RNA viruses, exemplified by the COVID-19 pandemic, pose a significant threat to global health. Their rapid mutation and host adaptability highlight the need for advanced tools for efficient viral studies and timely countermeasure development. Imaging technologies, such as cryo-electron microscopy and super-resolution microscopy, have been pivotal in advancing our understanding of viral structures, infection mechanisms, and virus-host interactions. However, each technique has limitations in the field of view or resolution. Recent advancements have focused on developing integrated multiscale imaging to better understand RNA virus pathogenesis. In this review, we examine recent progress in RNA virus imaging across molecular, cellular, and tissue scales, including cryo-electron tomography and correlative multiscale imaging, which link structural mapping with functional insights.

Palmitoylation is a reversible post-translational modification that enhances protein hydrophobicity and regulates cellular functions such as trafficking and signaling. In humans, this modification is catalyzed by 23 DHHC enzymes, but the mechanisms by which they recognize their substrates remain unclear. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein undergoes palmitoylation primarily by DHHC20 with subsequent modification by DHHC9 at 10 cytoplasmic tail (CT) cysteines, a modification crucial for membrane fusion and viral entry. Using AlphaFold2 modeling and site-directed mutagenesis, we identified three key components critical for efficient spike palmitoylation: (i) Lys1211 at the ectodomain-transmembrane domain (TMD) interface, likely facilitating electrostatic interactions with DHHC20's acidic residues; (ii) a stable trimeric TMD helix, where mutations at the trimer interface impair palmitoylation, in contrast to changes in outward-facing residues; and (iii) a conserved hydrophilic motif in the CT, located between acylated cysteine clusters, likely promoting optimal substrate positioning near DHHC20's catalytic site. Co-immunoprecipitation assays revealed that mutations in these residues disrupt spike-DHHC20 interactions, while leaving spike-DHHC9 binding unchanged, suggesting that they affect enzyme-substrate complex formation. Fusion assays revealed nuanced effects; while palmitoylation generally correlated positively with membrane fusion, certain exceptions highlighted the complex relationship between these processes. Mutations in the CT markedly reduce total spike palmitoylation but only modestly affect cell-cell fusion. Some substitutions in the TMD impair fusion with little change in overall acylation. Our findings elucidate the structural and biophysical determinants of spike palmitoylation and its distinct roles in membrane fusion, offering insights into SARS-CoV-2 pathogenesis and potential antiviral targets.

S-acylation is post-translational attachment of fatty acid residues, mostly palmitoyl-groups to cysteines. This lipid modification could influence protein oligomerization, localization, and topology within the membrane, and could play a regulatory role in cellular signaling. Improvements in the experimental techniques, specifically, MALDI-TOF MS, have elevated the study of S-acylation to a level of refined detail. It was possible to conclusively show that, beyond palmitate, other types of fatty acids, such as stearate and oleate could be attached to the specific binding sites. For example, fusion proteins of the enveloped viruses have been shown to attach stearates exclusively to the cysteine located at the C-terminal end of the transmembrane domain, while cysteines in the cytoplasmic domain have been modified with palmitates. In addition, emerging evidence suggests that the covalently linked fatty acids in the viral fusion proteins could recruit cholesterol into the viral membrane. We termed this preferential attachment of fatty acid residues of different types to the specific sites, depending on their localization, as “differential” S-acylation. Furthermore, MALDI-TOF MS detected attachment of unsaturated fatty acids (oleates) to the GNAI protein in response to stearate supplementation, shifting this regulatory protein out of the detergent-resistant membranes (lipid rafts) and, consequently, exerting antitumor effect. Solving the first 3D-structures of DHHC acyltransferases allowed proposing a mechanism of different fatty acids selection. However, it remains unclear whether preference for the acyl chain length is determined by the structure of the enzyme, protein substrate, or by the lipid composition of the membrane. In this review, we summarize the latest advances in the study of protein S-acylation with different types of fatty acids and substantiate significance of this post-translational modification from both fundamental and practical perspectives.

Equine arteritis virus (EAV) is a positive-stranded RNA virus of the Arteriviridae family. Its GP5/M dimer, the principal component of the viral envelope, mediates virus budding and serves as a key target for neutralizing antibodies. Using AlphaFold3, we predicted the 3D structure of the EAV GP5/M dimer and compared it to its homolog in porcine reproductive and respiratory syndrome virus (PRRSV). Both complexes share a conserved architecture comprising a short ectodomain, three helical transmembrane regions, and a β-sheet-rich endodomain. EAV GP5 features a longer ectodomain with four α-helices and a disulfide-linked β-sheet, which forms the most variable and surface-exposed region containing neutralizing epitopes. Adjacent conserved and variable N-glycosylation sites suggest immune evasion mechanisms involving antigenic drift and glycan shielding. Another epitope, located in a membrane-proximal helix, overlaps with known virulence and persistence determinants. The transmembrane domains are the most structurally conserved regions between EAV and PRRSV, characterized by tilted and kinked helices stabilized by hydrophilic interactions within the lipid bilayer. These findings provide molecular insights into the structural organization, immune targets, and virulence-associated features of the GP5/M dimer, offering a foundation for rational vaccine design against EAV.

Merbecovirus is a subgenus of betacoronaviruses and exhibits high genetic diversity with a capacity for cross-species transmission. However, beyond Middle East respiratory syndrome coronavirus (MERS-CoV), our knowledge of the ecology and pathogenic potential of these viruses remains limited. Merbecoviruses were once thought to rely exclusively on dipeptidyl peptidase 4 for cell entry, but recent discoveries have revealed that several members can also engage with angiotensin-converting enzyme 2 or aminopeptidase N, expanding their receptor repertoire and potential host range. Here we summarize recent advances in understanding of the receptor usage of merbecoviruses and examine how these insights inform pandemic preparedness and risk assessment. We discuss the development of targeted diagnostics, broad-spectrum antivirals and vaccines, including pan-coronavirus strategies. Together, these advances provide a foundation for predictive surveillance and rational countermeasure design, enabling earlier detection and more effective containment of future merbecovirus spillover events before they escalate into epidemics.

The natural host for avian influenza virus (AIV) is waterfowl. However, certain subtypes have breached species barriers, causing epizootics in many avian and mammalian species with occasional zoonotic infections in humans. The ongoing spread of highly pathogenic avian influenza (HPAI) A(H5N1) poses a significant and growing public health threat. Here, we discuss recent advances in viral detection and characterization technologies and their integration into the diagnostics and surveillance of AIV within a “One Health” framework.

Coronaviruses often cross species barriers, with receptor binding dictating their host range and zoonotic potential. Merbecoviruses, such as MERS-CoV, typically utilize DPP4 as their receptor, whereas Sarbecoviruses, like SARS-CoV, rely on ACE2. This study explores the receptor usage of four merbecoviruses identified in Vespertilionidae bats: HKU5, BtVs-SC2013, HKU25, and P. khulii-2011. Our findings reveal species-specific binding to bat ACE2: HKU5 binds exclusively to Pipistrellus abramus ACE2, P. khulii-2011 interacts solely with Murina aurata ACE2, BtVs-SC2013 recognizes ACE2 from Murina aurata and Myotis myotis, and HKU25 displays the broadest binding range. Beyond bats, BtVs-SC2013 binds to mink ACE2, while HKU25 interacts with both mink and pangolin ACE2, hinting at potential intermediate hosts for cross-species transmission. We also elucidated the mechanism behind HKU5's selective binding preference for P. abramus ACE2. Structural analysis and mutagenesis revealed that a carbohydrate attached at position 329 play a crucial role. Introducing the N-glycosylation site into P. abramus ACE2 eliminated binding, while its removal from P. pipistrellus ACE2, combined with two additional mutations, restored it. Moreover, we pinpointed key residues in mink ACE2 essential for binding the receptor-binding domain (RBD) of BtVs-SC2013 and HKU25. These findings illuminate the receptor usage and host specificity of bat merbecoviruses, enhancing our understanding of their potential for cross-species transmission and adaptation.

Alphaviruses are mosquito-transmitted viruses that cause severe zoonotic diseases. Their envelope glycoproteins, E1 and E2, undergo cysteine acylation, a process critical for virus infection but previously undefined mechanistically. Using the Getah virus as a model, we found that E1 is acylated at Cys433 in the endoplasmic reticulum (ER), a modification beneficial for virus entry. E2 follows a unique stepwise acylation pattern: Cys385 is acylated in the ER, while Cys395, Cys415, and Cys416 undergo interdependent acylation in the Golgi. Palmitoylation of E2 Cys415/416 proved essential for budding. Acylation also facilitated cholesterol incorporation into virions independently of lipid rafts. A small interfering RNA (siRNA) screen identified distinct acyltransferases for E1 and compartment-specific enzymes for E2. Depletion of Golgi-localized Asp-His-His-Cys (DHHC)11, which modifies Cys415/416, significantly inhibited replication of multiple alphaviruses. This study establishes the spatiotemporal model of alphavirus glycoprotein acylation and identifies DHHC11 as a conserved target for pan-alphavirus therapeutics, with potential implications for reducing the public health burden of alphavirus infections.

Porcine reproductive and respiratory syndrome virus (PRRSV), an Arteriviridae family enveloped RNA virus, is a major swine pathogen. Using yeast transformation-associated recombination (TAR) cloning, we efficiently generated infectious PRRSV and GFP-expressing clones, identifying transcription-regulating sequences as essential for stable foreign gene expression. Screening SARS-CoV-2 antivirals showed potent inhibition by the multitarget drug ribavirin, the polymerase inhibitors remdesivir and its metabolite GS-441524. Molnupiravir, targeting the polymerase by a different mechanism, showed reduced efficacy against PRRSV, while the protease inhibitor GC376 was ineffective. The AlphaFold-predicted structure of the PRRSV polymerase revealed conserved catalytic architecture with the SARS-CoV-2 polymerases, explaining cross-family inhibitor activity. In contrast, structural divergence in proteases correlated with GC376’s inefficacy. These findings underscore the utility of the TAR cloning for arterivirus engineering, with potential applications in vector vaccine development.

Despite accumulating evidence that bat-derived coronaviruses often require intermediate hosts to facilitate transmission to humans1, the potential role of fur animals in zoonotic coronavirus spillovers has largely been overlooked2. Here we report the isolation and characterization of a previously undescribed mink respiratory coronavirus (MRCoV) from farmed minks with pneumonia. Notably, MRCoV uses angiotensin-converting enzyme 2 (ACE2) as an entry receptor and can infect mink, bat, monkey and human cells. Cryo-electron microscopy analyses revealed that the MRCoV receptor-binding domain (RBD) binds to the same interface on ACE2 receptors as the RBD of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) despite structural differences. We identify the key determinants on the RBD of MRCoV and ACE2 that confer efficient binding. HKU5-33S, a bat coronavirus closely related to MRCoV, uses ACE2 of the bat Pipistrellus abramus for cell entry and requires only two amino acid substitutions to adapt to mink ACE2. SARS-CoV-2 protease and polymerase inhibitors potently block MRCoV infection, thereby indicating a potential therapeutic strategy. Collectively, these findings enhance our understanding of coronavirus receptor dynamics and highlight their zoonotic potential. Given the risks posed by fur farms as reservoirs for emerging pathogens, our study underscores the need for enhanced surveillance to mitigate future coronavirus outbreaks.

Protein palmitoylation is a lipid modification where a palmitoyl group is covalently attached via a thioester linkage to one or more cysteines on a substrate protein. This modification, catalyzed by a group of enzymes named DHHC enzymes after their conserved Asp-His-His-Cys motif, plays a significant role in regulating the localization, stability, and function of a wide range of cellular and viral proteins. By influencing how and where proteins interact within the cell, palmitoylation is essential for various cellular processes, including signaling pathways, membrane dynamics, and protein-protein interactions. Here, we describe the acyl-RAC assay, a biochemical technique designed to specifically enrich and analyze palmitoylated proteins from complex biological samples, such as cell lysates or tissue extracts. The assay begins by reducing and blocking free cysteine thiol groups on proteins, ensuring that only those thiols involved in thioester bonds with palmitates are accessible for downstream analysis. These thioester bonds are then cleaved to release the fatty acids from the cysteines, which are subsequently captured using thiopropyl Sepharose beads that bind to the newly exposed thiol groups. The captured proteins are eluted from the beads by breaking the bond between the thiol and the resin with reducing agents, and the proteins are then analyzed by SDS-PAGE followed by western blotting to identify and quantify them. The acyl-RAC assay's specificity for S-palmitoylated proteins makes it an invaluable tool for exploring this modification. It not only allows for the identification of previously unknown palmitoylated proteins, thereby deepening our understanding of palmitoylation in cellular processes and viral infections, but it also enables quantitative comparisons of protein palmitoylation under different experimental conditions or treatments. Key features • Allows identification of acylated proteins. • Quantitative analysis of S-palmitoylation levels under various conditions by western blot. • Requires at least seven days to complete.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major pathogen affecting pigs and belongs to the enveloped plus-stranded RNA virus family Arteriviridae. A unique feature of Arteriviruses is that the genes encoding the structural proteins overlap at their 3` and 5` ends. This impedes mutagenesis opportunities and precludes the binding of short peptides for antibody detection, as this would alter the amino acids encoded by the overlapping gene. In this study, we aimed to generate infectious PRRSV variants with separated genes encoding the minor glycoproteins Gp2, Gp3, and Gp4, accompanied by appended tags for detection. All recombinant genomes facilitate the release of infectious virus particles into the supernatant of transfected 293T cells, as evidenced by immunofluorescence of infected MARC-145 cells using anti-nucleocapsid antibodies. Furthermore, expression of Gp2-Myc and Gp3-HA was confirmed through immunofluorescence and western blot analysis with tag-specific antibodies. However, after two passages of Gp2-Myc and Gp3-HA viruses, the appended tags were completely removed as indicated by sequencing the viral genome. Recombinant viruses with separated Gp2 and Gp3 genes remained stable for at least nine passages, while those with Gp3 and Gp4 genes separated reverted to wild type after only four passages. Notably, this virus exhibited significantly reduced titers in growth assays. Furthermore, we introduced a tag to the C-terminus of Gp4. The Gp4-HA virus was consistently stable for at least 10 passages, and the HA-tag was detectable by western blotting and immunofluorescence.

The retransmissions of SARS-CoV-2 from several mammals - primarily mink and white-tailed deer - to humans have raised concerns for the emergence of a new animal-derived SARS-CoV-2 variant to worsen the pandemic. Here, we discuss animal species that are susceptible to natural or experimental infection with SARS-CoV-2 and can transmit the virus to mates or humans. We describe cutting-edge techniques to assess the impact of a mutation in the viral spike (S) protein on its receptor and on antibody binding. Our review of spike sequences of animal-derived viruses identified nine unique amino acid exchanges in the receptor-binding domain (RBD) that are not present in any variant of concern (VOC). These mutations are present in SARS-CoV-2 found in companion animals such as dogs and cats, and they exhibit a higher frequency in SARS-CoV-2 found in mink and white-tailed deer, suggesting that sustained transmissions may contribute to maintaining novel mutations. Four of these exchanges, such as Leu452Met, could undermine acquired immune protection in humans while maintaining high affinity for the human angiotensin-converting enzyme 2 (ACE2) receptor. Finally, we discuss important avenues of future research into animal-derived viruses with public health risks.

Alphaviruses are arboviruses transmitted by mosquitoes and are pathogenic to humans and livestock, causing a substantial public health burden. So far, several receptors have been identified for alphavirus entry; however, they cannot explain the broad host range and tissue tropism of certain alphaviruses, such as Getah virus (GETV), indicating the existence of additional receptors. Here we identify the evolutionarily conserved low-density lipoprotein receptor (LDLR) as a new cell entry factor for GETV, Semliki Forest virus (SFV), Ross River virus (RRV) and Bebaru virus (BEBV). Ectopic expression of LDLR facilitates cellular binding and internalization of GETV, which is mediated by the interaction between the E2-E1 spike of GETV and the ligand-binding domain (LBD) of LDLR. Antibodies against LBD block GETV infection in cultured cells. In addition, the GST-LBD fusion protein inhibits GETV infection both in vitro and in vivo. Notably, we identify the key amino acids in LDLR-LBD that played a crucial role in viral entry; specific mutations in the CR4 and CR5 domain of LDLR-LBD reduce viral entry to cells by more than 20-fold. These findings suggest that targeting the LDLR-LBD could be a potential strategy for the development of antivirals against multiple alphaviruses.

Various low-density lipoprotein receptors (LPRs) have been identified as entry factors for alphaviruses, and structures of the corresponding virion-receptor complexes have been determined. Here, we analyze the similarities and differences in the receptor binding modes of multiple alphaviruses to understand their ability to infect a wide range of hosts. We further discuss the challenges associated with the development of broad-spectrum treatment strategies against a diverse range of alphaviruses. Recently, studies have identified different low-density lipoprotein receptors as functional entry receptors for alphaviruses. In this Perspective, the authors compare the known alphavirus-receptor structures and discuss why it might be challenging to generate a broad-spectrum entry inhibitor.

In 2022, a boy with influenza-like symptoms was found to be infected with a novel H3N8 avian influenza virus in Henan province (1-4). Subsequently, in May 2022 H3N8 avian influenza virus caused a second human infection in Hunan province. The H3N8 human viruses originated from chickens (3, 5), but its RNA was also detected in nasopharyngeal swabs of cats and dogs and environmental samples collected in the patient's house (4). Of note, the full-length HA sequences from pets and human patients were identical, but revealed differences to avian H3N8 suggesting adaption to mammalian hosts.

Covalent attachment of the fatty acids palmitate or stearate to the cytoplasmic domain of viral glycoproteins is often crucial for viral replication. This has previously been studied for the hemagglutinin (HA) of Influenza A virus and the responsible enzymes have been identified, but similar studies have not been performed with HA of Influenza B virus, which contains palmitate linked to two cysteines. We show here that the modification is essential for virus replication since exchange of both cysteines or the cysteine located at the end of the cytoplasmic tail prevented the generation of viable viruses. Viruses with an exchange of the membrane-distal cysteine rapidly reverted back to wild-type virus. Blocking exit of proteins from the endoplasmic reticulum (ER) revealed that palmitoylation of HA of Influenza B virus occurs in the ER, whereas acylation of HA of Influenza A virus also in the Golgi. Infecting cells deficient in DHHC palmitoyltransferases revealed that HA of Influenza B virus is acylated by the ER-localized DHHCs 1, 2, 4 and 6, which are thus different from the enzymes previously identified for acylation of HA of Influenza A virus. A comparison of predicted and experimentally determined protein structures suggests that the exclusive acylation of the HA of Influenza B virus with palmitate is not a function of the responsible DHHCs and that the transmembrane region may be critical for the acylation of HA of Influenza A and B virus by different DHHCs.Importance Influenza viruses are a public health concern since they cause seasonal outbreaks and occasionally pandemics. Our study investigates the importance of a protein modification called "palmitoylation" in the replication of Influenza B virus. Palmitoylation involves attaching fatty acids to the viral protein hemagglutinin, and has previously been studied for Influenza A virus. We found that this modification is essential for the Influenza B virus to replicate, as mutating the sites where palmitate is attached prevented the virus from generating viable particles. Our experiments also showed that this modification occurs in the endoplasmic reticulum. We identified the specific enzymes responsible for this modification, which are different from those involved in palmitoylation of HA of Influenza A virus. Overall, our research illuminates the similarities and differences in fatty acid attachment to HA of Influenza A and B virus and identifies the responsible enzymes, which might be promising targets for antiviral therapy.

Swine pathogens have a long history of zoonotic transmission to humans, occasionally leading to sustained outbreaks or pandemics. Through a retrospective epidemiological study of swine populations in China, we describe novel lineages of porcine hemagglutinating encephalomyelitis virus (PHEV) complex coronaviruses (CoVs) that cause exclusively respiratory symptoms with no signs of the neurological symptoms typically associated with classical PHEV infection. Through large-scale epidemiological surveillance, we show that these novel lineages have circulated in at least eight provinces in southeastern China. Phylogenetic and recombination analyses of twenty-four genomes identified two major viral lineages causing respiratory symptoms with extensive recombination within them, between them, and between classical PHEV and the novel respiratory variant PHEV (rvPHEV) lineages. Divergence times among the sampled lineages in the PHEV virus complex date back to 1886-1958 (mean estimate 1928), with the two major rvPHEV lineages separating approximately 20 years later. Many rvPHEV viruses show amino acid substitutions at the carbohydrate-binding site of hemagglutinin esterase (HE) and/or have lost the cysteine required for HE dimerization. This resembles the early adaptation of human CoVs, where HE lost its hemagglutination ability to adapt to growth in the human respiratory tract. Our study represents the first report of the evolutionary history of rvPHEV circulating in swine and highlights the importance of characterizing CoV diversity and recombination in swine to identify pathogens with outbreak potential that could threaten swine farming.