Developing A Bitwise Macromolecular Assembly Simulator

Macromolecular machines play fundamental roles in many cellular tasks, from intracellular transport to protein synthesis and degradation. The majority of these machines must adopt a particular quaternary structure in order to function, and so understanding their assembly represents a critical component of our understanding of overall cellular physiology. Developing a theoretical and conceptual understanding of assembly has been hampered by the lack of general, efficient and scalable computational tools for simulating assembly processes. In this work, we develop a new framework that employs a bitwise representation of assembly intermediates. Using this framework, we have implemented a Bitwise Macromolecular Assembly Simulator (BMAS). This software leverages our binary representation of intermediates to perform most crucial computational steps using bitwise operators. This allows us to perform highly efficient Gillespie-style stochastic simulations of macromolecular assembly, resulting in a general simulation approach that is orders of magnitude faster than existing methods. Our approach is efficient enough to study of a wide variety of macromolecular machines, in addition to providing a tool that should assist in the design of novel self-assembling nanomaterials

Modeling reveals the strength of weak interactions in stacked-ring assembly.

Cells employ many large macromolecular machines for the execution and regulation of processes that are vital for cell and organismal viability. Interestingly, cells cannot synthesize these machines as functioning units. Instead, cells synthesize the molecular parts that must then assemble into the functional complex. Many important machines, including chaperones such as GroEL and proteases such as the proteasome, comprise protein rings that are stacked on top of one another. While there is some experimental data regarding how stacked-ring complexes such as the proteasome self-assemble, a comprehensive understanding of the dynamics of stacked-ring assembly is currently lacking. Here, we developed a mathematical model of stacked-trimer assembly and performed an analysis of the assembly of the stacked homomeric trimer, which is the simplest stacked-ring architecture. We found that stacked rings are particularly susceptible to a form of kinetic trapping that we term deadlock, in which the system gets stuck in a state where there are many large intermediates that are not the fully assembled structure but that cannot productively react. When interaction affinities are uniformly strong, deadlock severely limits assembly yield. We thus predicted that stacked rings would avoid situations where all interfaces in the structure have high affinity. Analysis of available crystal structures indicated that indeed the majority-if not all-of stacked trimers do not contain uniformly strong interactions. Finally, to better understand the origins of deadlock, we developed a formal pathway analysis and showed that, when all the binding affinities are strong, many of the possible pathways are utilized. In contrast, optimal assembly strategies utilize only a small number of pathways. Our work suggests that deadlock is a critical factor influencing the evolution of macromolecular machines and provides general principles for understanding the self-assembly efficiency of existing machines

Contribution of the C-terminal region within the catalytic core domain of HIV-1 integrase to yeast lethality, chromatin binding and viral replication

<p>Abstract</p> <p>Background</p> <p>HIV-1 integrase (IN) is a key viral enzymatic molecule required for the integration of the viral cDNA into the genome. Additionally, HIV-1 IN has been shown to play important roles in several other steps during the viral life cycle, including reverse transcription, nuclear import and chromatin targeting. Interestingly, previous studies have demonstrated that the expression of HIV-1 IN induces the lethal phenotype in some strains of <it>Saccharomyces cerevisiae</it>. In this study, we performed mutagenic analyses of the C-terminal region of the catalytic core domain of HIV-1 IN in order to delineate the critical amino acid(s) and/or motif(s) required for the induction of the lethal phenotype in the yeast strain HP16, and to further elucidate the molecular mechanism which causes this phenotype.</p> <p>Results</p> <p>Our study identified three HIV-1 IN mutants, V165A, A179P and KR186,7AA, located in the C-terminal region of the catalytic core domain of IN that do not induce the lethal phenotype in yeast. Chromatin binding assays in yeast and mammalian cells demonstrated that these IN mutants were impaired for the ability to bind chromatin. Additionally, we determined that while these IN mutants failed to interact with LEDGF/p75, they retained the ability to bind Integrase interactor 1. Furthermore, we observed that VSV-G-pseudotyped HIV-1 containing these IN mutants was unable to replicate in the C8166 T cell line and this defect was partially rescued by complementation with the catalytically inactive D64E IN mutant.</p> <p>Conclusion</p> <p>Overall, this study demonstrates that three mutations located in the C-terminal region of the catalytic core domain of HIV-1 IN inhibit the IN-induced lethal phenotype in yeast by inhibiting the binding of IN to the host chromatin. These results demonstrate that the C-terminal region of the catalytic core domain of HIV-1 IN is important for binding to host chromatin and is crucial for both viral replication and the promotion of the IN-induced lethal phenotype in yeast.</p

The HIV-1 Accessory Protein Vpu Downregulates Peroxisome Biogenesis

People living with HIV can experience accelerated aging and the development of neurological disorders. Recently, we reported that HIV-1 infection results in a dramatic loss of peroxisomes in macrophages and brain tissue. This is significant because (i) peroxisomes are important for the innate immune response and (ii) loss of peroxisome function is associated with cellular aging and neurodegeneration. Accordingly, understanding how HIV-1 infection causes peroxisome depletion may provide clues regarding how the virus establishes persistent infections and, potentially, the development of neurological disorders. Here, we show that the accessory protein Vpu is necessary and sufficient for the induction of microRNAs that target peroxisome biogenesis factors. The ability of Vpu to downregulate peroxisome formation depends on the Wnt/β-catenin pathway. Thus, in addition to revealing a novel mechanism by which HIV-1 uses intracellular signaling pathways to target antiviral signaling platforms (peroxisomes), we have uncovered a previously unknown link between the Wnt/β-catenin pathway and peroxisome homeostasis.Human immunodeficiency virus type 1 (HIV-1) establishes lifelong infections in humans, a process that relies on its ability to thwart innate and adaptive immune defenses of the host. Recently, we reported that HIV-1 infection results in a dramatic reduction of the cellular peroxisome pool. Peroxisomes are metabolic organelles that also function as signaling platforms in the innate immune response. Here, we show that the HIV-1 accessory protein Vpu is necessary and sufficient for the depletion of cellular peroxisomes during infection. Vpu induces the expression of four microRNAs that target mRNAs encoding proteins required for peroxisome formation and metabolic function. The ability of Vpu to downregulate peroxisomes was found to be dependent upon the Wnt/β-catenin signaling pathway. Given the importance of peroxisomes in innate immune signaling and central nervous system function, the roles of Vpu in dampening antiviral signaling appear to be more diverse than previously realized. Finally, our findings highlight a potential role for Wnt/β-catenin signaling in peroxisome homeostasis through modulating the production of biogenesis factors

West Nile virus infection causes endocytosis of a specific subset of tight junction membrane proteins.

West Nile virus (WNV) is a blood-borne pathogen that causes systemic infections and serious neurological disease in human and animals. The most common route of infection is mosquito bites and therefore, the virus must cross a number of polarized cell layers to gain access to organ tissue and the central nervous system. Resistance to trans-cellular movement of macromolecules between epithelial and endothelial cells is mediated by tight junction complexes. While a number of recent studies have documented that WNV infection negatively impacts the barrier function of tight junctions, the intracellular mechanism by which this occurs is poorly understood. In the present study, we report that endocytosis of a subset of tight junction membrane proteins including claudin-1 and JAM-1 occurs in WNV infected epithelial and endothelial cells. This process, which ultimately results in lysosomal degradation of the proteins, is dependent on the GTPase dynamin and microtubule-based transport. Finally, infection of polarized cells with the related flavivirus, Dengue virus-2, did not result in significant loss of tight junction membrane proteins. These results suggest that neurotropic flaviviruses such as WNV modulate the host cell environment differently than hemorrhagic flaviviruses and thus may have implications for understanding the molecular basis for neuroinvasion

Regional Scale Inversion of Chlorophyll Content of <i>Dendrocalamus giganteus</i> by Multi-Source Remote Sensing

The spectrophotometer method is costly, time-consuming, laborious, and destructive to the plant. Samples will be lost during the transportation process, and the method can only obtain sample point data. This poses a challenge to the estimation of chlorophyll content at the regional level. In this study, in order to improve the estimation accuracy, a new method of collaborative inversion of chlorophyll using Landsat 8 and Global Ecosystem Dynamics Investigation (GEDI) is proposed. Specifically, the chlorophyll content data set is combined with the preprocessed two remote-sensing (RS) factors to construct three regression models using a support vector machine (SVM), BP neural network (BP) and random forest (RF), and the better model is selected for inversion. In addition, the ordinary Kriging (OK) method is used to interpolate the GEDI point attribute data into the surface attribute data for modeling. The results showed the following: (1) The chlorophyll model of a single plant was y = 0.1373x1.7654. (2) The optimal semi-variance function models of pai, pgap_theta and pgap_theta_a3 are exponential models. (3) The top three correlations between the two RS data and the chlorophyll content were B2_3_SM, B2_3_HO, B2_5_EN and pai, pgap_theta, pgap_theta_a3. (4) The combination of the Landsat 8 imagery and GEDI resulted in the highest modeling accuracy, and RF had the best performance, with R2, RMSE and P values of 0.94, 0.18 g/m2 and 83.32%, respectively. This study shows that it is reliable to use Landsat 8 images and GEDI to retrieve the chlorophyll content of Dendrocalamus giganteus (D. giganteus), revealing the potential of multi-source RS data in the inversion of forest ecological parameters

Interplay between Zika Virus and Peroxisomes during Infection

Zika virus (ZIKV) has emerged as an important human pathogen that can cause congenital defects in the fetus and neurological conditions in adults. The interferon (IFN) system has proven crucial in restricting ZIKV replication and pathogenesis. The canonical IFN response is triggered by the detection of viral RNA through RIG-I like receptors followed by activation of the adaptor protein MAVS on mitochondrial membranes. Recent studies have shown that a second organelle, peroxisomes, also function as a signaling platforms for the IFN response. Here, we investigated how ZIKV infection affects peroxisome biogenesis and antiviral signaling. We show that ZIKV infection depletes peroxisomes in human fetal astrocytes, a brain cell type that can support persistent infection. The peroxisome biogenesis factor PEX11B was shown to inhibit ZIKV replication, likely by increasing peroxisome numbers and enhancing downstream IFN-dependent antiviral signaling. Given that peroxisomes play critical roles in brain development and nerve function, our studies provide important insights into the roles of peroxisomes in regulating ZIKV infection and potentially neuropathogenesis

MicroRNA-25/93 induction by Vpu as a mechanism for counteracting MARCH1-restriction on HIV-1 infectivity in macrophages

ABSTRACT The type 1 interferon-regulated E3 ubiquitin ligase MARCH1 reduces surface expression of HIV-1 envelope glycoproteins (Env) and their packaging into nascent virions, a condition that restricts viral infectivity. However, how HIV-1 counters this restriction, notably during infection of macrophages, remains unclear. Here, we show that the HIV-1 accessory protein Vpu increases the levels of microRNAs-25 and -93 to target MARCH1 mRNA. By recruiting β-TRCP, a component of the SCFβ-TRCP E3 ligase complex that targets phosphorylated β-catenin for degradation, Vpu increases β-catenin levels, which, in concert with TCF4/LEF, drives transcription of the MARCH1-targeting microRNAs. This potentiates HIV-1 infectivity as a result of increased Env incorporation into nascent virions. Pharmacological targeting of the β-catenin pathway inhibits Vpu-mediated upregulation of microRNAs-25 and -93 and restores MARCH1 restriction on HIV-1 infectivity. Overall, our findings highlight a novel mechanism by which HIV-1 counteracts MARCH1 by downregulating its expression via Vpu-mediated induction of microRNAs-25 and -93. IMPORTANCE In order to efficiently produce infectious viral particles, HIV must counter several restrictions exerted by host cell antiviral proteins. MARCH1 is a member of the MARCH protein family that restricts HIV infection by limiting the incorporation of viral envelope glycoproteins into nascent virions. Here, we identified two regulatory RNAs, microRNAs-25 and -93, induced by the HIV-1 accessory protein Vpu, that downregulate MARCH1 mRNA. We also show that Vpu induces these cellular microRNAs in macrophages by hijacking the cellular β-catenin pathway. The notion that HIV-1 has evolved a mechanism to counteract MARCH1 restriction on viral infectivity underlines the importance of MARCH1 in the host antiviral response

Internalization of JAM-1 is blocked by disrupting microtubules or inhibiting dynamin function.

<p>MDCK cells were infected with WNV and 24 hours later were treated with 10 µM nocodazole, 10 µM Dynasore or DMSO for a further 8 hours. Samples were then processed for indirect immunofluorescence using rabbit anti-JAM-1 and mouse anti-WNV NS3/2B. Primary antibodies were detected using donkey anti-mouse Alexa546 and donkey anti-rabbit Alexa488 secondary antibodies. Nuclei were counter stained with DAPI. Images were captured using a Leica TCS SP5 confocal scanning microscope. Size bars are 10 µm.</p