Has a protein ever been observed to develop

HIV - Anatomy of a Mass Murderer

The acquired immune deficiency AIDS, like the plague or smallpox, is one of the most devastating epidemics of mankind. AIDS is caused by the human immunodeficiency virus, or HIV for short. There is currently no end in sight to the threat posed by the virus. HIV succeeds in multiplying billions of times daily in the infected person's body and weakening the immune system to such an extent that it soon becomes powerless against otherwise largely harmless environmental germs. Millions of people have fallen victim to the virus so far. What makes the tiny virus capable of these misdeeds? Scientists from the Virology Department at the Hygiene Institute at Heidelberg University want to find answers to this question. The head, Hans-Georg Kräusslich, describes the molecular anatomy of HIV and explains which pathways the virus uses to fatally weaken the immune system.

It began seemingly unspectacular: On June 5, 1981, the magazine "Morbidity and Mortality Weekly Report" published a short note stating that five young men in Los Angeles had a very rare form of pneumonia and two had already died. Shortly afterwards, further noticeable clusters of rare diseases were observed. It soon became clear that it was not just a few cases, but a steadily spreading epidemic caused by a previously unknown pathogen that weakened the immune system. In 1983, a research team led by Luc Montagnier in Paris described a virus that had been isolated from the blood of sick people: it was later referred to as the "human immunodeficiency virus", or HIV for short.

As it turned out, the virus primarily attacks and destroys T helper cells, important members of the body's immune system. If a person has become infected with the virus, their immune system can withstand the virus attacks for the first time, so that the disease does not make itself felt at first. Over the course of several years, however, the regenerative power of the immune system is exhausted. The body's defenses no longer work, the disease AIDS (acquired immunodeficiency syndrome) breaks out: infections that are fended off by the healthy organism spread uncontrollably in AIDS patients and ultimately lead to death.

It is now clear that HIV is the cause of the disease AIDS. The evidence is clear and the perpetrator has been identified. But this knowledge could not stop the spread of the pathogen. To date, over 65 million people have become infected with HIV, and 25 million have already died of AIDS. About three million deaths occur every year. This makes AIDS the leading cause of death worldwide today. In late 2001, the World Health Organization estimated that five million people were newly infected with the virus during that one year. Using these numbers as a basis, it turns out that in the time it takes to read this article, about 150 people will be newly infected with HIV and 85 will die from AIDS.

The situation is currently particularly threatening in Central and South Africa. There, more than a third of the population in the most severely affected regions is infected with HIV. The number of new infections is also increasing dramatically in India and Eastern Europe. Since the epidemic predominantly falls victim to younger people of working age, the effects on the population structure and the economic and political development of the countries concerned are serious.

The AIDS epidemic is a huge global problem. An important measure to contain the disease is to prevent infection with HIV. This assumes that people are fully informed about the risk of infection. However, the current situation shows that this strategy has only limited success. Even a protective vaccine is currently not in sight, despite intensive efforts. It was recently described that even in an HIV-infected person, in whom the first infection had triggered a good response from the immune system, a second infection with a variant of the virus occurred. This raises doubts whether a vaccine that protects against all HIV variants can ever be developed. Given this situation and the millions of people already infected, an urgent goal of biomedical research is and remains to find additional drugs that can effectively fight the virus and control the disease.

Much like catching a murderer, fighting the deadly virus requires different disciplines to work together and complement one another. The solitary detective who solitary gathers traces at the scene of the crime and draws infallible conclusions from them only exists in the novel. In reality, investigators who collect clues or question witnesses are supported by teams of specialists: forensic biologists, forensic biologists or computer experts use their specialist knowledge to help solve the case. In a similar way, scientists from different disciplines have to work together to find ways in which HIV can be suppressed: The findings of the treating physicians about how the disease affects the organism and the effects and side effects of different treatment methods are supported by studies of the Basic medical research supplemented. Similar to coroners, cell biologists, immunologists and pathologists deal with the fate of the victim: They examine the consequences of the virus infection for the individual cell, the immune system and the entire organism. Virologists, molecular and structural biologists, biochemists and biophysicists are more interested in the inner workings of the "perpetrator" HIV. They are trying to analyze his "character" in order to use this knowledge to make him vulnerable.

At first glance, this task seems manageable. Compared to other pathogens, the HI virus is extremely simple. Its genome, with almost 10,000 individual components, is around 20 times smaller than that of a herpes virus and 300,000 times smaller than the human genetic molecule DNA. HIV only needs nine proteins to induce the infected cell to produce new viruses that destroy the host cell, attack other cells - and in the longer term cause AIDS.

However, it is precisely the simple structure of HIV that harbors the problems associated with researching and combating it: in order for the virus to reproduce with so few proteins of its own, it must use numerous functions of the host cell. These cellular functions are very complex - and in some cases have not yet been studied. In addition, the cellular functions are usually out of the question as targets for antiviral therapy, because this would damage the host cell itself. Another problem is that the viruses and the mischief they cause cannot be directly observed. The deadly pathogen measures just 150 nanometers in diameter (around 1 / 7000th of a millimeter). It can therefore not be made visible with a normal light microscope, but only with an electron microscope. In order to elucidate the exact structure of a virus particle and its procedure in the infected cell, modern biophysical and cell biological techniques are necessary.

HIV recognizes its target by certain molecules that sit on the outer skin - the membrane - of human cells. It docks to these surface molecules. In addition to the surface molecule CD4, which is found on many human immune cells, the virus also needs a so-called coreceptor in order to penetrate the cell. One of these coreceptors is built incorrectly in some people because the genes that carry the instructions for its correct construction are changed (mutated). People with this mutation are largely, if not completely, immune from infection with HIV. After the successful docking, the virus fuses with the cell membrane, and the genetic information of the virus reaches the inside of the cell.

The genetic material of the virus is a ribonucleic acid (RNA). It differs chemically from human genetic information (DNA = deoxy-RNA). This difference makes it necessary that the virus's RNA must first be rewritten in DNA. To do this, the virus has brought its own "translator", an enzyme called "reverse transcriptase". After transcription has taken place, the viral DNA reaches the nucleus of the cell in a previously little known way. There it meets the chromosomes. More or less by chance, the viral DNA is incorporated into one of the human chromosomes.

From now on, the genome of the virus is viewed and treated by the cell like a normal human gene: Once integrated, the viral genetic material remains in the cell until it dies. Before this, the genetic information of the virus is passed on to the daughter cells that are created every time the cell divides. Once integrated, the virus genome cannot be "cut out" again. Therefore, even those patients in whom the virus replication can be completely suppressed with medication cannot really be cured at the moment. HIV has therefore developed an ideal strategy to survive in the long term: the genome of the virus remains deep inside the cell in hibernation, as it were, without the intruder being noticed by the organism's surveillance system.

But that's not all: the activation of the virus genome built into the human genome causes the cell to produce new viral RNA and new viral proteins. The components of the virus reach the inside of the cell membrane in a way that is as yet hardly known. There, the virus proteins collect with the RNA to form spherical particles. The cell membrane then turns inside out together with the particle it encloses. Finally, membrane-coated particles detach from the cell. This process is aptly called "virus budding" by experts. It can be clearly seen with an electron microscope.

The new particles released contain all of the components of HIV. However, the particles are unable to infect other cells. They are still "immature" and have to be converted into a "mature", that is, an infectious virus, in a final stage of development. This virus maturation is an extremely interesting process: It begins with a virus' own enzyme - a so-called protease - cutting up proteins of the virus at predetermined interfaces inside the still immature particles. This is followed by a dramatic rearrangement of all components contained in the virus. This rearrangement ends with the development of a mature infectious virus with the typical conical internal structure. The two groups of drugs available to date intervene at crucial points in virus replication and maturation: The "reverse transcriptase inhibitors" block the "translator", the enzyme reverse transcriptase; the so-called protease inhibitors inhibit the "scissors", the protease enzyme, and thus prevent the virus from spreading further.

In order to find further therapeutic starting points, the structure of the virus must be examined down to the smallest detail. It is also important to understand how the individual components of the virus interact with one another. This information can only be provided by basic research.

The dramatic rearrangement described above is particularly interesting for us: most of the components inside the virus change their position and are assigned to a new location: A stable, ordered structure is transformed into a completely different - also ordered - structure. This reorganization, which takes place hidden inside the tiny virus ball, is extremely complex. It is roughly comparable to a completely overcrowded tram in which the passengers change their seats from one order - for example the order in which they boarded - to another order - e.g. that according to increasing age - without being allowed to open the doors of the car . This would probably not work among humans - but the virus has developed strategies that have the high degree of order and coordination necessary for such a process.

This complex "shifting" is controlled by the molecular interactions between the virus' own structural proteins. We are currently trying to understand precisely these interactions. Our goal is to specifically disrupt both particle formation and its maturation and thus to create new approaches for drug therapy. The most important step here is to elucidate the anatomy of the virus - i.e. to know the architecture of immature and mature virus particles at the molecular level.

The methods that are usually used to elucidate molecular structures - X-ray crystallography and nuclear magnetic resonance spectroscopy - are not suitable for this project: HIV particles are too complex and do not have the strictly ordered structure necessary for crystallography. An electron microscope picture clearly shows the structure of the virus, but does not provide all the details and does not provide a three-dimensional insight. For these reasons, we examined mature and immature HIV particles using a special technique called "cryo-electron microscopy". This method allows the virus to be viewed directly in the frozen state - without chemical pretreatment, without staining and without thin sections. At first you don't see much in these pictures. However, since these are projection images of a regular structure, they can be computationally evaluated in such a way that a model of the spatial structure of the virus is obtained.

The resolution is not so great that you could see individual atoms. But it makes individual virus components - the protein molecules - visible. Since the fine structure of some of these proteins is already known in atomic resolution, we can try to fit this information into the overall architecture that we have determined with the help of cryo-electron microscopy. In this way, we want to get a picture of the inside of the virus that - ideally - is accurate down to the atom.

During our research, we discovered that, for all the precision required to assemble them, HIV particles look very different. They are of different sizes and - if you look at the details - they are built differently. Obviously, they are not always designed according to the same assembly instructions with the same number of components. The assembly seems to be based more on a construction plan that can be designed variably. This variability is good for the virus: it helps it to adapt quickly to changing environmental conditions. For us researchers, this variability is less advantageous: it makes it more difficult to develop precisely tailored inhibitors. And even if an inhibitor fits perfectly, the virus can possibly evade it through a rapid change.

Cryo-electron microscopy can - together with other methods - provide images of complete HIV particles in high resolution. However, these images are still far too complex for a detailed analysis. To get a clearer picture, we tried to recreate the inner structure - the capsid - of the mature and immature virus from the individual building blocks in the test tube. Before that, we examined which proteins and which sections of the protein are necessary for this. We then produced the identified building blocks in large quantities using genetic engineering methods and then tried to construct ordered particles from these building blocks that correspond to the capsid inside the virus. In this way it was actually possible to produce ordered particles. Surprisingly, this worked so well that our "artificial" capsids and those derived from mature and immature viruses were so similar that none of the methods available to us could distinguish them.

The particles reproduced in the test tube now help us to understand the molecular anatomy of the complex virus. In addition, the simplified systems can be used to search for active substances that can inhibit virus formation. Of course, the "replicas" from the test tube are far from being viruses - they lack the genome, the membrane covering and the replication apparatus of the virus. Nevertheless, our results show that it is possible to simulate certain functions of the virus with the help of a few, precisely known individual components. They show us a way in which we could succeed in fully deciphering the blueprint for HIV.

However, an image of the pathogen that is true to the original even down to its atomic details will always be a static image. The rigid image does not make it possible to study the dynamic processes during the formation and transport of viruses out of the cell or the infestation of a new cell. We see a "photograph" of the virus, a snapshot with extremely fine resolution - but no film from beginning to end. The "snapshots" must therefore be combined with the dynamic processes in order to capture the complete dramaturgy of the attack.

To do this, we marked the virus with a dye.This allows us to accompany individual components of the virus on their journey into, through and out of the cell. This is made possible by genetic engineering methods with which we smuggled the genetic information for a protein, which originally came from a sea jellyfish, into the genetic makeup of the virus. This protein has a special property: it glows green - it fluoresces - as soon as you illuminate it with ultraviolet light. With the help of fluorescent protein and modern fluorescence optical methods, we can follow in real time in living cells how the viruses are formed from their individual components and how they penetrate the cells.

Using this method, we are likely to accumulate enormous amounts of data. In addition, it can be assumed that the viral transport routes are very variable. A meaningful interpretation of the complex results will therefore probably only be possible with the help of specially developed mathematical models and computer simulations.

But here, too, the limit has not yet been reached: In the meantime, further virus components marked with different colored partners have been developed with which it is possible to observe further details "live and in color". With these methods, we have a good chance of being able to accompany the deadly virus on its complete journey through the cell. Knowing his exact route is linked to the hope of one day being able to irrevocably thwart his fateful paths with new, effective strategies.

Author:
Prof. Dr. Hans-Georg Kräusslich,
Virology Department, Hygiene Institute,
Im Neuenheimer Feld 324, 69120 Heidelberg,
Tel: (0 62 21) 56-50 02; Fax: (0 62 21) 56-5003;
e-mail: [email protected]