Baking, brewing…and virology: a fresh look under the hood of hepatitis B virus!

In the 1960s Barry Blumberg and colleagues demonstrated that an infectious agent was responsible for hepatitis B. The discovery of hepatitis B virus (HBV) was undoubtedly a milestone for the development of reliable diagnostics, a vaccine and antiviral therapies.
Published in Microbiology
Baking, brewing…and virology: a fresh look under the hood of hepatitis B virus!
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HBV replication in cultured cells
Human hepatoma cells harboring modified hepatitis B virus cccDNA are labeled by green fluorescent proteins. Blue oval shapes are cell nuclei labeled by fluorescent Hoechst dye, which stains DNA. Nuclei without green fluorescent protein are cells that do not contain hepatitis B virus cccDNA.

Nonetheless, a estimated 2 billion people have become exposed to HBV. Unfortunately, 90% of children will progress to chronicity whereas those who contract the virus in adulthood have a ca 90% of clearing HBV. Chronic hepatitis B can lead to severe liver disease including fibrosis, cirrhosis or hepatocellular carcinoma (HCC). In fact, the great majority of HCCs world-wide can be attributed to HBV.

The first hepatitis B vaccine became available in the early 1980s and its wide-spread administration proved to be successful in reducing viral transmission. However, the vaccine can only prevent new infections but has no effect on already established chronic infections. The advent of antivirals targeting the HBV reverse transcriptase was critical for suppressing viremia in chronically infected patients. However, current treatment rarely leads to a cure and even patients on continuous antiviral treatment remain at an elevated risk of developing HCC.

With at least 257 million chronic HBV carriers there is a continued urgent need for developing more effective therapies. Progress towards curative treatments has been hampered by the incomplete understanding of the viral life-cycle. HBV is a very small enveloped virus that encodes its genetic information as DNA. The form of its genome that the virion carries into the host cell is referred to as relaxed circular DNA (rcDNA) and is unusually structured. In order for the virus to establish persistence, rcDNA needs to be converted into a form referred to as covalently closed circular DNA (cccDNA) in the nucleus of the hepatocyte, the only cell type in the body that supports HBV infection. Currently, it is only poorly understood how exactly cccDNA is formed.

To address this fundamental question, we took an arguably unconventional approach and utilized yeast to decipher the replicative mechanism of this virus. One may wonder, why yeast? The above mentioned rcDNA needs to be “repaired” at multiple sites to form cccDNA. HBV itself only encodes four gene products, and we know that none of them are capable of catalyzing such “repair” steps. Consequently, we argued that HBV relies on host enzymes for the rc- to cccDNA conversion. Yeast has proven to be one of the best models to study cellular replicative mechanisms as it can be genetically manipulated with ease, propagates rapidly and has a plethora of sophisticated tools already available for its study. Importantly, many fundamental processes are nearly identical across yeast and human cells, and thus we argued that the essential machinery may be conserved. Remarkably, cellular extracts from yeast fully supported the conversion of HBV rc- to cccDNA, thereby validating this model as suitable for studying viral persistence. Using this method, we screened several dozens of cellular repair factors and ultimately identified five core components of the DNA lagging strand synthesis as essential for cccDNA formation

The yeast and human versions of all the five repair factors operate in the same way, and we further demonstrated that all five recombinant human versions of the cellular factors we identified constitute a minimal set of proteins that support repair of rcDNA to form cccDNA.   Collectively, our work breaks new ground in understanding key aspect of the HBV replicative cycle, and – more importantly - provides new tools for deciphering the precise mechanism of cccDNA formation. Conceivably, this knowledge could contribute to the development of more effective therapies against this dreadful disease.

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