Biology subjects

Bajorek, M.

Publications and source records attributed to Bajorek, M..

8 recordsLinked to original sources

bioRxiv2026

The actin-binding protein drebrin interacts with the respiratory syncytial virus matrix protein and promotes virus particle release.

Respiratory syncytial virus (RSV) is an enveloped virus that assembles and buds at the plasma membrane, producing filamentous infectious particles. Unlike many other enveloped viruses, a substantial fraction of RSV virions remains associated with the surface of infected cells, limiting our understanding of the membrane-scission mechanisms responsible for particle release. Here, we identified the actin-binding protein drebrin as a cellular interactor of the RSV matrix (M) protein, both in vitro and in infected cells. We demonstrated that RSV infection induces the relocalization of drebrin to actin-rich structures at the cell periphery associated with viral filaments. Using a Transwell-based assay to quantify infectious particles release without mechanically perturbing cells, we confirmed that RSV release is independent of the endosomal sorting complex required for transport (ESCRT) pathway and showed that drebrin depletion reduced RSV release, whereas drebrin overexpression enhanced it. Since drebrin depletion did not significantly affect overall infectious virus production, our results suggest that drebrin may act during the final step leading to membrane scission and RSV particle release. Finally, proteomics analysis identified Transmembrane 9 superfamily member 2 (TM9SF2) as a drebrin-associated protein specifically during infection. TM9SF2 depletion reduced RSV particle release and impaired the RSV-induced relocalization of drebrin to the plasma membrane. Together, these findings identify drebrin and TM9SF2 as host factors regulating RSV release and support a model in which TM9SF2 promotes the recruitment of drebrin to viral budding sites.

microbiology↗

bioRxiv2025

A dual interaction between RSV NS1 and MED25 ACID domain reshapes antiviral responses

Respiratory syncytial virus (RSV), the most common cause of bronchiolitis and pneumonia in infants, elicits a remarkably weak innate immune response. This is partly due to the type I interferon (IFN-I) antagonism of the non-structural RSV NS1 protein. It was recently suggested that NS1 could modulate host transcription via an interaction with the MED25 subunit of the Mediator complex. Previous work emphasized the role of the NS1 C-terminal helix 3 for recruitment of the MED25 ACID domain, a target of transcription factors (TFs). Here we show that the NS1 /{beta} core domain binds to MED25 ACID, and acts cooperatively with NS1 3 to achieve nanomolar affinity. This strong interaction is rationalized by the dual NS1 binding site on MED25 ACID predicted by AlphaFold3, which overlaps with the two canonical binding interfaces of TF transactivation domains (TADs), H1 and H2. By NMR, we confirmed that the NS1 /{beta} core domain targets the H2 interface. Several single amino acid NS1 /{beta} core domain mutations displayed reduced affinity for MED25 ACID, both in vitro and in cellula, at a comparable extent to the deletion of NS1 3. These mutations resulted in attenuated replication of recombinant RSV (rRSV) and increased expression of several antiviral interferon-stimulated genes (ISG) in interferon-competent cells. In MED25 knockdown cells, rRSV-mCherry replication was further attenuated, in line with the upregulation of IFT1 and ISG15 ISGs. The difference between WT and NS1 mutant rRSV-mCherry was partially lost, suggesting that RSV uses MED25 to control antiviral responses, by a mechanism involving the NS1-MED25 ACID complex. The strong interaction and the extended binding surface of NS1 on MED25 ACID provide evidence for a mechanism, where NS1 blocks access of transcription factors to MED25 ACID, and thereby MED25 mediated transcription activation. Author SummaryRespiratory syncytial virus (RSV) is a major pathogen for acute lower respiratory infections in infants and in the elderly. RSV elicits a remarkably weak immune response. It has developed a unique strategy to counteract the immune system, by encoding two small multifunctional proteins, RSV NS1 and NS2. NS1 is involved in interferon antagonism in the cytosol. Recently NS1 was shown to modulate host transcription in the nucleus. However, the mechanisms underpinning this function are not fully clear. Here we focus on the interplay between NS1 and the cellular MED25 coactivator protein, which can contribute to the antiviral response by activating several innate immune response genes. The MED25 C-terminal ACtivator Interacting Domain (ACID), a target of cellular transcription factors (TF), is a key feature for this function. To investigate the impact of MED25 hijacking by NS1, we combined in vitro biophysical experiments and cellular assays to probe the relationship between the stability of the NS1-MED25 ACID complex and RSV replication as well as antiviral responses. Our results suggest that this interaction is correlated with antiviral response antagonism, probably by hindering TFs to interact with MED25-ACID. This knowledge might pave the way for antiviral strategies aimed at stimulating appropriate immune responses.

pathology↗

bioRxiv2023

Specific targeting and clustering of Phosphatidylserine lipids by RSV M protein is critical for virus particle production

Human Respiratory Syncytial virus (RSV) is the leading cause of infantile bronchiolitis in the developed world and of childhood deaths in resource-poor settings. The elderly and the immunosuppressed are also affected. It is a major unmet target for vaccines and anti-viral drugs. RSV assembles and buds from the host cell plasma membrane by forming infections viral particles which are mostly filamentous. A key interaction during RSV assembly is the interaction of plasma membrane lipids with the Matrix (M) protein layer forming at assembly sites. Although the structure of RSV M protein dimer is known, it is unclear how the viral M proteins interact with certain plasma membrane lipids to promote viral assembly. Here, we demonstrate that M proteins cluster at the plasma membrane by selectively binding with phosphatidylserine (PS). Our in vitro studies suggest that M binds PS lipid as dimers and M mutant with a phosphomimetic substitution inhibits interaction with PS lipids. The presence of other negatively charged lipids like PI(4, 5)P2 does not affect RSV M ability to bind, while cholesterol negatively affects M interaction with lipids. Moreover, we show that the initial binding of the RSV M protein with lipids is independent of the cytoplasmic tail of fusion (F) glycoprotein (FCT). It is the first in vitro study of M interaction with plasma membrane lipids. M binding to plasma membrane may represent a viable therapeutic strategy for small molecules that will block viral spread.

biochemistry↗

bioRxiv2023

A role for the proteasome alpha2 subunit N-tail in substrate processing

The proteolytic active sites of the 26S proteasome are sequestered within the catalytic chamber of its 20S core particle (CP). Access to this chamber is through a narrow channel defined by the outer subunits. Free 20S CP is found in a latent state in which the N-termini of neighboring subunits form a gate blocking access to the channel. Entry of substrates can be facilitated by the attachment of the activators or regulatory particles, which can rearrange the blocking subunit N-terminal residues. In order to determine the specific physiological role of individual elements working in concert within the gate, we constructed a set of truncations or single-site mutations in each of the participating N-terminal tails. We report herein that whereas only a few N-termini are important for maintaining a closed gate, all seven N-termini participate in the open gate. Specifically, an invariant tyrosine (Y) in each subunit forms a hydrogen bond with a conserved aspartate (D) in the N-terminal tail of its counterclockwise neighbor, with the exception of the 1-2 pair leaving a gap in the ring circumference. A third residue (X) of this YD(X) motif aligns the open channel; specifically, the phenylalanine (F) at this position of the 2 subunit comes in direct contact with the translocating substrate. Consequently, deletion of this 2 N-terminal tail slows down proteolysis despite the appearance of an open gate state. We conclude that the YD(X) motif in N-terminal tail of subunits plays an important role in gating the proteasome and in processing the substrate.

biochemistry↗

bioRxiv2021

Respiratory Syncytial virus NS1 protein targets the transactivator binding domain of MED25

Respiratory syncytial virus has evolved a unique strategy to evade host immune response by coding for two non-structural proteins NS1 and NS2. Recently it was shown that in infected cells, nuclear NS1 could be involved in transcription regulation of host genes linked to innate immune response, via an interaction with chromatin and the Mediator complex. Here we identified the MED25 Mediator subunit as an NS1 interactor in a yeast two-hybrid screen. We demonstrate that NS1 directly interacts with MED25 in vitro and in cellula, and that this interaction involves the C-terminal 3 helix of NS1 and the MED25 ACID domain. More specifically we showed by NMR that the NS1 3 sequence primarily binds to the MED25 ACID H2 face, which is a transactivation domain (TAD) binding site for transcription regulators such as ATF6, a master regulator of ER stress response activated upon viral infection. Moreover, we found out that the NS1 3 helix could compete with ATF6 TAD binding to MED25. This finding points to a mechanism of NS1 interfering with innate immune response by impairing recruitment by cellular TADs of the Mediator via MED25 and hence transcription of specific genes by RNA polymerase II. ImportanceHuman RSV is the leading cause of infantile bronchiolitis in the world and one of the major causes of childhood deaths in resource-poor settings. It is a major unmet target for vaccines and anti-viral drugs. RSV non-structural protein NS1 is known to antagonize the cellular immune response and was recently shown to be involved in transcription regulation of infected cells. However, the exact mechanism of this regulation is not well defined. Here we show that nuclear NS1 interacts directly with the Mediator subunit MED25 and is able to compete with a cellular transcription activator, which is activated during viral infection. We hypothesize that this interaction may underlie regulation of the expression of genes involved in the innate immune response.

microbiology↗

bioRxiv2021

Characterization of the interaction domains between the phosphoprotein and the nucleocapsid of human Metapneumovirus

Human metapneumovirus (HMPV) causes severe respiratory diseases in young children. The HMPV RNA genome is encapsidated by the viral nucleoprotein, forming an RNA-N complex (NNuc), which serves as template for genome replication and mRNA transcription by the RNA-dependent RNA polymerase (RdRp). The RdRp is formed by the association of the large polymerase subunit (L), which has RNA polymerase, capping and methyltransferase activities, and the tetrameric phosphoprotein (P). P plays a central role in the RdRp complex by binding to NNuc and L, allowing the attachment of the L polymerase to the nucleocapsid template. During infection these proteins concentrate in cytoplasmic inclusion bodies (IBs) where viral RNA synthesis occurs. By analogy to the closely related pneumovirus respiratory syncytial virus (RSV), it is likely that the formation of IBs depends on the interaction between P and NNuc. However, the HMPV P-NNuc interaction still remains to characterize. Here, we finely characterized the binding domains involved in HMPV P and NNuc interaction by studying binding between recombinant proteins, combined with the use of a functional assay of the polymerase complex activity and the study of the recruitment of these proteins to IBs by immunofluorescence. We show that the last 6 C-terminal residues of HMPV P are necessary and sufficient for binding to NNuc, that P binds the N-terminal domain of N (NNTD), and identified conserved N residues critical for the interaction. Our results allowed to propose a structural model of the HMPV P-NNuc interaction. IMPORTANCELike respiratory syncytial virus (RSV), human metapneumovirus (HMPV) is a leading cause of severe respiratory infections in children but also affects human populations of all ages worldwide. Nowadays, no vaccine or efficient antiviral treatments are available for these two pneumoviruses. A better understanding of the molecular mechanisms involved in viral replication could help the design or discovery of specific antiviral compounds. In this work we have investigated the interaction between two major viral proteins involved in HMPV RNA synthesis, the N and P proteins. We finely characterized their domains of interaction, in particular a pocket on the surface of the N protein that could be used as a template for the design of compounds interfering with N-P complexes and viral replication.

microbiology↗

bioRxiv2020

Tetramerization of Phosphoprotein is essential for Respiratory Syncytial virus budding while its N terminal region mediates direct interactions with the Matrix protein.

It was shown previously that the Matrix (M), Phosphoprotein (P), and the Fusion (F) proteins of Respiratory syncytial virus (RSV) are sufficient to produce virus-like particles (VLPs) that resemble the RSV infection-induced virions. However, the exact mechanism and interactions among the three proteins are not known. This work examines the interaction between P and M during RSV assembly and budding. We show that M interacts with P in the absence of other viral proteins in cells using a Split Nano Luciferase assay. By using recombinant proteins, we demonstrate a direct interaction between M and P. By using Nuclear Magnetic Resonance (NMR) we identify three novel M interaction sites on P, namely site I in the N2 region, site II in the 115-125 region, and the oligomerization domain (OD). We show that the OD, and likely the tetrameric structural organization of P, is required for virus-like filament formation and VLP release. Although sites I and II are not required for VLP formation, they appear to modulate P levels in RSV VLPs. ImportanceHuman RSV is the commonest cause of infantile bronchiolitis in the developed world and of childhood deaths in resource-poor settings. It is a major unmet target for vaccines and anti-viral drugs. The lack of knowledge of RSV budding mechanism presents a continuing challenge for VLP production for vaccine purpose. We show that direct interaction between P and M modulates RSV VLP budding. This further emphasizes P as a central regulator of RSV life cycle, as an essential actor for transcription and replication early during infection and as a mediator for assembly and budding in the later stages for virus production.

microbiology↗

bioRxiv2018

The ESCRT-III proteins IST1 and CHMP1B assemble around nucleic acids

ESCRT-III proteins can promote inside-out or outside-in membrane tubulation and fission. In addition, several observations suggest that ESCRT factors may also associate with nucleic acids during development, different stages of the cell cycle, and during retro-transposition of parasitic nucleic acids like LINE1 elements. Two ESCRT-III subunits, IST1 (aka CHMP8) and CHMP1B, can coassemble as an external protein coat around liposomes in vitro and around recycling endosomal tubules in living cells. Here we show that recombinant IST1 and CHMP1B can also copolymerize into double stranded filaments that surround nucleic acids. Electron cryo-microscopy reconstructions of nucleic acid-bound IST1-CHMP1B copolymers revealed that the polynucleotides track along a binding groove formed between filaments of the inner CHMP1B strand. The well-ordered structures also reveal that the C-terminal tails of CHMP1B subunits extrude through the outer IST1 layer to the tube exterior. As a result, the MIT domain binding motifs of both CHMP1B and IST1 are arrayed on the outer surface of the copolymer, where they could bind and recruit MIT domain-containing co-factors, such as the SPASTIN ATPase or the USP8 ubiquitin protease. Our structure raises the possibility that ESCRT-III proteins may form nucleic acid complexes in mammalian cells.

biochemistry↗