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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.

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.

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., Author summary Herpes simplex virus (HSV)-1 is a highly prevalent human virus that causes life-long infections. While the most common symptom of HSV-1 infection is orofacial lesions (‘cold sores’), HSV-1 infection can also cause fatal encephalitis and it is a leading cause of infectious blindness. The HSV-1 genome encodes many proteins that dramatically remodel the environment of infected cells to promote virus replication and spread, including enzymes that add phosphate groups (kinases) to cellular and viral proteins in order to fine-tune their function. Here we identify that pUL21 is an HSV-1 protein that binds directly to protein phosphatase 1 (PP1), a highly abundant cellular enzyme that removes phosphate groups from proteins. We demonstrate that pUL21 stimulates the specific dephosphorylation of both cellular and viral proteins, including a component of the viral nuclear egress complex that is essential for efficient assembly of new HSV-1 particles. Furthermore, our in vitro evolution experiments demonstrate that pUL21 antagonises the activity of the HSV-1 kinase pUS3. Our work highlights the precise control that herpesviruses exert upon the protein environment within infected cells, and specifically the careful balance of kinase and phosphatase activity that HSV-1 requires for optimal replication and spread.

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.

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.

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.