provide a useful tool for protein delivery. Indeed, Tat-mediated protein transduction has spawned a new field, contributing to the many citations of this paper.
As for HIV biology, a follow-up study (EMBO J. 10: 1733, 1991) suggested that Tat binds tightly but nonspecifically to many cell types and becomes endocytosed. Other studies suggest that additional entry pathways may exist, especially when Tat peptides are fused to other proteins to facilitate their delivery. Given the lack of a specific cell surface receptor, the biological roles of extracellular Tat, its location, and its ability to circulate systemically remain poorly understood. However, it is clear that extracellular Tat can elicit many types of effects in tissue culture cells, can alter expression of cellular genes, and can stimulate virus replication, especially when Tat is released from infected cells and enters neighboring cells. Thus, exploring the functions of extracellular Tat remains an active area of investigation.
If extracellular Tat does, indeed, contribute to HIV biology and pathogenesis, it provides a potential therapeutic target. Many HIV-infected individuals raise strong antibody responses to Tat, and there appears to be a correlation between strong response and slower disease progression, prompting ongoing attempts to develop Tat-based vaccines. From the perspective of a basic researcher, it would be just desserts if what started as a simple control experiment might eventually connect to strategies to control HIV infection.
About the author: Alan Frankel is a Professor at University of California San Francisco.
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