Identification of Viral and Cellular Proteins in Proximity to the HSV-2 pUL16 Tegument Protein in Infected Cells

Mapping in-cell protein contact sites reveals hijacking of paraspeckles during influenza A virus infection

Influenza A virus (IAV) hijacks host cellular machinery during infection but many host–virus protein interactions remain uncharacterized, particularly in their native context. Here, we applied in-cell cross-linking mass spectrometry, integrated with structural modelling and functional assays, to map protein–protein contact sites in IAV-infected human cells. This revealed previously unrecognized virus–host interactions linked to spatially organized processes. We identified host factors linked to the maturation of distinct glycoforms of the viral surface glycoprotein haemagglutinin through the membrane-bound endoplasmic reticulum–Golgi system. In the nucleus, we observed the progressive disassembly of paraspeckles (phase-separated membraneless compartments) across multiple cell lines. Mechanistically, viral nucleoprotein and non-structural protein 1 interact with host paraspeckle proteins, the viral endonuclease PA-X degrades long non-coding RNA housed within paraspeckles and viral RNA polymerase II is inhibited to drive paraspeckle disruption, which releases host factors that facilitate IAV replication. These findings uncover mechanisms by which IAV exploits and remodels host compartments during infection. In-cell cross-linking mass spectrometry maps protein contacts in influenza-infected human cells, revealing how the virus hijacks host membrane-trafficking factors and dismantles nuclear paraspeckles to enhance replication.

Nuclear activities and interactome of the NS5 protein of tick-borne encephalitis virus

ABSTRACT Orthoflaviviruses are RNA viruses responsible for significant diseases in humans, domesticated animals, and wildlife. Their NS5 protein is central in viral replication, functioning both as an RNA-dependent RNA polymerase and a methyltransferase, while also modulating cellular processes, including the interferon response. Although viral replication is cytoplasmic, the NS5 protein of several mosquito-borne orthoflaviviruses cycles between the cytoplasm and the nucleus of infected human cells. However, the nuclear localization and function of NS5 of tick-borne orthoflaviviruses, such as tick-borne encephalitis virus (TBEV), remain poorly understood. Microscopy analysis and cell fractionation revealed that the NS5 protein of TBEV localized to both the cytoplasm and nucleoplasm of infected cells. Mutagenesis studies identified critical residues required for its nuclear targeting. Mutating these residues in a TBEV replicon abolished viral replication. Immunoprecipitation-mass spectrometry analyses performed in two human cell lines infected with TBEV recovered 352 NS5 partners. Among them, 187 were nuclear or partially nuclear. By integrating our interactome data with that of Powassan virus (POWV), another tick-borne orthoflavivirus, we refined a list of 20 high-confidence NS5 partners, including splicing factors and chromatin modulators. Functional analysis revealed that seven of these nuclear partners significantly modulated viral replication, further underscoring the importance of nuclear NS5 in the viral life cycle. Our work advances our understanding of the nuclear function of the NS5 proteins of tick-borne orthoflaviviruses. IMPORTANCE Tick-borne orthoflaviviruses are emerging globally, spreading across Europe, Asia, and North America, where they infect humans, domesticated animals, and wildlife. These viruses produce a protein called NS5, which drives viral replication and helps evade the innate immune response. We observed that the NS5 protein of tick-borne encephalitis virus (TBEV) localized both in the cytoplasm and nucleoplasm of infected human cells. We identified the specific residues responsible for its nuclear addressing and showed that it interacts with numerous nuclear proteins, including some involved in regulating gene expression. Seven of these nuclear partners significantly influenced viral replication, highlighting the importance of NS5’s nuclear activity. This work sheds light on how tick-borne orthoflaviviruses manipulate host cells, deepening our understanding of their replication strategies. Tick-borne orthoflaviviruses are emerging globally, spreading across Europe, Asia, and North America, where they infect humans, domesticated animals, and wildlife. These viruses produce a protein called NS5, which drives viral replication and helps evade the innate immune response. We observed that the NS5 protein of tick-borne encephalitis virus (TBEV) localized both in the cytoplasm and nucleoplasm of infected human cells. We identified the specific residues responsible for its nuclear addressing and showed that it interacts with numerous nuclear proteins, including some involved in regulating gene expression. Seven of these nuclear partners significantly influenced viral replication, highlighting the importance of NS5’s nuclear activity. This work sheds light on how tick-borne orthoflaviviruses manipulate host cells, deepening our understanding of their replication strategies.

Conserved herpesvirus protein kinase (CHPK)-mediated phosphorylation of viral proteins associated with nucleocytoplasmic trafficking during natural infection

Structural Overview of Herpesvirus Tegument Proteins

Virion-wide interactome mapping of HSV-1 reveals maturation-dependent remodeling and convergent organization of herpesvirus tegument networks

Herpesvirus virions form by remodeling of intracellular virus-host interaction networks into evolutionarily conserved particle architectures. Here, we define a virion-wide spatial and quantitative protein proximity map of herpes simplex virus 1 (HSV-1) by combining cross-linking mass spectrometry with quantitative proteomics. Integration with intracellular interaction maps reveals that maturation acts as a selective filter, transforming broad virus-host associations into an organized virion network. This process depletes biosynthetic and nuclear components while enriching interactions involved in tegument organization and envelope acquisition around the viral protein UL49. Comparison with analogous maps of human cytomegalovirus (HCMV) identifies HSV-1-UL49 and HCMV-UL32 as functionally equivalent network hubs despite lacking evolutionary relatedness. Both hubs converge on shared phosphoregulatory host factors, short linear interaction motifs, and liquid-liquid phase separation. At the virion surface, the host complement regulator CD59 protects particles from complement-mediated inactivation. Together, these findings show how conserved organizational principles shape virus-specific virion interaction networks during herpesvirus maturation.

Beyond the Capsid: How Can Post-Translational Modifications Modulate the Multifunctionality of the Orthoflavivirus Capsid Protein?

The orthoflavivirus capsid (C) protein is a multifunctional protein that plays essential roles throughout the viral life cycle. Besides viral RNA encapsidation for nucleocapsid assembly, it associates with lipid droplets, interacts with host proteins, and translocates to the nucleus, although its nuclear functions are still poorly understood. How these diverse activities are coordinated remains an open question. Post-translational modifications (PTMs), which are key regulators of protein function, have emerged as critical modulators of the infection cycle in many RNA viruses. However, little is known about the occurrence and functional significance of PTMs in orthoflavivirus C proteins. Here, we review the current evidence on PTMs in orthoflavivirus C proteins and integrate insights from studies of other RNA viruses to propose mechanisms by which PTMs may regulate C protein function. To complement this review, we performed a comparative in silico analysis of predicted PTM sites in the C proteins of dengue, Zika, West Nile, and Japanese encephalitis viruses. By integrating PTM predictions with experimentally validated modification sites, residue conservation, and structural mapping, we identified conserved regulatory hotspots that represent promising targets for future experimental validation. Together, these findings highlight PTMs as an underexplored regulatory mechanism in orthoflavivirus capsid biology and provide a framework for future mechanistic investigations.