Encapsidated hepatitis B virus reverse transcriptase is poised on an ordered RNA lattice.

Assembly of a hepatitis B virus (HBV) virion begins with the formation of an RNA-filled core composed of a symmetrical capsid (built of core protein), viral pregenomic RNA, and viral reverse transcriptase. To generate the circular dsDNA genome of HBV, reverse transcription requires multiple template switches within the confines of the capsid. To date, most anti-HBV therapeutics target this reverse transcription process. The detailed molecular mechanisms of this crucial process are poorly understood because of the lack of structural information. We hypothesized that capsid, RNA, and viral reverse transcriptase would need a precise geometric organization to accomplish reverse transcription. Here we present the asymmetric structure of authentic RNA-filled cores, determined to 14.5-Å resolution from cryo-EM data. Capsid and RNA are concentric. On the interior of the RNA, we see a distinct donut-like density, assigned to viral reverse transcriptase, which pins the viral pregenomic RNA to the capsid inner surface. The observation of a unique ordered structure inside the core suggests that assembly and the first steps of reverse transcription follow a single, determinate pathway and strongly suggests that all subsequent steps in DNA synthesis do as well.

Insight into the mechanism of inhibition of adeno-associated virus by the Mre11/Rad50/Nbs1 complex.

UNLABELLED: Adeno-associated virus (AAV) is a dependent virus of the family Parvoviridae. The gene expression and replication of AAV and derived recombinant AAV (rAAV) vectors are severely limited (>10-fold) by the cellular DNA damage-sensing complex made up of Mre11, Rad50, and Nbs1 (MRN). The AAV genome does not encode the means to circumvent this block to productive infection but relies on coinfecting helper virus to do so. Using adenovirus helper proteins E1B55k and E4orf6, which enhance the transduction of AAV via degradation of MRN, we investigated the mechanism through which this DNA damage complex inhibits gene expression from rAAV. We tested the substrate specificity of inhibition and the contribution of different functions of the MRN complex. Our results demonstrate that both single- and double-stranded rAAV vectors are inhibited by MRN, which is in contrast to the predominant model that inhibition is the result of a block to second-strand synthesis. Exploring the contribution of known functions of MRN, we found that inhibition of rAAV does not require downstream DNA damage response factors, including signaling kinases ATM and ATR. The nuclease domain of Mre11 appears to play only a minor role in inhibition, while the DNA binding domain makes a greater contribution. Additionally, mutation of the inverted terminal repeat of the rAAV genome, which has been proposed to be the signal for interaction with MRN, is tolerated by the mechanism of inhibition. These results articulate a model of inhibition of gene expression in which physical interaction is more important than enzymatic activity and several key downstream damage repair factors are dispensable. IMPORTANCE: Many viruses modulate the host DNA damage response (DDR) in order to create a cellular environment permissive for infection. The MRN complex is a primary sensor of damage in the cell but also responds to invading viral genomes, often posing a block to infection. AAV is greatly inhibited by MRN and dependent on coinfecting helper virus, such as adenovirus, to remove this factor. Currently, the mechanism through which MRN inhibits AAV and other viruses is poorly understood. Our results reform the predominant model that inhibition of rAAV by MRN is due to limiting second-strand DNA synthesis. Instead, a novel mechanism of inhibition of gene expression independent of a block in rAAV DNA synthesis or downstream damage factors is indicated. These findings have clear implications for understanding this restriction to transduction of AAV and rAAV vectors, which have high therapeutic relevance and likely translate to other viruses that must navigate the DDR.

Viral vectors for gene delivery to the central nervous system.

The potential benefits of gene therapy for neurological diseases such as Parkinson's, Amyotrophic Lateral Sclerosis (ALS), Epilepsy, and Alzheimer's are enormous. Even a delay in the onset of severe symptoms would be invaluable to patients suffering from these and other diseases. Significant effort has been placed in developing vectors capable of delivering therapeutic genes to the CNS in order to treat neurological disorders. At the forefront of potential vectors, viral systems have evolved to efficiently deliver their genetic material to a cell. The biology of different viruses offers unique solutions to the challenges of gene therapy, such as cell targeting, transgene expression and vector production. It is important to consider the natural biology of a vector when deciding whether it will be the most effective for a specific therapeutic function. In this review, we outline desired features of the ideal vector for gene delivery to the CNS and discuss how well available viral vectors compare to this model. Adeno-associated virus, retrovirus, adenovirus and herpesvirus vectors are covered. Focus is placed on features of the natural biology that have made these viruses effective tools for gene delivery with emphasis on their application in the CNS. Our goal is to provide insight into features of the optimal vector and which viral vectors can provide these features.

Roles of the envelope proteins in the amplification of covalently closed circular DNA and completion of synthesis of the plus-strand DNA in hepatitis B virus.

Covalently closed circular DNA (cccDNA), the nuclear form of hepatitis B virus (HBV), is synthesized by repair of the relaxed circular (RC) DNA genome. Initially, cccDNA is derived from RC DNA from the infecting virion, but additional copies of cccDNA are derived from newly synthesized RC DNA molecules in a process termed intracellular amplification. It has been shown that the large viral envelope protein limits the intracellular amplification of cccDNA for duck hepatitis B virus. The role of the envelope proteins in regulating the amplification of cccDNA in HBV is not well characterized. The present report demonstrates regulation of synthesis of cccDNA by the envelope proteins of HBV. Ablation of expression of the envelope proteins led to an increase (>6-fold) in the level of cccDNA. Subsequent restoration of envelope protein expression led to a decrease (>50%) in the level of cccDNA, which inversely correlated with the level of the envelope proteins. We found that the expression of L protein alone or in combination with M and/or S proteins led to a decrease in cccDNA levels, indicating that L contributes to the regulation of cccDNA. Coexpression of L and M led to greater regulation than either L alone or L and S. Coexpression of all three envelope proteins was also found to limit completion of plus-strand DNA synthesis, and the degree of this effect correlated with the level of the proteins and virion secretion.

Integrating research into a molecular cloning course to address the evolving biotechnology landscape.

The rapid development of molecular biotechnology presents a curricular challenge for educators trying to provide students with relevant coursework. A comprehensive biology education should also include opportunities for students to develop intellectual and technical skills through authentic research experiences. Integrating relevant and interesting research projects into their classes, however, can be a challenging task for instructors. To address these varied demands, we redesigned our existing molecular cloning course to incorporate an independent research project assessing calcium signaling. In the revised course, students use traditional and recombination-based cloning strategies to generate bacterial and mammalian expression vectors encoding CaMPARI, a novel fluorescent calcium indicator. Bacterially-expressed CaMPARI is used in protein quantification and purification assays. Students must also design their own research project evaluating the effect of chemotherapeutic agents on calcium signaling in a mammalian system. Revised and novel labs were designed to be modular, facilitating their integration into the course over 2 years. End-of-semester student evaluations were compared between years revealing a significant difference in students' perception of the course's difficulty between years. This change in attitude highlights potential pedagogical considerations that must be examined when introducing new material and activities into existing courses. Since calcium signaling is important for cellular process across diverse species, instructors may be able to develop research projects within their respective areas of interest. Integration of authentic research experiences into the curriculum is challenging; however, the framework described here provides a versatile structure that can be adapted to merge diverse instructor interests with evolving educational needs.

Unique Down to Our Microbes-Assessment of an Inquiry-Based Metagenomics Activity.

Metagenomics is an important method for studying microbial life. However, undergraduate exposure to metagenomics is hindered by associated software, computing demands, and dataset access. In this inquiry-based activity designed for introductory life science majors and nonmajors, students perform an investigation of the bacterial communities inhabiting the human belly button and associated metagenomics data collected through a citizen science project and visualized using an open-access bioinformatics tool. The activity is designed for attainment of the following student learning outcomes: defining terms associated with metagenomics analyses, describing the biological impact of the microbiota on human health, formulating a hypothesis, analyzing and interpreting metagenomics data to compare microbiota, evaluating a specific hypothesis, and synthesizing a conceptual model as to why bacterial populations vary. This activity was implemented in six introductory biology and biotechnology courses across five institutions. Attainment of student learning outcomes was assessed through completion of a quiz and students' presentations of their findings. In presentations, students demonstrated their ability to develop novel hypotheses and analyze and interpret metagenomic data to evaluate their hypothesis. In quizzes, students demonstrated their ability to define key terms and describe the biological impact of the microbiota on human health. Student learning gains assessment also revealed that students perceived gains for all student learning outcomes. Collectively, our assessment demonstrates achievement of the learning outcomes and supports the utility of this inquiry-based activity to engage undergraduates in the scientific process via analyses of metagenomics datasets and associated exploration of a microbial community that lives on the human body.

Recombinant adeno-associated virus: clinical application and development as a gene-therapy vector.

Gene therapy is gaining momentum as a method of treating human disease. Initially conceived as a strategy to complement defective genes in monogenic disorders, the scope of gene therapy has expanded to encompass a variety of applications. Likewise, the molecular tools for gene delivery have evolved and diversified to meet these various therapeutic needs. Recombinant adeno-associated virus (rAAV) has made significant strides toward clinical application with an excellent safety profile and successes in several clinical trials. This review covers the basic biology of rAAV as a gene therapy vector as well as its advantages compared with other methods of gene delivery. The status of clinical trials utilizing rAAV is also discussed in detail. In conclusion, methods of engineering the vector to overcome challenges identified from these trials are covered, with emphasis on modification of the viral capsid to increase the tissue/cell-specific targeting and transduction efficiency.

Development of cell cultures that express hepatitis B virus to high levels and accumulate cccDNA.

Establishment of an infection with hepatitis B virus (HBV) requires synthesis and maintenance of a covalently closed circular DNA (cccDNA) form of the viral genome in the nucleus of host cells. To facilitate the investigation of the synthesis of cccDNA, cell cultures were developed that express HBV to high levels. Cell lines derived from hepatoma cells Huh7 and HepG2 were created that express Epstein-Barr virus (EBV) nuclear antigen-1 and a fusion protein of the Tet repressor and Kox1 transcriptional repression domain stably. Transfection of these cell lines with an expression plasmid for HBV that contains the origin of plasmid replication of EBV (oriP) led to increases in the intracellular levels of HBV core protein ( approximately 8- to 51-fold) and encapsidated HBV DNA ( approximately 3- to 12-fold) in comparison to Huh7 and HepG2 cells. Virion production was also increased ( approximately 3- to 12-fold) in these cell cultures and an increase in the level of cccDNA ( approximately 3-fold) was observed in the Huh7-derived cell lines. In addition, these cell lines maintained the HBV expression plasmid upon selection and expressed HBV conditionally. Thus, these cell cultures exhibit several features that facilitate study of the synthesis of cccDNA and other aspects of replication of HBV.