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Nucleocytoplasmic large DNA viruses (NCLDVs) are a group of large viruses that infect a wide range of hosts, from animals to protists. These viruses are grouped together in NCLDV based on genomic sequence analyses. They share a set of essential genes for virion morphogenesis and replication. Most NCLDVs generally have large physical sizes while their morphologies vary in different families, such as icosahedral, brick, or oval shape, raising the question of the possible regulatory factor on their morphogenesis. The capsids of icosahedral NCLDVs are assembled from small building blocks, named capsomers, which are the trimeric form of the major capsid proteins. Note that the capsids of immature poxvirus are spherical even though they are assembled from capsomers that share high structural conservation with those icosahedral NCLDVs. The recently published high resolution structure of NCLDVs, Paramecium bursaria Chlorella virus 1 and African swine fever virus, described the intensive network of minor capsid proteins that are located underneath the capsomers. Among these minor proteins is the elongated tape measure protein (TmP) that spans from one icosahedral fivefold vertex to another. In this study, we focused on the critical roles that TmP plays in the assembly of icosahedral NCLDV capsids, answering a question raised in a previously proposed spiral mechanism. Interestingly, basic local alignment search on the TmPs showed no significant hits in poxviruses, which might be the factor that differentiates poxviruses and icosahedral NCLDVs in their morphogenesis. [ABSTRACT FROM AUTHOR]

One quarter of the world's population are infected by Mycobacterium tuberculosis (Mtb), which is a leading death-causing bacterial pathogen. Recent evidence has demonstrated that two virulence factors, ESAT-6 and CFP-10, play crucial roles in Mtb's cytosolic translocation. Many efforts have been made to study the ESAT-6 and CFP-10 proteins. Some studies have shown that ESAT-6 has an essential role in rupturing phagosome. However, the mechanisms of how ESAT-6 interacts with the membrane have not yet been fully understood. Recent studies indicate that the ESAT-6 disassociates with CFP-10 upon their interaction with phagosome membrane, forming a membrane-spanning pore. Based on these observations, as well as the available structure of ESAT-6, ESAT-6 is hypothesized to form an oligomer for membrane insertion as well as rupturing. Such an ESAT-6 oligomer may play a significant role in the tuberculosis infection. Therefore, deeper understanding of the oligomerization of ESAT-6 will establish new directions for tuberculosis treatment. However, the structure of the oligomer of ESAT-6 is not known. Here, we proposed a comprehensive approach to model the complex structures of ESAT-6 oligomer inside a membrane. Several computational tools, including MD simulation, symmetrical docking, MM/PBSA, are used to obtain and characterize such a complex structure. Results from our studies lead to a well-supported hypothesis of the ESAT-6 oligomerization as well as the identification of essential residues in stabilizing the ESAT-6 oligomer which provide useful insights for future drug design targeting tuberculosis. The approach in this research can also be used to model and study other cross-membrane complex structures. Results from our studies modeled the ESAT-6 oligomerization and identified essential residues in stabilizing the ESAT-6 oligomer. This study provides useful insights for future drug design targeting Mycobacterium tuberculosis (Mtb). The approach in this research can also be used to model and study other cross-membrane complex structures. [ABSTRACT FROM AUTHOR]

At the molecular level, the circadian clock is regulated by a time delayed transcriptional-translational feedback loop in which the core proteins interact with each other rhythmically to drive daily biological rhythms. The C-terminal domain of a key clock protein PER2 (PER2c) plays a critically important role in the loop, not only for its interaction with the binding partner CRY proteins but also for the CRY/PER complex's translocation from the cytosol to the nucleus. Previous circular dichroism (CD) spectroscopic studies have shown that mouse PER2c (mPER2c) is less structured in solution by itself but folded into stable secondary structures upon interaction with mouse CRYs. To understand the stability and folding of human PER2c (hPER2c), we expressed and purified hPER2c. Three oligomerization forms of recombinant hPER2c were identified and thoroughly characterized through a combination of biochemical and biophysical techniques. Different to mPER2c, both thermal unfolding DLS and CD analyses suggested that all forms of hPER2c have very stable secondary structures in solution by themselves with melting temperatures higher than the physiological body temperature, indicating that hPER2c does not require CRY to fold. Furthermore, we examined the effects of EDTA, salt concentration, and a reducing agent on hPER2c folding and oligomerization. The ability of hPER2c forming oligomers reflects the potential role of hPER2c in the assembly of circadian rhythm core protein complexes. [ABSTRACT FROM AUTHOR]

African swine fever virus (ASFV) is the causative pathogen of the recent African swine fever epidemic, with devastating impacts on economy. A recent study by Wang et al. reveals the multilayer structural details of ASFV at near-atomic resolution, which provides interesting insights about giant virus assembly and paves the way for vaccine development. [ABSTRACT FROM AUTHOR]

In the last three decades, many giant DNA viruses have been discovered. Giant viruses present a unique and essential research frontier for studies of self-assembly and regulation of supramolecular assemblies. The question on how these giant DNA viruses assemble thousands of proteins so accurately to form their protein shells, the capsids, remains largely unanswered. Revealing the mechanisms of giant virus assembly will help to discover the mysteries of many self-assembly biology problems. Paramecium bursaria Chlorella virus-1 (PBCV-1) is one of the most intensively studied giant viruses. Here, we implemented a multi-scale approach to investigate the interactions among PBCV-1 capsid building units called capsomers. Three binding modes with different strengths are found between capsomers around the relatively flat area of the virion surface at the icosahedral 2-fold axis. Furthermore, a capsomer structure manipulation package is developed to simulate the capsid assembly process. Using these tools, binding forces among capsomers were investigated and binding funnels were observed that were consistent with the final assembled capsid. In addition, total binding free energies of each binding mode were calculated. The results helped to explain previous experimental observations. Results and tools generated in this work established an initial computational approach to answer current unresolved questions regarding giant virus assembly mechanisms. Results will pave the way for studying more complicated process in other biomolecular structures. [ABSTRACT FROM AUTHOR]

Summary: Cylindrospermopsis (Raphidiopsis) raciborskii is an invasive, filamentous, nitrogen‐fixing cyanobacterium that forms frequent blooms in freshwater habitats. While viruses play key roles in regulating the abundance, production and diversity of their hosts in aquatic ecosystems, the role(s) of viruses in the ecology of C. raciborskii is almost unexplored. Progress in this field has been hindered by the absence of a characterized virus–host system in C. raciborskii. To bridge this gap, we sequenced the genome of CrV‐01T, a previously isolated cyanosiphovirus, and its host, C. raciborskii strain Cr2010. Analyses suggest that CrV‐01T represents a distinct clade of siphoviruses infecting, and perhaps lysogenizing, filamentous cyanobacteria. Its genome contains unique features that include an intact CRISPR array and a 12 kb inverted duplication. Evidence suggests CrV‐01T recently gained the ability to infect Cr2010 and recently lost the ability to form lysogens. The cyanobacterial host contains a CRISPR‐Cas system with CRISPR spacers matching protospacers within the inverted duplication of the CrV‐01T genome. Examination of metagenomes demonstrates that viruses with high genetic identity to CrV‐01T, but lacking the inverted duplication, are present in C. raciborskii blooms in Australia. The unique genomic features of the CrV/Cr2010 system offers opportunities to investigate in more detail virus–host interactions in an ecologically important bloom‐forming cyanobacterium. [ABSTRACT FROM AUTHOR]

R526 --> 648.7 --> Circadian rhythm is the internal clock in the body that cycles in approximately 24 hours. It controls many physiological changes such as the sleep‐wake cycle and the body‐temperature fluctuation. In humans, every single cell has its own circadian rhythm that is synchronized by a master clock in the brain. At the molecular level, circadian rhythms of individual cells are controlled by the transcriptional‐translational feedback loop (TTFL). This loop is initialized by the heterodimerization of circadian locomotor outputs cycle kaput (CLOCK) and brain muscle ARNT‐like 1 (BMAL1). The long‐term goal of the project is to study the structure and the function of the full‐length human CLOCK/BMAL1 complex. Human CLOCK and BMAL1 genes have been cloned into a bacterial expression system. In order to reach the long‐term goal, it is necessary to optimize the condition of expression and purification of the CLOCK/BMAL1 complex to a high quality and homogeneity. In this study, concentration of the inducer, induction time, cell density, and lysis methods have been optimized through multiple series of expression trials. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS‐PAGE) and Western Blotting have been used to assess the quantity and quality of CLOCK/BMAL1 expression. Protein expressed using the optimized conditions has been subjected to purification experiments. Expression and purification of the CLOCK/BMAL1 proteins will build a solid foundation for the structural and functional studies, which will deepen our understanding of the molecular mechanism of circadian rhythm that will facilitate the design of therapeutic reagents to treat circadian related diseases such as sleep‐wake disorder and jet lags. [ABSTRACT FROM AUTHOR]