Monday, December 30, 2024
12/30/2024
PROJECT SUMMARY Hepatitis B virus (HBV) infection is a global public health concern. Despite effective vaccines to prevent this disease, current approved treatment rarely leads to a complete cure. There is an unmet medical need for developing new therapeutics for HBV infection that can lead to a sustained response. Targeting HBV capsid assembly process has become an emerging strategy for developing new antiviral treatment for HBV. However, many efforts have been made by using different capsid protein (Cp) constructs expressed in Escherichia coli to mimic or reconstitute native-like viral particles. Yet, these structures cannot correctly represent the native HBV conformations due to the lacks of nucleic acid binding domain of the Cp, viral genome, and viral enzymes, all of which are required for viral replication. Furthermore, the lack of post translational modifications of the Cp also hampers the interpretation between observed Cp structures to the biomedical data obtained from the mammalian cell culture system or experimental animals. To date there is no available high-resolution native HBV structures, which is a major gap in knowledge of the HBV field. In this proposal, we aim to use cryo-electron microscope (cryo-EM) to directly characterize the structures of native HBV capsids and virions from human cells. In Aim 1, we will determine the high-resolution structures of purified intracellular HBV capsids with different types of viral genome. We will address the key questions concerning the structural dynamics of HBV capsids during genome maturation. We will also determine the structure of the HBV reverse transcriptase (RT) and its location during reverse transcription to help understand its mode of action (whether the RT is static or moves). In Aim 2, we will investigate the high-resolution structures of secreted HBV virions. This aim will address the questions concerning how HBV capsids interact with the viral envelop proteins. Finally, experimental findings from these two Aims will be integrated to elucidate capsid dynamics during HBV replication and illuminate the molecular determinant(s) of HBV envelopment. This proposal is expected to solve 4 types of intracellular HBV capsid structures (empty, RNA-filled, single- stranded DNA-filled, and mature partially double-stranded DNA-filled capsids) and 3 secreted enveloped HBV virion structures (empty, mature, and prematurely secreted virions) using cryo-EM to define the conformational changes of the capsid during viral replication, particularly in the context of different viral genome forms and interactions between the capsid and surface proteins. The methodology exploits appropriate mutations of Cp and RT to ensure obtaining homogenous particles of the various types as described above, which can be further computationally classified to minimize cross-contamination. Understanding the native HBV structures will provide valuable new information for HBV biology and guide the design of novel antiviral drugs in the future. The project is anticipated to impact fields ranging from HBV, molecular virology, antiviral drug development, and macromolecular structure and function.
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