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This study provides crucial insights into the molecular mechanisms of nucleocapsid assembly and transport in filoviruses, specifically Marburg virus (MARV) and Ebola virus (EBOV). Using advanced live-cell imaging, we uncovered how the conserved PPxPxY motif in nucleoprotein (NP) mediates its interactions with VP30, thereby regulating nucleocapsid formation and viral replication. Notably, while VP30’s role differs between MARV and EBOV, the underlying mechanism of NP-VP30 interaction via this motif appears conserved across filoviruses, making it a promising target for broad-spectrum antiviral strategies. These findings deepen our understanding of nucleocapsid protein compatibility and virus-host interactions, offering new avenues for therapeutic intervention against these deadly pathogens.

A crucial step of HBV (Hepatitis B Virus) virion morphogenesis is the envelopment of the nucleocapsid by the viral envelope proteins, which is triggered by an interaction between the HBV core protein and the large HBV envelope protein. To document this protein–protein interaction, we co-expressed core and large HBV envelope (LHBs) in Huh-7 cells and subjected the cells to microscopy examination by Fluorescence Resonance Energy Transfer (FRET) and Transmission Electron Microscopy (TEM). Our results show that the sole expression of the core protein leads to assembly of capsids that remain individually isolated within the whole cell, but particularly within the nucleus. In the presence of LHBs, capsids were observed as large clusters in a membrane rich region peripheral to the nucleus. In this context, core-LHBs complex co-localize with markers of the late endosome/multivesicular bodies, this co-localization being driven by LHBs. These results thus show that LHBs binds to the core proteins when preassembled into capsid, at membranes of the late endosome, where the inner capsid and the outer envelope meet to assemble a virion.

Satellite tobacco necrosis virus (STNV) is one of the smallest viruses known. Its genome encodes only its coat protein (CP) subunit, relying on the polymerase of its helper virus TNV for replication. The genome has been shown to contain a cryptic set of dispersed assembly signals in the form of stem-loops that each present a minimal CP-binding motif AXXA in the loops. The genomic fragment encompassing nucleotides 1–127 is predicted to contain five such packaging signals (PSs). We have used mutagenesis to determine the critical assembly features in this region. These include the CP-binding motif, the relative placement of PS stem-loops, their number, and their folding propensity. CP binding has an electrostatic contribution, but assembly nucleation is dominated by the recognition of the folded PSs in the RNA fragment. Mutation to remove all AXXA motifs in PSs throughout the genome yields an RNA that is unable to assemble efficiently. In contrast, when a synthetic 127-nt fragment encompassing improved PSs is swapped onto the RNA otherwise lacking CP recognition motifs, assembly is partially restored, although the virus-like particles created are incomplete, implying that PSs outside this region are required for correct assembly. Swapping this improved region into the wild-type STNV1 sequence results in a better assembly substrate than the viral RNA, producing complete capsids and outcompeting the wild-type genome in head-to-head competition. These data confirm details of the PS-mediated assembly mechanism for STNV and identify an efficient approach for production of stable virus-like particles encapsidating nonnative RNAs or other cargoes.

The coronavirus’ (CoV) membrane (M) protein is the driving force during assembly, but this process remains poorly characterized. Previously, we described two motifs in the C-tail of the Middle East respiratory syndrome CoV (MERS-CoV) M protein involved in its endoplasmic reticulum (ER) exit ( 211 DxE 213 ) and trans-Golgi network (TGN) retention ( 199 KxGxYR 204 ). Here, their function in virus assembly was investigated by two different virus-like particle (VLP) assays and by mutating both motifs in an infectious MERS-CoV cDNA clone. It was shown that the 199 KxGxYR 204 motif was essential for VLP and infectious virus assembly. Moreover, the mislocalization of the M protein induced by mutation of this motif prevented M–E interaction. Hampering the ER export of M by mutating its 211 DxE 213 motif still allowed the formation of nucleocapsid-empty VLPs, but prevented the formation of fully assembled VLPs and infectious particles. Taken together, these data show that the MERS-CoV assembly process highly depends on the correct intracellular trafficking of its M protein, and hence that not only specific protein–protein interacting motifs but also correct subcellular localization of the M protein in infected cells is essential for virus formation and should be taken into consideration when studying the assembly process.

Surface tubular elements (STEs) are critical components of poxvirus mature virions and play a role in suppressing host cell protein synthesis. In this study, we isolated and purified STEs from native poxvirus virions and subsequently determined their core composition and high-resolution architecture. We identified that STE is mainly composed of membrane proteins A14 and A17, along with phospholipid molecules. Within the repeat structural unit of STE, A14 proteins form two homodimers within the repeating unit, with A17 monomers flanking either side. Phospholipid molecules are distributed within the A14-A14 and A14-A17 interfaces. Our study not only revealed the molecular structures of A14 and A17 but also further emphasized that the reticulon-like and highly oligomerized characteristics of A17 provide membrane curvature, while the A14-A17-phospholipid network stabilizes the tubular structure. We proposed a hypothetical model that A17 drives changes in viral membrane curvature during maturation. These findings enhance our understanding of poxvirus biology and may guide therapeutic strategies against poxvirus infections.

Protein N-myristoylation is an important fatty acylation catalyzed by N-myristoyltransferases (NMTs), which are ubiquitous enzymes in eukaryotes. Specifically, attachment of a myristoyl group is vital for proteins participating in various biological functions, including signal transduction, cellular localization, and oncogenesis. Recent studies have revealed unexpected mechanisms indicating that protein N-myristoylation is involved in host defense against microbial and viral infections. In this review, we describe the current understanding of protein N-myristoylation (mainly focusing on myristoyl switches) and summarize its crucial roles in regulating innate immune responses, including TLR4-dependent inflammatory responses and demyristoylation-induced innate immunosuppression during Shigella flexneri infection. Furthermore, we examine the role of myristoylation in viral assembly, intracellular host interactions, and viral spread during human immunodeficiency virus-1 (HIV-1) infection. Deeper insight into the relationship between protein N-myristoylation and innate immunity might enable us to clarify the pathogenesis of certain infectious diseases and better harness protein N-myristoylation for new therapeutics.The relationship between protein N-myristoylation and innate immunity has emerged as an exciting area of research. Here, the recent progress of protein N-myristoylation and modulation in innate immune responses, including TLR4 inflammatory responses, demyristoylation-induced innate immunosuppression during Shigella flexneri infection, and human immunodeficiency virus-1 (HIV-1) assembly, intracellular host interactions and spread, is summarized.

In 2019, a new pandemic virus belonging to the betacoronavirus family emerged, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This new coronavirus appeared in Wuhan, China, and is responsible for severe respiratory pneumonia in humans, namely, coronavirus disease 2019 (COVID-19). In 2019, a new pandemic virus belonging to the betacoronavirus family emerged, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This new coronavirus appeared in Wuhan, China, and is responsible for severe respiratory pneumonia in humans, namely, coronavirus disease 2019 (COVID-19). Having infected almost 200 million people worldwide and caused more than 4.1 million deaths as of today, this new disease has raised a significant number of questions about its molecular mechanism of replication and, in particular, how infectious viral particles are produced. Although viral entry is well characterized, the full assembly steps of SARS-CoV-2 have still not been fully described. Coronaviruses, including SARS-CoV-2, have four main structural proteins, namely, the spike glycoprotein (S), the membrane glycoprotein (M), the envelope protein (E), and the nucleocapsid protein (N). All these proteins have key roles in the process of coronavirus assembly and budding. In this review, we gathered the current knowledge about betacoronavirus structural proteins involved in viral particle assembly, membrane curvature and scission, and then egress in order to suggest and question a coherent model for SARS-CoV-2 particle production and release.

To generate infectious viral particles, viruses must specifically select their genomic RNA from milieu that contains a complex mixture of cellular or non-genomic viral RNAs. In this review, we focus on the role of viral encoded RNA structures in genome packaging. We first discuss how packaging signals are constructed from local and long-range base pairings within viral genomes, as well as inter-molecular interactions between viral and host RNAs. Then, how genome packaging is regulated by the biophysical properties of RNA. Finally, we examine the impact of RNA packaging signals on viral evolution.

Herpesvirus virions contain a unique tegument layer sandwiched between the capsid and lipid envelope and composed of multiple copies of about two dozen viral proteins. However, little is known about the structure of the tegument or how it is assembled. Here, we show that a conserved tegument protein UL11 from herpes simplex virus 1, a prototypical alphaherpesvirus, is an intrinsically disordered protein that undergoes liquid-liquid phase separation in vitro . Through sequence analysis, we find intrinsically disordered regions of different lengths in all HSV-1 tegument proteins. We hypothesize that intrinsic disorder is a common characteristic of tegument proteins and propose a new model of tegument as a biomolecular condensate. A distinguishing morphological feature of all herpesviruses is the multiprotein tegument layer located between the nucleocapsid and lipid envelope of the virion. Tegument proteins play multiple roles in viral replication, including viral assembly, but we do not yet understand their individual functions or how the tegument is assembled and organized. UL11, the smallest tegument protein, is important for several distinct processes in replication, including efficient virion morphogenesis and cell-cell spread. However, the mechanistic understanding of its role in these and other processes is limited in part by the scant knowledge of its biochemical and structural properties. Here, we report that UL11 from herpes simplex virus 1 (HSV-1) is an intrinsically disordered, conformationally dynamic protein that undergoes liquid-liquid phase separation (LLPS) in vitro . Intrinsic disorder may underlie the ability of UL11 to exert multiple functions and bind multiple partners. Sequence analysis suggests that not only all UL11 homologs but also all HSV-1 tegument proteins contain intrinsically disordered regions of different lengths. The presence of intrinsic disorder, and potentially, the ability to form LLPS, may thus be a common feature of the tegument proteins. We hypothesize that tegument assembly may involve the formation of a biomolecular condensate, driven by the heterogeneous mixture of intrinsically disordered tegument proteins. IMPORTANCE Herpesvirus virions contain a unique tegument layer sandwiched between the capsid and lipid envelope and composed of multiple copies of about two dozen viral proteins. However, little is known about the structure of the tegument or how it is assembled. Here, we show that a conserved tegument protein UL11 from herpes simplex virus 1, a prototypical alphaherpesvirus, is an intrinsically disordered protein that undergoes liquid-liquid phase separation in vitro . Through sequence analysis, we find intrinsically disordered regions of different lengths in all HSV-1 tegument proteins. We hypothesize that intrinsic disorder is a common characteristic of tegument proteins and propose a new model of tegument as a biomolecular condensate.

Late assembly (L) domains are conserved sequences that are necessary for the late steps of viral replication, acting like cellular adaptors to engage the ESCRT membrane fission machinery that promote virion release. These short sequences, whose mutation or deletion produce the accumulation of immature virions at the plasma membrane, were firstly identified within retroviral Gag precursors, and in a further step, also in structural proteins of many other enveloped RNA viruses including arenaviruses, filoviruses, rhabdoviruses, reoviruses, and paramyxoviruses. Three classes of L domains have been identified thus far (PT/SAP, YPXnL/LXXLF, and PPxY), even if it has recently been suggested that other motifs could act as L domains. Here, we summarize the current state of knowledge of the different types of L domains and their cellular partners in the budding events of RNA viruses, with a particular focus on retroviruses.