A short piece of RNA or DNA, surrounded by a protein container and maybe a membrane - a good description of a virus. Viruses have a very simple structure and this is precisely the reason why they attack their host organisms - they require the chemical environment of a living cell for reproduction. Virologists in Prof. Dr. Michael Nassal’s group at the University Medical Centre in Freiburg are investigating the molecular mechanisms used by the hepatitis B virus to reproduce. The researchers’ work might in future be able to help the three to four hundred million people worldwide who suffer from chronic hepatitis B infections. The researchers are especially interested in a viral protein that ‘pulls itself up by its own bootstraps’.
Approximately two billion people worldwide have probably been in contact with this virus. This assumption is based on the molecular traces the hepatitis B virus (HBV) leaves in blood samples. Approximately 350 million people worldwide suffer from chronic HBV infections, which can lead to hepatitis, liver cirrhosis or even to liver cancer. The situation is especially menacing in Asia, southern Africa and in parts of Latin America. But even Europe suffers from the problem that current anti-HBV drugs quickly lose their effect. So-called nucleoside analogues, for example, are still able to effectively prevent the replication of the viruses, but the same viruses begin to adapt to a given drug within a few months. “We are therefore looking for completely new inhibition mechanisms,” said Prof. Dr. Michael Nassal of the Department of Gastroenterology, Hepatology, Endocrinology and Infectiology at the University Medical Centre in Freiburg. “But this requires us to understand in detail how HBV reproduces and how it replicates its genome.”
Human HB viruses are extremely host-specific; they almost exclusively attack the liver cells (hepatocytes) of people and apes. Packed into a lipid membrane and inside a protective protein container (capsid), HB viruses have a partially double-stranded circular, though not entirely closed, DNA genome. The genome is relatively small and consists of only about 3000 base pairs. HB viruses are among the smallest autonomously reproducing viruses.
Infection starts at the membrane of a liver cell. One or more unknown receptors recognise specific surface structures on the viral envelope and mediate the inward transfer of the virus. When the virus has entered the cell, it discards its envelope and is present as a blank capsid. The viruses use another, still unknown, mechanism to reach the nuclear membrane and release their DNA into the cell nucleus. “What happens now is completely unclear,” said Nassal. “The only thing we know is that the structure of the viral genome changes.” The DNA forms a completely closed double-strand ring (cccDNA, circular covalently closed DNA). Nassal and his colleagues assume that the enzymes of the cellular DNA repair system play a role in forming this ring. The normal function of these enzymes is to repair errors in the host’s own DNA. They also realise that part of the viral genome is present as a single strand and thus close the gaps – a fatal error. One of the researchers’ projects focuses on identifying the enzymes and the mechanisms that play a role in this gap closing. It is essential for the researchers to understand how cccDNA is generated since it plays a key role in the viral reproduction cycle. cccDNA is not only transferred to both daughter cells when the cell divides, but also serves as a template for the cell’s own transcription enzymes. Amongst other things, these enzymes produce messenger RNAs (mRNAs) of the seven viral proteins that remain outside of the nucleus, where they serve as the constituents for new virus particles. Another major product is what is known as pre-genomic RNA (pgRNA), which comprises the entire genetic information of the DNA genome. This exits the cell nucleus and can be packaged into new capsids. However, there is still one problem with this process: the pgRNA is not at this point available as DNA. The problem is solved by another protein, in which Nassal and his team are particularly interested.The particular enzyme of interest is also found in retroviruses such as HIV and is called reverse transcriptase. It is one of the products of pgRNA, produced from pgRNA by the host’s protein biosynthesis enzymes. The enzyme is able to revert the normal process of transcription by transcribing RNA into DNA. Many years ago, Nassal and his team identified the steps induced by this unique molecule. They used an experimental system in which the isolated components could be analysed in a test tube away from the uncontrollable environment of the cell interior. These experiments revealed that the reverse transcriptase interacted with the protein constituents of the capsid; replication can only take place inside the capsid. But the reverse transcriptase needs to bind to a specific site in the pgRNA structure, a hairpin loop known as epsilon. This binding is required for the exclusive packaging of the viral pgRNA (not cellular RNAs or other viral messenger RNAs). Replication cannot start without this binding. Support comes once again from the host cell: so-called chaperons, which normally help proteins to fold correctly, appear to mediate the formation of this complex.
"The HBV reverse transcriptase is different from all other related retroviral enzymes," said Nassal explaining that all known reverse transcriptases function in a similar way: they use an RNA as template and pair it with complementary DNA constituents, leading to the generation of a negative copy of the RNA strand, which consists of DNA. In order to initiate base pairing, the enzymes need a short start sequence, which is known as primer, which another cellular enzyme needs to adapt to the RNA template. Reverse transcriptases are only able to prolong existing sequences. However, the HBV reverse transcriptase ‘pulls itself up by its own bootstraps' like Baron Münchhausen: it generates its own primer by using an additional protein domain, the so-called terminal protein (TP). This protein piece uses a short sequence of the epsilon loop as template and pairs it with DNA constituents. The resulting DNA piece is then placed at the other end of the pgRNA where it is used as primer for the actual replication process.
In the future, Nassal and his team plan to clarify the precise 3D structure of the complex consisting of reverse transcriptase and pgRNA. They already know that the binding is achieved by several intermediary steps and that energy is needed for this process. It is difficult to find out which spatial changes occur during this process. Therefore, they have joined forces with biophysicists who are experts in imaging methods and they are hoping that this cooperation will bring them a step further towards an understanding. This is of course pure basic research. "However, in order to be able to develop truly new drugs against this virus, we need to understand the basic mechanisms," said Nassal. Nassal and his team are also pursuing other projects that focus on the role of cellular chaperons for the replication of the virus, how the binding of reverse transcriptase to the capsid constituents can be prevented, and how cccDNA is generated in the nucleus. Maybe in a few years' time, there will be some new approaches for the development of drugs.
Further information: Prof. Dr. Michael NassalFreiburg University Medical CentreInternal Medicine II / Molecular BiologyHugstetter Str. 55D-79106 FreiburgTel/Fax: +49 (0)761/270-3507E-mail: nassal(at)ukl.uni-freiburg.de