Correction: pUL21 is a viral phosphatase adaptor that promotes herpes simplex virus replication and spread.

[This corrects the article DOI: 10.1371/journal.ppat.1009824.].

pUL21 is a viral phosphatase adaptor that promotes herpes simplex virus replication and spread.

The herpes simplex virus (HSV)-1 protein pUL21 is essential for efficient virus replication and dissemination. While pUL21 has been shown to promote multiple steps of virus assembly and spread, the molecular basis of its function remained unclear. Here we identify that pUL21 is a virus-encoded adaptor of protein phosphatase 1 (PP1). pUL21 directs the dephosphorylation of cellular and virus proteins, including components of the viral nuclear egress complex, and we define a conserved non-canonical linear motif in pUL21 that is essential for PP1 recruitment. In vitro evolution experiments reveal that pUL21 antagonises the activity of the virus-encoded kinase pUS3, with growth and spread of pUL21 PP1-binding mutant viruses being restored in adapted strains where pUS3 activity is disrupted. This study shows that virus-directed phosphatase activity is essential for efficient herpesvirus assembly and spread, highlighting the fine balance between kinase and phosphatase activity required for optimal virus replication.

Lipid-specific oligomerization of the Marburg virus matrix protein VP40 is regulated by two distinct interfaces for virion assembly.

Marburg virus (MARV) is a lipid-enveloped virus harboring a negative-sense RNA genome, which has caused sporadic outbreaks of viral hemorrhagic fever in sub-Saharan Africa. MARV assembles and buds from the host cell plasma membrane where MARV matrix protein (mVP40) dimers associate with anionic lipids at the plasma membrane inner leaflet and undergo a dynamic and extensive self-oligomerization into the structural matrix layer. The MARV matrix layer confers the virion filamentous shape and stability but how host lipids modulate mVP40 oligomerization is mostly unknown. Using in vitro and cellular techniques, we present a mVP40 assembly model highlighting two distinct oligomerization interfaces: the (N-terminal domain [NTD] and C-terminal domain [CTD]) in mVP40. Cellular studies of NTD and CTD oligomerization interface mutants demonstrate the importance of each interface in matrix assembly. The assembly steps include protein trafficking to the plasma membrane, homo-multimerization that induced protein enrichment, plasma membrane fluidity changes, and elongations at the plasma membrane. An ascorbate peroxidase derivative (APEX)-transmission electron microscopy method was employed to closely assess the ultrastructural localization and formation of viral particles for wildtype mVP40 and NTD and CTD oligomerization interface mutants. Taken together, these studies present a mechanistic model of mVP40 oligomerization and assembly at the plasma membrane during virion assembly that requires interactions with phosphatidylserine for NTD-NTD interactions and phosphatidylinositol-4,5-bisphosphate for proper CTD-CTD interactions. These findings have broader implications in understanding budding of lipid-enveloped viruses from the host cell plasma membrane and potential strategies to target protein-protein or lipid-protein interactions to inhibit virus budding.

SH3 Domain-Containing Protein 2 Plays a Crucial Role at the Step of Membrane Tubulation during Cell Plate Formation.

During cytokinesis in plants, trans-Golgi network-derived vesicles accumulate at the center of dividing cells and undergo various structural changes to give rise to the planar cell plate. However, how this conversion occurs at the molecular level remains elusive. In this study, we report that SH3 Domain-Containing Protein 2 (SH3P2) in Arabidopsis thaliana plays a crucial role in converting vesicles to the planar cell plate. SH3P2 RNAi plants showed cytokinesis-defective phenotypes and produced aggregations of vesicles at the leading edge of the cell plate. SH3P2 localized to the leading edge of the cell plate, particularly the constricted or curved regions of the cell plate. The BAR domain of SH3P2 induced tubulation of vesicles. SH3P2 formed a complex with dynamin-related protein 1A (DRP1A) and affected DRP1A accumulation to the cell plate. Based on these results, we propose that SH3P2 functions together with DRP1A to convert the fused vesicles to tubular structures during cytokinesis.

A pyrene-based two-photon excitable fluorescent probe to visualize nuclei in live cells.

The two-photon absorption properties of a pyrene-pyridinium dye (1) were studied for potential application in two-photon spectroscopy. When probe 1 was used in cellular two-photon fluorescence microscopy imaging, it allowed the visualization of nuclei in live cells with a relatively low probe concentration (such as 1 µM). Spectroscopic evidence further revealed that probe 1 interacted with DNA as an intercalator. The proposed DNA intercalation properties of probe 1 were consistent with the experimental findings that suggested that the observed nucleus staining ability is dependent on the substituents on the pyridinium fragment of the probe.

Lysosome imaging in cancer cells by pyrene-benzothiazolium dyes: An alternative imaging approach for LAMP-1 expression based visualization methods to avoid background interference.

A series of pyrene-benzothiazolium dyes (1a-1d) were experimentally investigated to study their internalization mechanism into cellular lysosomes as well as their potential imaging applications for live cell imaging. The lysosome selectivity of the probes was further compared by using fluorescently tagged lysosome associated membrane protein-1 (LAMP-1) expression-dependent visualization in both normal (COS-7, HEK293) and cancer (A549, Huh 7.5) cell lines. These probes were successfully employed as reliable lysosome markers in tumor cell models, thus providing an attractive alternative to LAMP-1 expression-dependent visualization methods. One advantage of these probes is the elimination of significant background fluorescence arising from fluorescently tagged protein expression on the cell surface when cells were transfected with LAMP-1 expression plasmids. Probes exhibited remarkable ability to stain cellular lysosomes for long-term experiments (up to 24 h) and the highly lipophilic nature of the probe design allowed their accumulation in hydrophobic regions of the cellular lysosomes. Experimental evidences indicated that the probes are likely to be internalized into lysosomes via endocytosis and accumulated in the hydrophobic regions of the lysosomes rather than in the acidic lysosomal lumen. These probes also demonstrated significant stability and lysosome staining for fixed cell imaging applications as well. Lastly, the benzothiazolium moiety of the probes was identified as the key component for lysosome selectivity.

Bright red-emitting highly reliable styryl probe with large Stokes shift for visualizing mitochondria in live cells under wash-free conditions.

Bright red-emitting pyridinium cyanine based styryl probe 2 is synthesized in good yields. Probe 2 demonstrated a large Stokes' shift (Δλ ≈ 128 nm, 4227 cm-1 in DCM) and excellent fluorescent quantum yield (ϕfl ≈ 0.2 - 0.7) due to strong Intra-molecular charge transfer (ICT). Probe 2 found to exhibit exceptional selectivity for cellular mitochondria in both normal (COS-7) and cancer (A549) cell lines. Probe 2 is readily applicable as a "wash-free" dye to visualize mitochondria as it does not require post-staining washing prior to imaging. Styryl probe 2 also showed an excellent biocompatibility as the calculated LC50 (lethal concentration, 50%) value was > 20 µM. Probe 2 emission did not show any interferences from anionic species or other biological molecules. Probe 2 is readily excitable (λex ∼460 and λem ∼618 nm) with the available laser (454 nm) in commercial microscopes and thus it can be a useful probe for mitochondrial tracking in live cells.

Detection of lipid-induced structural changes of the Marburg virus matrix protein VP40 using hydrogen/deuterium exchange-mass spectrometry.

Marburg virus (MARV) is a lipid-enveloped virus from the Filoviridae family containing a negative sense RNA genome. One of the seven MARV genes encodes the matrix protein VP40, which forms a matrix layer beneath the plasma membrane inner leaflet to facilitate budding from the host cell. MARV VP40 (mVP40) has been shown to be a dimeric peripheral protein with a broad and flat basic surface that can associate with anionic phospholipids such as phosphatidylserine. Although a number of mVP40 cationic residues have been shown to facilitate binding to membranes containing anionic lipids, much less is known on how mVP40 assembles to form the matrix layer following membrane binding. Here we have used hydrogen/deuterium exchange (HDX) mass spectrometry to determine the solvent accessibility of mVP40 residues in the absence and presence of phosphatidylserine and phosphatidylinositol 4,5-bisphosphate. HDX analysis demonstrates that two basic loops in the mVP40 C-terminal domain make important contributions to anionic membrane binding and also reveals a potential oligomerization interface in the C-terminal domain as well as a conserved oligomerization interface in the mVP40 N-terminal domain. Lipid binding assays confirm the role of the two basic patches elucidated with HD/X measurements, whereas molecular dynamics simulations and membrane insertion measurements complement these studies to demonstrate that mVP40 does not appreciably insert into the hydrocarbon region of anionic membranes in contrast to the matrix protein from Ebola virus. Taken together, we propose a model by which association of the mVP40 dimer with the anionic plasma membrane facilitates assembly of mVP40 oligomers.

Crystal Structure of Marburg Virus VP40 Reveals a Broad, Basic Patch for Matrix Assembly and a Requirement of the N-Terminal Domain for Immunosuppression.

UNLABELLED: Marburg virus (MARV), a member of the filovirus family, causes severe hemorrhagic fever with up to 90% lethality. MARV matrix protein VP40 is essential for assembly and release of newly copied viruses and also suppresses immune signaling in the infected cell. Here we report the crystal structure of MARV VP40. We found that MARV VP40 forms a dimer in solution, mediated by N-terminal domains, and that formation of this dimer is essential for budding of virus-like particles. We also found the N-terminal domain to be necessary and sufficient for immune antagonism. The C-terminal domains of MARV VP40 are dispensable for immunosuppression but are required for virus assembly. The C-terminal domains are only 16% identical to those of Ebola virus, differ in structure from those of Ebola virus, and form a distinct broad and flat cationic surface that likely interacts with the cell membrane during virus assembly. IMPORTANCE: Marburg virus, a cousin of Ebola virus, causes severe hemorrhagic fever, with up to 90% lethality seen in recent outbreaks. Molecular structures and visual images of the proteins of Marburg virus are essential for the development of antiviral drugs. One key protein in the Marburg virus life cycle is VP40, which both assembles the virus and suppresses the immune system. Here we provide the molecular structure of Marburg virus VP40, illustrate differences from VP40 of Ebola virus, and reveal surfaces by which Marburg VP40 assembles progeny and suppresses immune function.

Investigation of the Lipid Binding Properties of the Marburg Virus Matrix Protein VP40.

UNLABELLED: Marburg virus (MARV), which belongs to the virus family Filoviridae, causes hemorrhagic fever in humans and nonhuman primates that is often fatal. MARV is a lipid-enveloped virus that during the replication process extracts its lipid coat from the plasma membrane of the host cell it infects. MARV carries seven genes, one of which encodes its matrix protein VP40 (mVP40), which regulates the assembly and budding of the virions. Currently, little information is available on mVP40 lipid binding properties. Here, we have investigated the in vitro and cellular mechanisms by which mVP40 associates with lipid membranes. mVP40 associates with anionic membranes in a nonspecific manner that is dependent upon the anionic charge density of the membrane. These results are consistent with recent structural determination of mVP40, which elucidated an mVP40 dimer with a flat and extensive cationic lipid binding interface. IMPORTANCE: Marburg virus (MARV) is a lipid-enveloped filamentous virus from the family Filoviridae. MARV was discovered in 1967, and yet little is known about how its seven genes are used to assemble and form a new viral particle in the host cell it infects. The MARV matrix protein VP40 (mVP40) underlies the inner leaflet of the virus and regulates budding from the host cell plasma membrane. In vitro and cellular assays in this study investigated the mechanism by which mVP40 associates with lipids. The results demonstrate that mVP40 interactions with lipid vesicles or the inner leaflet of the plasma membrane are electrostatic but nonspecific in nature and are dependent on the anionic charge density of the membrane surface. Small molecules that can disrupt lipid trafficking or reduce the anionic charge of the plasma membrane interface may be useful in inhibiting assembly and budding of MARV.

Roles of NS1 Protein in Flavivirus Pathogenesis.

Flaviviruses such as dengue, Zika, and West Nile viruses are highly concerning pathogens that pose significant risks to public health. The NS1 protein is conserved among flaviviruses and is synthesized as a part of the flavivirus polyprotein. It plays a critical role in viral replication, disease progression, and immune evasion. Post-translational modifications influence NS1's stability, secretion, antigenicity, and interactions with host factors. NS1 protein forms extensive interactions with host cellular proteins allowing it to affect vital processes such as RNA processing, gene expression regulation, and cellular homeostasis, which in turn influence viral replication, disease pathogenesis, and immune responses. NS1 acts as an immune evasion factor by delaying complement-dependent lysis of infected cells and contributes to disease pathogenesis by inducing endothelial cell damage and vascular leakage and triggering autoimmune responses. Anti-NS1 antibodies have been shown to cross-react with host endothelial cells and platelets, causing autoimmune destruction that is hypothesized to contribute to disease pathogenesis. However, in contrast, immunization of animal models with the NS1 protein confers protection against lethal challenges from flaviviruses such as dengue and Zika viruses. Understanding the multifaceted roles of NS1 in flavivirus pathogenesis is crucial for effective disease management and control. Therefore, further research into NS1 biology, including its host protein interactions and additional roles in disease pathology, is imperative for the development of strategies and therapeutics to combat flavivirus infections successfully. This Review provides an in-depth exploration of the current available knowledge on the multifaceted roles of the NS1 protein in the pathogenesis of flaviviruses.

Bright NIR-Emitting Styryl Pyridinium Dyes with Large Stokes' Shift for Sensing Applications.

Two NIR-emitting donor-π-acceptor (D-π-A) type regioisomeric styryl pyridinium dyes (1a-1b) were synthesized and studied for their photophysical performance and environment sensitivity. The two regioisomers, 1a and 1b, exhibited interesting photophysical properties including, longer wavelength excitation (λex ≈ 530-560 nm), bright near-infrared emission (λem ≈ 690-720 nm), high-fluorescence quantum yields (ϕfl ≈ 0.24-0.72) large Stokes' shift (∆λ ≈ 150-240 nm) and high-environmental sensitivity. Probe's photophysical properties were studied in different environmental conditions such as polarity, viscosity, temperature, and concentration. Probes (1a-1b) exhibited noticeable changes in absorbance, emission and Stokes' shift while responding to the changes in physical environment. Probe 1b exhibited a significant bathochromic shift in optical spectra (∆λ ≈ 20-40 nm) compared to its isomer 1a, due to the regio-effect. Probes (1a-1b) exhibited an excellent ability to visualize bacteria (Bacillus megaterium, Escherichia coli), and yeast (Saccharomyces cerevisiae) via fluorescence microscopy.

Mutation of Hydrophobic Residues in the C-Terminal Domain of the Marburg Virus Matrix Protein VP40 Disrupts Trafficking to the Plasma Membrane.

Marburg virus (MARV) is a lipid-enveloped negative sense single stranded RNA virus, which can cause a deadly hemorrhagic fever. MARV encodes seven proteins, including VP40 (mVP40), a matrix protein that interacts with the cytoplasmic leaflet of the host cell plasma membrane. VP40 traffics to the plasma membrane inner leaflet, where it assembles to facilitate the budding of viral particles. VP40 is a multifunctional protein that interacts with several host proteins and lipids to complete the viral replication cycle, but many of these host interactions remain unknown or are poorly characterized. In this study, we investigated the role of a hydrophobic loop region in the carboxy-terminal domain (CTD) of mVP40 that shares sequence similarity with the CTD of Ebola virus VP40 (eVP40). These conserved hydrophobic residues in eVP40 have been previously shown to be critical to plasma membrane localization and membrane insertion. An array of cellular experiments and confirmatory in vitro work strongly suggests proper orientation and hydrophobic residues (Phe281, Leu283, and Phe286) in the mVP40 CTD are critical to plasma membrane localization. In line with the different functions proposed for eVP40 and mVP40 CTD hydrophobic residues, molecular dynamics simulations demonstrate large flexibility of residues in the EBOV CTD whereas conserved mVP40 hydrophobic residues are more restricted in their flexibility. This study sheds further light on important amino acids and structural features in mVP40 required for its plasma membrane localization as well as differences in the functional role of CTD amino acids in eVP40 and mVP40.

Red-emitting pyrene-benzothiazolium: unexpected selectivity to lysosomes for real-time cell imaging without alkalinizing effect.

A series of pyrene-benzothiazolium probes were synthesized. By replacing the pyridinium with a benzothiazolium unit, the selectivity of pyrene-derivatives is found to switch from nuclear to cellular lysosomes. New probes do not require proton participation and exhibit high biocompatibility and long-term imaging ability.

Bright red-emitting pyrene derivatives with a large Stokes shift for nucleus staining.

A highly fluorescent red-emitting pyrene derivative was synthesized and found to exhibit a large Stokes shift (λem ≈ 610 nm, Δλ ∼ 130 nm, Δν ∼ 4597 cm-1). The probe molecule showed remarkable selectivity to stain the nucleus in both live and fixed cells, with higher sensitivity than commercial dye DRAQ5.