Progression of herpesvirus infection remodels mitochondrial organization and metabolism.

Viruses target mitochondria to promote their replication, and infection-induced stress during the progression of infection leads to the regulation of antiviral defenses and mitochondrial metabolism which are opposed by counteracting viral factors. The precise structural and functional changes that underlie how mitochondria react to the infection remain largely unclear. Here we show extensive transcriptional remodeling of protein-encoding host genes involved in the respiratory chain, apoptosis, and structural organization of mitochondria as herpes simplex virus type 1 lytic infection proceeds from early to late stages of infection. High-resolution microscopy and interaction analyses unveiled infection-induced emergence of rough, thin, and elongated mitochondria relocalized to the perinuclear area, a significant increase in the number and clustering of endoplasmic reticulum-mitochondria contact sites, and thickening and shortening of mitochondrial cristae. Finally, metabolic analyses demonstrated that reactivation of ATP production is accompanied by increased mitochondrial Ca2+ content and proton leakage as the infection proceeds. Overall, the significant structural and functional changes in the mitochondria triggered by the viral invasion are tightly connected to the progression of the virus infection.

Visible Light-Induced Specific Protein Reaction Delineates Early Stages of Cell Adhesion.

Light is well-established for control of bond breakage but not for control of specific bond formation in complex environments. We previously engineered the diffusion-limited reactivity of the SpyTag003 peptide with its protein partner SpyCatcher003 through spontaneous isopeptide bond formation. This system enables precise and irreversible assembly of biological building blocks with applications from biomaterials to vaccines. Here we establish a system for the rapid control of this amide bond formation with visible light. We have generated a caged SpyCatcher003, which allows light triggering of covalent bond formation to SpyTag003 in mammalian cells. Photocaging is achieved through site-specific incorporation of an unnatural coumarin-lysine at the reactive site of SpyCatcher003. We showed a uniform specific reaction in cell lysate upon light activation. We then used the spatiotemporal precision of a 405 nm confocal laser for uncaging in seconds, probing the earliest events in mechanotransduction by talin, the key force sensor between the cytoskeleton and the extracellular matrix. Reconstituting talin induced rapid biphasic extension of lamellipodia, revealing the kinetics of talin-regulated cell spreading and polarization. Thereafter we determined the hierarchy of the recruitment of key components for cell adhesion. Precise control over site-specific protein reaction with visible light creates diverse opportunities for cell biology and nanoassembly.

Visible light-induced specific protein reaction delineates early stages of cell adhesion.

Light is well established for control of bond breakage, but not for control of specific bond formation in complex environments. We previously engineered diffusion-limited reactivity of SpyTag003 peptide with its protein partner SpyCatcher003 through spontaneous transamidation. This system enables precise and irreversible assembly of biological building blocks, with applications from biomaterials to vaccines. Here, we establish a system for rapid control of this amide bond formation with visible light. We have generated a caged SpyCatcher003, which allows light triggering of covalent bond formation to SpyTag003 in mammalian cells. Photocaging is achieved through site-specific incorporation of an unnatural coumarin-lysine at the reactive site of SpyCatcher003. We showed uniform specific reaction in cell lysate upon light activation. We then used the spatiotemporal precision of a 405 nm confocal laser for uncaging in seconds, probing the earliest events in mechanotransduction by talin, the key force sensor between the cytoskeleton and extracellular matrix. Reconstituting talin induced rapid biphasic extension of lamellipodia, revealing the kinetics of talin-regulated cell spreading and polarization. Thereafter we determined the hierarchy of recruitment of key components for cell adhesion. Precise control over site-specific protein reaction with visible light creates diverse opportunities for cell biology and nanoassembly.

Talin variant P229S compromises integrin activation and associates with multifaceted clinical symptoms.

Adhesion of cells to the extracellular matrix (ECM) must be exquisitely coordinated to enable development and tissue homeostasis. Cell-ECM interactions are regulated by multiple signalling pathways that coordinate the activation state of the integrin family of ECM receptors. The protein talin is pivotal in this process, and talin's simultaneous interactions with the cytoplasmic tails of the integrins and the plasma membrane are essential to enable robust, dynamic control of integrin activation and cell-ECM adhesion. Here, we report the identification of a de novo heterozygous c.685C>T (p.Pro229Ser) variant in the TLN1 gene from a patient with a complex phenotype. The mutation is located in the talin head region at the interface between the F2 and F3 domains. The characterization of this novel p.P229S talin variant reveals the disruption of adhesion dynamics that result from disturbance of the F2-F3 domain interface in the talin head. Using biophysical, computational and cell biological techniques, we find that the variant perturbs the synergy between the integrin-binding F3 and the membrane-binding F2 domains, compromising integrin activation, adhesion and cell migration. Whilst this remains a variant of uncertain significance, it is probable that the dysregulation of adhesion dynamics we observe in cells contributes to the multifaceted clinical symptoms of the patient and may provide insight into the multitude of cellular processes dependent on talin-mediated adhesion dynamics.

Surface Modification of Bioactive Glass Promotes Cell Attachment and Spreading.

Phosphate glasses have several advantages over traditional silicate-based bioglasses but are inferior in the crucial step of cell attachment to their surface. Here, as a proof of concept, we analyze fibroblast attachment to the phosphate glass surface subjected to basic treatment and silanization. Silicate (S53P4)- and phosphate (Sr50)-based bioactive glasses were either untreated or surface-treated with basic buffer and functionalized with silane. The surface-treated samples were studied as such and after fibronectin was adsorbed on to their surface. With both glass types, surface treatment enhanced fibroblast adhesion and spreading in comparison to the untreated glass. The surface-treated Sr50 glass allowed for cell adhesion, proliferation, and spreading to a similar extent as seen with S53P4 and borosilicate control glasses. Here, we show that surface treatment of bioactive glass can be used to attract cell adhesion factors found in the serum and promote cell-material adhesion, both important for efficient tissue integration.

Avidin-Conjugated Nanofibrillar Cellulose Hydrogel Functionalized with Biotinylated Fibronectin and Vitronectin Promotes 3D Culture of Fibroblasts.

The future success of physiologically relevant three-dimensional (3D) cell/tissue models is dependent on the development of functional biomaterials, which can provide a well-defined 3D environment instructing cellular behavior. To establish a platform to produce tailored hydrogels, we conjugated avidin (Avd) to anionic nanofibrillar cellulose (aNFC) and demonstrated the use of the resulting Avd-NFC hydrogel for 3D cell culture, where Avd-NFC allows easy functionalization via biotinylated molecules. Avidin was successfully conjugated to nanocellulose and remained functional, as demonstrated by electrophoresis and titration with fluorescent biotin. Rheological analysis indicated that Avd-NFC retained shear-thinning and gel-forming properties. Topological characterization using AFM revealed the preserved fiber structure and confirmed the binding of biotinylated vitronectin (B-VN) on the fiber surface. The 3D cell culture experiments with mouse embryonic fibroblasts demonstrated the performance of Avd-NFC hydrogels functionalized with biotinylated fibronectin (B-FN) and B-VN. Cells cultured in Avd-NFC hydrogels functionalized with B-FN or B-VN formed matured integrin-mediated adhesions, indicated by phosphorylated focal adhesion kinase. We observed significantly higher cell proliferation rates when biotinylated proteins were bound to the Avd-NFC hydrogel compared to cells cultured in Avd-NFC alone, indicating the importance of the presence of adhesive sites for fibroblasts. The versatile Avd-NFC allows the easy functionalization of hydrogels with virtually any biotinylated molecule and may become widely utilized in 3D cell/tissue culture applications.

Cancer associated talin point mutations disorganise cell adhesion and migration.

Talin-1 is a key component of the multiprotein adhesion complexes which mediate cell migration, adhesion and integrin signalling and has been linked to cancer in several studies. We analysed talin-1 mutations reported in the Catalogue of Somatic Mutations in Cancer database and developed a bioinformatics pipeline to predict the severity of each mutation. These predictions were then assessed using biochemistry and cell biology experiments. With this approach we were able to identify several talin-1 mutations affecting integrin activity, actin recruitment and Deleted in Liver Cancer 1 localization. We explored potential changes in talin-1 signalling responses by assessing impact on migration, invasion and proliferation. Altogether, this study describes a pipeline approach of experiments for crude characterization of talin-1 mutants in order to evaluate their functional effects and potential pathogenicity. Our findings suggest that cancer related point mutations in talin-1 can affect cell behaviour and so may contribute to cancer progression.

Extrusion-Based Bioprinting of Multilayered Nanocellulose Constructs for Cell Cultivation Using In Situ Freezing and Preprint CaCl2 Cross-Linking.

Extrusion-based bioprinting with a preprint cross-linking agent and an in situ cooling stage provides a versatile method for the fabrication of 3D structures for cell culture. We added varying amounts of calcium chloride as a precross-linker into native nanofibrillated cellulose (NFC) hydrogel prior to 3D bioprinting to fabricate structurally stable multilayered constructs without the need for a separate cross-linking bath. To further enhance their stability, we bioprinted the multilayered structures onto an in situ temperature-controlled printing stage at 25, 0, and -10 °C. The extruded and subsequently freeze-dried volumetric constructs maintained their structures after being immersed into a cell culture medium. The ability to maintain the shape after immersion in cell media is an essential feature for the fabrication of stem cell-based artificial organs. We studied the viability and distribution of mouse embryonic fibroblast cells into the hydrogels using luminescence technique and confocal microscopy. Adding CaCl2 increased the stability of the multilayered nanocellulose structures, making them suitable for culturing cells inside the 3D hydrogel environment. Lower stage temperature considerably improved the structural stability of the 3D printed structures, however, had no effect on cell viability.

Human Enterovirus Group B Viruses Rely on Vimentin Dynamics for Efficient Processing of Viral Nonstructural Proteins.

We report that several viruses from the human enterovirus group B cause massive vimentin rearrangements during lytic infection. Comprehensive studies suggested that viral protein synthesis was triggering the vimentin rearrangements. Blocking the host cell vimentin dynamics with ß, ß'-iminodipropionitrile (IDPN) did not significantly affect the production of progeny viruses and only moderately lowered the synthesis of structural proteins such as VP1. In contrast, the synthesis of the nonstructural proteins 2A, 3C, and 3D was drastically lowered. This led to attenuation of the cleavage of the host cell substrates PABP and G3BP1 and reduced caspase activation, leading to prolonged cell survival. Furthermore, the localization of the proteins differed in the infected cells. Capsid protein VP1 was found diffusely around the cytoplasm, whereas 2A and 3D followed vimentin distribution. Based on protein blotting, smaller amounts of nonstructural proteins did not result from proteasomal degradation but from lower synthesis without intact vimentin cage structure. In contrast, inhibition of Hsp90 chaperone activity, which regulates P1 maturation, lowered the amount of VP1 but had less effect on 2A. The results suggest that the vimentin dynamics regulate viral nonstructural protein synthesis while having less effect on structural protein synthesis or overall infection efficiency. The results presented here shed new light on differential fate of structural and nonstructural proteins of enteroviruses, having consequences on host cell survival.IMPORTANCE A virus needs the host cell in order to replicate and produce new progeny viruses. For this, the virus takes over the host cell and modifies it to become a factory for viral proteins. Irrespective of the specific virus family, these proteins can be divided into structural and nonstructural proteins. Structural proteins are the building blocks for the new progeny virions, whereas the nonstructural proteins orchestrate the takeover of the host cell and its functions. Here, we have shown a mechanism that viruses exploit in order to regulate the host cell. We show that viral protein synthesis induces vimentin cages, which promote production of specific viral proteins that eventually control apoptosis and host cell death. This study specifies vimentin as the key regulator of these events and indicates that viral proteins have different fates in the cells depending on their association with vimentin cages.

Approaching infinite affinity through engineering of peptide-protein interaction.

Much of life's complexity depends upon contacts between proteins with precise affinity and specificity. The successful application of engineered proteins often depends on high-stability binding to their target. In recent years, various approaches have enabled proteins to form irreversible covalent interactions with protein targets. However, the rate of such reactions is a major limitation to their use. Infinite affinity refers to the ideal where such covalent interaction occurs at the diffusion limit. Prototypes of infinite affinity pairs have been achieved using nonnatural reactive groups. After library-based evolution and rational design, here we establish a peptide-protein pair composed of the regular 20 amino acids that link together through an amide bond at a rate approaching the diffusion limit. Reaction occurs in a few minutes with both partners at low nanomolar concentration. Stopped flow fluorimetry illuminated the conformational dynamics involved in docking and reaction. Hydrogen-deuterium exchange mass spectrometry gave insight into the conformational flexibility of this split protein and the process of enhancing its reaction rate. We applied this reactive pair for specific labeling of a plasma membrane target in 1 min on live mammalian cells. Sensitive and specific detection was also confirmed by Western blot in a range of model organisms. The peptide-protein pair allowed reconstitution of a critical mechanotransmitter in the cytosol of mammalian cells, restoring cell adhesion and migration. This simple genetic encoding for rapid irreversible reaction should provide diverse opportunities to enhance protein function by rapid detection, stable anchoring, and multiplexing of protein functionality.

Slow Infection due to Lowering the Amount of Intact versus Empty Particles Is a Characteristic Feature of Coxsackievirus B5 Dictated by the Structural Proteins.

Enterovirus B species typically cause a rapid cytolytic infection leading to efficient release of progeny viruses. However, they are also capable of persistent infections in tissues, which are suggested to contribute to severe chronic states such as myocardial inflammation and type 1 diabetes. In order to understand the factors contributing to differential infection strategies, we constructed a chimera by combining the capsid proteins from fast-cytolysis-causing echovirus 1 (EV1) with nonstructural proteins from coxsackievirus B5 (CVB5), which shows persistent infection in RD cells. The results showed that the chimera behaved similarly to parental EV1, leading to efficient cytolysis in both permissive A549 and semipermissive RD cells. In contrast to EV1 and the chimera, CVB5 replicated slowly in permissive cells and showed persistent infection in semipermissive cells. However, there was no difference in the efficiency of uptake of CVB5 in A549 or RD cells in comparison to the chimera or EV1. CVB5 batches constantly contained significant amounts of empty capsids, also in comparison to CVB5's close relative CVB3. During successive passaging of batches containing only intact CVB5, increasing amounts of empty and decreasing amounts of infective capsids were produced. Our results demonstrate that the increase in the amount of empty particles and the lowering of the amount of infective particles are dictated by the CVB5 structural proteins, leading to slowing down of the infection between passages. Furthermore, the key factor for persistent infection is the small amount of infective particles produced, not the high number of empty particles that accumulate.IMPORTANCE Enteroviruses cause several severe diseases, with lytic infections that lead to rapid cell death but also persistent infections that are more silent and lead to chronic states of infection. Our study compared a cytolytic echovirus 1 infection to persistent coxsackievirus B5 infection by making a chimera with the structural proteins of echovirus 1 and the nonstructural proteins of coxsackievirus B5. Coxsackievirus B5 infection was found to lead to the production of a high number of empty viruses (empty capsids) that do not contain genetic material and are unable to continue the infection. Coinciding with the high number of empty capsids, the amount of infective virions decreased. This characteristic property was not observed in the constructed chimera virus, suggesting that structural proteins are in charge of these phenomena. These results shed light on the mechanisms that may cause persistent infections. Understanding events leading to efficient or inefficient infections is essential in understanding virus-caused pathologies.

Talin-mediated force transmission and talin rod domain unfolding independently regulate adhesion signaling.

Talin protein is one of the key components in integrin-mediated adhesion complexes. Talins transmit mechanical forces between ß-integrin and actin, and regulate adhesion complex composition and signaling through the force-regulated unfolding of talin rod domain. Using modified talin proteins, we demonstrate that these functions contribute to different cellular processes and can be dissected. The transmission of mechanical forces regulates adhesion complex composition and phosphotyrosine signaling even in the absence of the mechanically regulated talin rod subdomains. However, the presence of the rod subdomains and their mechanical activation are required for the reinforcement of the adhesion complex, cell polarization and migration. Talin rod domain unfolding was also found to be essential for the generation of cellular signaling anisotropy, since both insufficient and excess activity of the rod domain severely inhibited cell polarization. Utilizing proteomics tools, we identified adhesome components that are recruited and activated either in a talin rod-dependent manner or independently of the rod subdomains. This study clarifies the division of roles between the force-regulated unfolding of a talin protein (talin 1) and its function as a physical linker between integrins and the cytoskeleton.

Echovirus 1 internalization negatively regulates epidermal growth factor receptor downregulation.

We have demonstrated previously that the human picornavirus Echovirus 1 (EV1) triggers an infectious internalization pathway that follows closely, but seems to stay separate, from the epidermal growth factor receptor (EGFR) pathway triggered by epidermal growth factor (EGF). Here, we confirmed by using live and confocal microscopy that EGFR and EV1 vesicles are following intimately each other but are distinct entities with different degradation kinetics. We show here that despite being sorted to different pathways and located in distinct endosomes, EV1 inhibits EGFR downregulation. Simultaneous treatment with EV1 and EGF led to an accumulation of EGFR in cytoplasmic endosomes, which was evident already 15 min p.i. and more pronounced after 2 hr p.i. EV1 treatment led to reduced downregulation, which was proven by increased total cellular amount of EGFR. Confocal microscopy studies revealed that EGFR accumulated in large endosomes, presumably macropinosomes, which were not positive for markers of the early, recycling, or late endosomes/lysosomes. Interestingly, EV1 did not have a similar blocking effect on bulk endosomal trafficking or transferrin recycling along the clathrin pathway suggesting that EV1 did not have a general effect on cellular trafficking pathways. Importantly, EGF treatment increased EV1 infection and increased cell viability during infection. Simultaneous EV1 and EGF treatment seemed to moderately enhance phosphorylation of protein kinase C α. Furthermore, similar phenotype of EGFR trafficking could be produced by phorbol 12-myristate 13-acetate treatment, further suggesting that activated protein kinase C α could be contributing to EGFR phenotype. These results altogether demonstrate that EV1 specifically affects EGFR trafficking, leading to EGFR downregulation, which is beneficial to EV1 infection.

Infectious Entry Pathway of Enterovirus B Species.

Enterovirus B species (EV-B) are responsible for a vast number of mild and serious acute infections. They are also suspected of remaining in the body, where they cause persistent infections contributing to chronic diseases such as type I diabetes. Recent studies of the infectious entry pathway of these viruses revealed remarkable similarities, including non-clathrin entry of large endosomes originating from the plasma membrane invaginations. Many cellular factors regulating the efficient entry have recently been associated with macropinocytic uptake, such as Rac1, serine/threonine p21-activated kinase (Pak1), actin, Na/H exchanger, phospholipace C (PLC) and protein kinase Cα (PKCα). Another characteristic feature is the entry of these viruses to neutral endosomes, independence of endosomal acidification and low association with acidic lysosomes. The biogenesis of neutral multivesicular bodies is crucial for their infection, at least for echovirus 1 (E1) and coxsackievirus A9 (CVA9). These pathways are triggered by the virus binding to their receptors on the plasma membrane, and they are not efficiently recycled like other cellular pathways used by circulating receptors. Therefore, the best "markers" of these pathways may be the viruses and often their receptors. A deeper understanding of this pathway and associated endosomes is crucial in elucidating the mechanisms of enterovirus uncoating and genome release from the endosomes to start efficient replication.

6-o- and N-sulfated syndecan-1 promotes baculovirus binding and entry into Mammalian cells.

Baculoviruses are insect-specific viruses commonly found in nature. They are not able to replicate in mammalian cells but can transduce them when equipped with an appropriate mammalian cell active expression cassette. Although the viruses have been studied in several types of mammalian cells from different origins, the receptor that baculovirus uses to enter or interact with mammalian cells has not yet been identified. Due to the wide tropism of the virus, the receptor has been suggested to be a generally found cell surface molecule. In this article, we investigated the interaction of baculovirus and mammalian cell surface heparan sulfate proteoglycans (HSPG) in more detail. Our data show that baculovirus requires HSPG sulfation, particularly N- and 6-O-sulfation, to bind to and transduce mammalian cells. According to our results, baculovirus binds specifically to syndecan-1 (SDC-1) but does not interact with SDC-2 to SDC-4 or with glypicans. Competition experiments performed with SDC-1 antibody or recombinant SDC-1 protein inhibited baculovirus binding, and SDC-1 overexpression enhanced baculovirus-mediated transduction. In conclusion, we show that SDC-1, a commonly found cell surface HSPG molecule, has a role in the binding and entry of baculovirus in vertebrate cells. The results presented here reveal important aspects of baculovirus entry and can serve as a basis for next-generation baculovirus vector development for gene delivery.

Cell susceptibility to baculovirus transduction and echovirus infection is modified by protein kinase C phosphorylation and vimentin organization.

Some cell types are more susceptible to viral gene transfer or virus infection than others, irrespective of the number of viral receptors or virus binding efficacy on their surfaces. In order to characterize the cell-line-specific features contributing to efficient virus entry, we studied two cell lines (Ea.hy926 and MG-63) that are nearly nonpermissive to insect-specific baculovirus (BV) and the human enterovirus echovirus 1 (EV1) and compared their characteristics with those of a highly permissive (HepG2) cell line. All the cell lines contained high levels of viral receptors on their surfaces, and virus binding was shown to be efficient. However, in nonpermissive cells, BV and its receptor, syndecan 1, were unable to internalize in the cells and formed large aggregates near the cell surface. Accordingly, EV1 had a low infection rate in nonpermissive cells but was still able to internalize the cells, suggesting that the postinternalization step of the virus was impaired. The nonpermissive and permissive cell lines showed differential expression of syntenin, filamentous actin, vimentin, and phosphorylated protein kinase C subtype α (pPKCα). The nonpermissive nature of the cells could be modulated by the choice of culture medium. RPMI medium could partially rescue infection/transduction and concomitantly showed lower syntenin expression, a modified vimentin network, and altered activities of PKC subtypes PKCα and PKCε. The observed changes in PKCα and PKCε activation caused alterations in the vimentin organization, leading to efficient BV transduction and EV1 infection. This study identifies PKCα, PKCε, and vimentin as key factors affecting efficient infection and transduction by EV1 and BV, respectively.

Clathrin-independent entry of baculovirus triggers uptake of E. coli in non-phagocytic human cells.

The prototype baculovirus, Autographa californica multiple nucleopolyhedrovirus, an insect pathogen, holds great potential as a gene therapy vector. To develop transductional targeting and gene delivery by baculovirus, we focused on characterizing the nature and regulation of its uptake in human cancer cells. Baculovirus entered the cells along fluid-phase markers from the raft areas into smooth-surfaced vesicles devoid of clathrin. Notably, regulators associated with macropinocytosis, namely EIPA, Pak1, Rab34, and Rac1, had no significant effect on viral transduction, and the virus did not induce fluid-phase uptake. The internalization and nuclear uptake was, however, affected by mutants of RhoA, and of Arf6, a regulator of clathrin-independent entry. Furthermore, the entry of baculovirus induced ruffle formation and triggered the uptake of fluorescent E. coli bioparticles. To conclude, baculovirus enters human cells via a clathrin-independent pathway, which is able to trigger bacterial uptake. This study increases our understanding of virus entry strategies and gives new insight into baculovirus-mediated gene delivery in human cells.