Biology subjects

Petrich, A.

Publications and source records attributed to Petrich, A..

8 recordsLinked to original sources

bioRxiv2024

Antiviral Mechanisms and Preclinical Evaluation of Amantadine Analogs that Continue to Inhibit Influenza A Viruses with M2 S31N-Based Drug Resistance

To better manage seasonal and pandemic influenza infections, new drugs are needed with enhanced activity against contemporary amantadine- and rimantadine-resistant influenza A virus (IAV) strains containing the S31N variant of the viral M2 ion channel (M2S31N). Here we tested 36 amantadine analogs against a panel of viruses containing either M2S31N or the parental, M2 S31 wild-type variant (M2WT). We found that several analogs, primarily those with sizeable lipophilic adducts, inhibited up to three M2S31N-containing viruses with activities at least 5-fold lower than rimantadine, without inhibiting M2S31N proton currents or modulating endosomal pH. While M2WT viruses in passaging studies rapidly gained resistance to these analogs through the established M2 mutations V27A and/or A30T, resistance development was markedly slower for M2S31N viruses and did not associate with additional M2 mutations. Instead, a subset of analogs, exemplified by 2-propyl-2-adamantanamine (38), but not 2-(1-adamantyl)piperidine (26), spiro[adamantane-2,2-pyrrolidine] (49), or spiro[adamantane-2,2-piperidine] (60), inhibited cellular entry of infectious IAV following pre-treatment and/or H1N1 pseudovirus entry. Conversely, an overlapping subset of the most lipophilic analogs including compounds 26, 49, 60, and others, disrupted viral M2-M1 protein colocalization required for intracellular viral assembly and budding. Finally, a pilot toxicity study in mice demonstrated that 38 and 49 were tolerated at doses approaching those of amantadine. Together, these results indicate that amantadine analogs act on multiple, complementary mechanisms to inhibit replication of M2S31N viruses. Highlights- Current IAVs have M2 mutations that confer resistance to amantadine and rimantadine - Several amantadine analogs inhibit these viruses without acting on M2 proton currents - Alternative antiviral targets include IAV entry and M2-M1 protein colocalization - Amantadine analogs are also tolerated in mice - Future amantadine antivirals could simultaneously act on multiple IAV mechanisms

microbiology↗

bioRxiv2024

P-BODY AND CYTOSKELETON REMODELING BY ORTHOHANTAVIRUSES.

Orthohantaviruses, are emerging zoonotic pathogens causing life-threatening diseases in humans. The orthohantavirus genome consists of three RNA segment (vRNAs) of negative polarity, which are encapsidated by the viral nucleoprotein (N). To date, the precise subcellular behavior of vRNAs and N has not been fully elucidated. Here, we present a comprehensive analysis of orthohantavirus infections using Fluorescence in situ Hybridization (FISH) and multiple sequential FISH (Mu-Seq FISH), which enables simultaneous detection of viral RNAs, viral mRNAs, N, and cellular factors. Our experiments revealed distinct patterns of viral RNA clustering with varying degrees of N association. Moreover, we found a significant spatial correlation of virus vRNAs and N with cellular processing (P)-bodies, underlining their key role in orthohantavirus replication. Throughout the course of an infection, we observed an increasing dominance of N expression, while concomitantly P-body numbers grew significantly. We also found indications for a preferential 5-end degradation of viral mRNAs in P-bodies. Furthermore, we report that orthohantavirus infection is accompanied by a significant redistribution of cellular components: while filamentous actin and microtubules become enriched in the perinuclear region, P-bodies move to the cell periphery. Finally, co-localization analyses suggest a formation of viral factories containing N, vRNAs, and viral mRNAs, indicating an intricate orthohantavirus assembly hierarchy. AUTHOR SUMMARYIn this study we used advanced imaging techniques to observe the dynamics of viral components and key cellular structures during orthohantavirus infections. Our experiments show that orthohantaviruses cause significant changes within the cell, particularly involving P-bodies and components of the cytoskeleton, such as actin and microtubules. We also provided comprehensive spatiotemporal maps of orthohantaviral components, including visualization of the viral nucleoprotein, genomic RNA and mRNAs. Finally, we found indications for a 5end degradation of virus mRNA in P-bodies, thus adding to our understanding of intracellular host-pathogen crosstalk. In summary, our work highlights the intricate relationship between viruses and host cells, emphasizing the dynamic changes that occur during orthohantaviral infections.

microbiology↗

bioRxiv2023

Influenza A virus infection alters lipid packing and surface electrostatic potential of the host plasma membrane

The pathogenesis of influenza A viruses (IAVs) is influenced by several factors, including IAV strain origin and reassortment, tissue tropism and host type. While such factors were mostly investigated in the context of virus entry, fusion and replication, little is known about the viral-induced changes to the host lipid membranes which might be relevant in the context of virion assembly. In this work, we applied several biophysical fluorescence microscope techniques (i.e., Forster energy resonance transfer, generalized polarization imaging and scanning fluorescence correlation spectroscopy) to quantify the effect of infection by two IAV strains of different origin on the plasma membrane (PM) of avian and human cell lines. We found that IAV infection affects the membrane charge of the inner leaflet of the PM. Moreover, we showed that IAV infection impacts lipid-lipid interactions by decreasing membrane fluidity and increasing lipid packing. Because of such alterations, diffusive dynamics of membrane-associated proteins are hindered. Taken to-gether, our results indicate that the infection of avian and human cell lines with IAV strains of different origins had similar effects on the biophysical properties of the PM.

biophysics↗

bioRxiv2023

Benchmarking of novel green fluorescent proteins for the quantification of protein oligomerization in living cells

Protein-protein-interactions play an important role in several cellular functions. Quantitative non-invasive techniques are applied in living cells to evaluate such interactions, thereby providing a broader understanding of complex biological processes. Fluorescence fluctuation spectroscopy describes a group of quantitative microscopy approaches for the characterization of molecular interactions at single cell resolution. Through the obtained molecular brightness, it is possible to determine the oligomeric state of proteins. This is usually achieved by fusing fluorescent proteins (FPs) to the protein of interest. Recently, the number of novel green FPs has increased, with consequent improvements to the quality of fluctuation-based measurements. The photophysical behavior of FPs is influenced by multiple factors (including photobleaching, protonation-induced "blinking" and long-lived dark states). Assessing these factors is critical for selecting the appropriate fluorescent tag for live cell imaging applications. In this work, we focus on novel green FPs that are extensively used in live cell imaging. A systematic performance comparison of several green FPs in living cells under different pH conditions using Number & Brightness (N&B) analysis and scanning fluorescence correlation spectroscopy was performed. Our results show that the new FP Gamillus exhibits higher brightness at the cost of lower photostability and fluorescence probability (pf), especially at lower pH. mGreenLantern, on the other hand, thanks to a very high pf, is best suited for multimerization quantification at neutral pH. At lower pH, mEGFP remains apparently the best choice for multimerization investigation. These guidelines provide the information needed to plan quantitative fluorescence microscopy involving these FPs, both for general imaging or for Protein-protein-interactions quantification via fluorescence fluctuation-based methods.

biophysics↗

bioRxiv2021

Influenza A M2 recruits M1 to the plasma membrane: a fluorescence fluctuation microscopy study

Influenza A virus (IAV) is a respiratory pathogen that causes seasonal epidemics with significant mortality. One of the most abundant proteins in IAV particles is the matrix protein 1 (M1), which is essential for the virus structural stability. M1 organizes virion assembly and budding at the plasma membrane (PM), where it interacts with other viral components. The recruitment of M1 to the PM as well as its interaction with the other viral envelope proteins (hemagglutinin (HA), neuraminidase, matrix protein 2 (M2)) is controversially discussed in previous studies. Therefore, we used fluorescence fluctuation microscopy techniques (i.e., scanning fluorescence cross-correlation spectroscopy and Number and Brightness) to quantify the oligomeric state of M1 and its interactions with other viral proteins in co-transfected as well as infected cells. Our results indicate that M1 is recruited to the PM by M2, as a consequence of the strong interaction between the two proteins. In contrast, only a weak interaction between M1 and HA was observed. M1-HA interaction occurred only in the case that M1 was already bound to the PM. We therefore conclude that M2 initiates the assembly of IAV by recruiting M1 to the PM, possibly allowing its further interaction with other viral proteins. Statement of SignificanceInfluenza A virus (IAV) is a pathogen responsible for epidemics and occasional pandemics and, therefore, a significant burden on health systems. To develop innovative therapeutic approaches, a deeper understanding of the viral replication cycle is needed. For example, during the formation of new virions in infected cells, several viral components must assemble at the plasma membrane, but the molecular interactions involved in this process are not clearly understood. In this work, we use quantitative fluorescence microscopy methods to monitor the interplay between several viral proteins in live cell models. Our results underline the importance of the interactions between two specific proteins (M1 and M2) and shed light on the first steps in IAV assembly.

biophysics↗

bioRxiv2020

Multi-color fluorescence fluctuation spectroscopy in living cells via spectral detection

Signaling pathways in biological systems rely on specific interactions between multiple biomolecules. Fluorescence fluctuation spectroscopy provides a powerful toolbox to quantify such interactions directly in living cells. Cross-correlation analysis of spectrally separated fluctuations provides information about inter-molecular interactions but is usually limited to two fluorophore species. Here, we present scanning fluorescence spectral correlation spectroscopy (SFSCS), a versatile approach that can be implemented on commercial confocal microscopes, allowing the investigation of interactions between multiple protein species at the plasma membrane. We demonstrate that SFSCS enables cross-talk-free cross-correlation, diffusion and oligomerization analysis of up to four protein species labeled with strongly overlapping fluorophores. As an example, we investigate the interactions of influenza A virus (IAV) matrix protein 2 with two cellular host factors simultaneously. We furthermore apply raster spectral image correlation spectroscopy for the simultaneous analysis of up to four species and determine the stoichiometry of ternary IAV polymerase complexes in the cell nucleus.

biophysics↗

bioRxiv2020

Effect of Erufosine on Membrane Lipid Order in Breast Cancer Cell Models

Alkylphospholipids are a novel class of antineoplastic drugs showing remarkable therapeutic potential. Among them, Erufosine (EPC3) is a promising drug for the treatment of several types of tumors. While EPC3 is supposed to exert its function by interacting with lipid membranes, the exact molecular mechanisms involved are not known yet. In this work, we applied a combination of several fluorescence microscopy and analytical chemistry approaches (i.e., scanning fluorescence correlation spectroscopy, line-scan fluorescence correlation spectroscopy, generalized polarization imaging, as well as thin layer and gas chromatography) to quantify the effect of EPC3 in biophysical models of the plasma membrane, as well as in cancer cell lines. Our results indicate that EPC3 affects lipid-lipid interactions in cellular membranes by decreasing lipid packing and increasing membrane disorder and fluidity. As a consequence of these alterations in the lateral organization of lipid bilayers, the diffusive dynamics of membrane proteins are also significantly increased. Taken together, these findings suggest that the mechanism of action of EPC3 might be linked to its effects on fundamental biophysical properties of lipid membranes, as well as lipid metabolism in cancer cells.

biophysics↗

bioRxiv2019

Macropinocytosis and Clathrin-Dependent Endocytosis Play Pivotal Roles for the Infectious Entry of Puumala Virus.

Viruses from the taxonomic family Hantaviridae are encountered as emerging pathogens causing two life-threatening human zoonoses: hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS) with case fatalities of up to 50%. Here we comprehensively investigated entry of the Old-World Hantavirus, Puumala virus (PUUV), into mammalian cells, showing that upon treatment with pharmacological inhibitors of macropinocytosis and clathrin-mediated endocytosis, PUUV infections are significantly reduced. We demonstrated that the inhibitors did not interfere with viral replication and that RNA interference, targeting cellular mediators of macropinocytosis, is able to decrease PUUV infection levels significantly. Moreover, we established lipophilic tracer staining of PUUV virus particles and showed co-localization of stained virions and markers of macropinocytic uptake. Cells treated with lysosomotrophic agents were shown to exhibit an increased resistance to infection, confirming previous data suggesting that a low pH-dependent step is involved in PUUV infection. Finally, we observed a significant increase in the fluid-phase uptake of cell infected with PUUV, indicative of a virus-triggered promotion of macropinocytosis.\n\nAuthor SummaryThe Hantaviridae family comprises a very diverse group of virus species and is considered an emerging global public health threat. Human pathogenic hantaviruses are primarily rodent-borne. Zoonosis is common with more than 150,000 annually registered cases and a case fatality index of up to 50%. Individual hantavirus species differ significantly in terms of their pathogenicity, but also their cell biology and host-pathogen interactions. In this study, we focused on the most prevalent pathogenic hantavirus in Europe, Puumala virus (PUUV), and investigated the entry and internalization of PUUV virions into mammalian cells. We showed that both, clathrin-mediated endocytosis and macropinocytosis, are cellular pathways exploited by the virus to establish productive infections and demonstrated that pharmacological inhibition of macropinocytosis or its targeted knockdown using RNA interference significantly reduced viral infections. We also found indications for an increase of macropinocytic uptake upon PUUV infections, suggesting that the virus triggers specific cellular mechanisms in order to promote its own internalization and facilitate infections.

microbiology↗