-
Brain Communications 2024Neurodegeneration in the autoimmune disease multiple sclerosis still poses a major therapeutic challenge. Effective drugs that target the inflammation can only partially...
Neurodegeneration in the autoimmune disease multiple sclerosis still poses a major therapeutic challenge. Effective drugs that target the inflammation can only partially reduce accumulation of neurological deficits and conversion to progressive disease forms. Diet and the associated gut microbiome are currently being discussed as crucial environmental risk factors that determine disease onset and subsequent progression. In people with multiple sclerosis, supplementation of the short-chain fatty acid propionic acid, as a microbial metabolite derived from the fermentation of a high-fiber diet, has previously been shown to regulate inflammation accompanied by neuroprotective properties. We set out to determine whether the neuroprotective impact of propionic acid is a direct mode of action of short-chain fatty acids on CNS neurons. We analysed neurite recovery in the presence of the short-chain fatty acid propionic acid and butyric acid in a reverse-translational model of human-induced primary neurons differentiated from people with multiple sclerosis-derived induced pluripotent stem cells. We found that recovery of damaged neurites is induced by propionic acid and butyric acid. We could also show that administration of butyric acid is able to enhance propionic acid-associated neurite recovery. Whole-cell proteome analysis of induced primary neurons following recovery in the presence of propionic acid revealed abundant changes of protein groups that are associated with the chromatin assembly, translational, and metabolic processes. We further present evidence that these alterations in the chromatin assembly were associated with inhibition of histone deacetylase class I/II following both propionic acid and butyric acid treatment, mediated by free fatty acid receptor signalling. While neurite recovery in the presence of propionic acid is promoted by activation of the anti-oxidative response, administration of butyric acid increases neuronal ATP synthesis in people with multiple sclerosis-specific induced primary neurons.
PubMed: 38894951
DOI: 10.1093/braincomms/fcae182 -
Plants (Basel, Switzerland) May 2024In plants, the ubiquitin (Ub)-26S proteasome system (UPS) regulates numerous biological functions by selectively targeting proteins for ubiquitylation and degradation....
In plants, the ubiquitin (Ub)-26S proteasome system (UPS) regulates numerous biological functions by selectively targeting proteins for ubiquitylation and degradation. However, the regulation of Ub itself on plant growth and development remains unclear. To demonstrate a possible impact of Ub supply, as seen in animals and flies, we carefully analyzed the growth and developmental phenotypes of two different () gene overexpression plants of . One is transformed with (designated ), driven by the cauliflower mosaic virus promoter, while the other expresses (designated ), driven by the endogenous promoter of . We discovered that and had contrasting seed yields. Compared to wildtype (WT), the former exhibited a reduced seed yield, while the latter showed an increased seed production that was attributed to enhanced growth vigor and an elevated silique number per plant. However, reduced seed sizes were common in both and . Differences in the activity and size of the 26S proteasome assemblies in the two transgenic plants were also notable in comparison with WT, suggestive of a contributory role of expression in proteasome assembly and function. Collectively, our findings demonstrated that exogenous expression of recombinant Ub may optimize plant growth and development by influencing the UPS activities via structural variance, expression patterns, and abundance of free Ub supply.
PubMed: 38891294
DOI: 10.3390/plants13111485 -
Scientific Reports Jun 2024We report the first cryoEM structure of the Hendra henipavirus nucleoprotein in complex with RNA, at 3.5 Å resolution, derived from single particle analysis of a...
We report the first cryoEM structure of the Hendra henipavirus nucleoprotein in complex with RNA, at 3.5 Å resolution, derived from single particle analysis of a double homotetradecameric RNA-bound N protein ring assembly exhibiting D14 symmetry. The structure of the HeV N protein adopts the common bi-lobed paramyxoviral N protein fold; the N-terminal and C-terminal globular domains are bisected by an RNA binding cleft containing six RNA nucleotides and are flanked by the N-terminal and C-terminal arms, respectively. In common with other paramyxoviral nucleocapsids, the lateral interface between adjacent N and N protomers involves electrostatic and hydrophobic interactions mediated primarily through the N-terminal arm and globular domains with minor contribution from the C-terminal arm. However, the HeV N multimeric assembly uniquely identifies an additional protomer-protomer contact between the N N-terminus and N C-terminal arm linker. The model presented here broadens the understanding of RNA-bound paramyxoviral nucleocapsid architectures and provides a platform for further insight into the molecular biology of HeV, as well as the development of antiviral interventions.
Topics: Cryoelectron Microscopy; Hendra Virus; Nucleoproteins; Nucleocapsid; Models, Molecular; RNA, Viral; Nucleocapsid Proteins
PubMed: 38890308
DOI: 10.1038/s41598-024-58243-z -
Open Biology Jun 2024We present a novel small molecule antiviral chemotype that was identified by an unconventional cell-free protein synthesis and assembly-based phenotypic screen for...
We present a novel small molecule antiviral chemotype that was identified by an unconventional cell-free protein synthesis and assembly-based phenotypic screen for modulation of viral capsid assembly. Activity of PAV-431, a representative compound from the series, has been validated against infectious viruses in multiple cell culture models for all six families of viruses causing most respiratory diseases in humans. In animals, this chemotype has been demonstrated efficacious for porcine epidemic diarrhoea virus (a coronavirus) and respiratory syncytial virus (a paramyxovirus). PAV-431 is shown to bind to the protein 14-3-3, a known allosteric modulator. However, it only appears to target the small subset of 14-3-3 which is present in a dynamic multi-protein complex whose components include proteins implicated in viral life cycles and in innate immunity. The composition of this target multi-protein complex appears to be modified upon viral infection and largely restored by PAV-431 treatment. An advanced analog, PAV-104, is shown to be selective for the virally modified target, thereby avoiding host toxicity. Our findings suggest a new paradigm for understanding, and drugging, the host-virus interface, which leads to a new clinical therapeutic strategy for treatment of respiratory viral disease.
Topics: Antiviral Agents; Humans; Animals; 14-3-3 Proteins; Multiprotein Complexes; Host-Pathogen Interactions; Cell Line
PubMed: 38889796
DOI: 10.1098/rsob.230363 -
PLoS Pathogens Jun 2024The majority of rod-shaped and some filamentous plant viruses encode a cysteine-rich protein (CRP) that functions in viral virulence; however, the roles of these CRPs in...
The majority of rod-shaped and some filamentous plant viruses encode a cysteine-rich protein (CRP) that functions in viral virulence; however, the roles of these CRPs in viral infection remain largely unknown. Here, we used barley stripe mosaic virus (BSMV) as a model to investigate the essential role of its CRP in virus morphogenesis. The CRP protein γb directly interacts with BSMV coat protein (CP), the mutations either on the His-85 site in γb predicted to generate a potential CCCH motif or on the His-13 site in CP exposed to the surface of the virions abolish the zinc-binding activity and their interaction. Immunogold-labeling assays show that γb binds to the surface of rod-shaped BSMV virions in a Zn2+-dependent manner, which enhances the RNA binding activity of CP and facilitates virion assembly and stability, suggesting that the Zn2+-dependent physical association of γb with the virion is crucial for BSMV morphogenesis. Intriguingly, the tightly binding of diverse CRPs to their rod-shaped virions is a general feature employed by the members in the families Virgaviridae (excluding the genus Tobamovirus) and Benyviridae. Together, these results reveal a hitherto unknown role of CRPs in the assembly and stability of virus particles, and expand our understanding of the molecular mechanism underlying virus morphogenesis.
Topics: Zinc; Virion; Capsid Proteins; Virus Assembly; Plant Viruses; Plant Diseases; Cysteine; Viral Proteins; Morphogenesis
PubMed: 38885273
DOI: 10.1371/journal.ppat.1012311 -
PLoS Pathogens Jun 2024Influenza viruses transcribe and replicate their genome in the nucleus of the infected cells, two functions that are supported by the viral RNA-dependent RNA-polymerase...
Influenza viruses transcribe and replicate their genome in the nucleus of the infected cells, two functions that are supported by the viral RNA-dependent RNA-polymerase (FluPol). FluPol displays structural flexibility related to distinct functional states, from an inactive form to conformations competent for replication and transcription. FluPol machinery is constituted by a structurally-invariant core comprising the PB1 subunit stabilized with PA and PB2 domains, whereas the PA endonuclease and PB2 C-domains can pack in different configurations around the core. To get insights into the functioning of FluPol, we selected single-domain nanobodies (VHHs) specific of the influenza A FluPol core. When expressed intracellularly, some of them exhibited inhibitory activity on type A FluPol, but not on the type B one. The most potent VHH (VHH16) binds PA and the PA-PB1 dimer with an affinity below the nanomolar range. Ectopic intracellular expression of VHH16 in virus permissive cells blocks multiplication of different influenza A subtypes, even when induced at late times post-infection. VHH16 was found to interfere with the transport of the PA-PB1 dimer to the nucleus, without affecting its handling by the importin β RanBP5 and subsequent steps in FluPol assembly. Using FluPol mutants selected after passaging in VHH16-expressing cells, we identified the VHH16 binding site at the interface formed by PA residues with the N-terminus of PB1, overlapping or close to binding sites of two host proteins, ANP32A and RNA-polymerase II RPB1 subunit which are critical for virus replication and transcription, respectively. These data suggest that the VHH16 neutralization is likely due to several activities, altering the import of the PA-PB1 dimer into the nucleus as well as inhibiting specifically virus transcription and replication. Thus, the VHH16 binding site represents a new Achilles' heel for FluPol and as such, a potential target for antiviral development.
Topics: Single-Domain Antibodies; Humans; Antiviral Agents; Influenza A virus; Virus Replication; Animals; RNA-Dependent RNA Polymerase; Viral Proteins; Influenza, Human; HEK293 Cells; Dogs; Madin Darby Canine Kidney Cells
PubMed: 38875296
DOI: 10.1371/journal.ppat.1011642 -
Emerging Microbes & Infections Dec 2024The global outbreak of Mpox, caused by the monkeypox virus (MPXV), has attracted international attention and become another major infectious disease event after...
The global outbreak of Mpox, caused by the monkeypox virus (MPXV), has attracted international attention and become another major infectious disease event after COVID-19. The mRNA cap N7 methyltransferase (RNMT) of MPXV methylates the N7 position of the added guanosine to the 5'-cap structure of mRNAs and plays a vital role in evading host antiviral immunity. MPXV RNMT is composed of the large subunit E1 and the small subunit E12. How E1 and E12 of MPXV assembly remains unclear. Here, we report the crystal structures of E12, the MTase domain of E1 with E12 (E1-E12) complex, and the E1-E12-SAM ternary complex, revealing the detailed conformations of critical residues and the structural changes upon E12 binding to E1. Functional studies suggest that E1 N-terminal extension (Asp545-Arg562) and the small subunit E12 play an essential role in the binding process of SAM. Structural comparison of the AlphaFold2-predicted E1, E1-E12 complex, and the homologous D1-D12 complex of vaccinia virus (VACV) indicates an allosteric activating effect of E1 in MPXV. Our findings provide the structural basis for the MTase activity stimulation of the E1-E12 complex and suggest a potential interface for screening the anti-poxvirus inhibitors.
Topics: Methyltransferases; Monkeypox virus; Viral Proteins; Crystallography, X-Ray; RNA Caps; Models, Molecular; Humans; Protein Conformation; Protein Binding; RNA, Messenger
PubMed: 38873898
DOI: 10.1080/22221751.2024.2369193 -
Heliyon Jun 2024Biocontainment regulations restrict the research on NiV to BSL-4 laboratories, thus limiting the mechanistic studies related to viral entry and allied pathogenesis....
Biocontainment regulations restrict the research on NiV to BSL-4 laboratories, thus limiting the mechanistic studies related to viral entry and allied pathogenesis. Understanding the precise process of viral-particle production and host cell entry is critical for designing targeted therapies or particle-based vaccines. In this study, we have synthesized HiBiT-tagged-NiV-VLPs to ease BSL-2 particle handling. We propose a simple yet effective approach of generating substantial amount of HiBiT-tagged NiV-VLPs by co-expressing viral structural proteins in HEK293T cells. Though homologous to parent virus, the incapacitated replication potential facilitates a BSL-2 handling of these particles. The inclusion of a highly sensitive HiBiT tag on these VLPs allows for a quick detection of viral binding and entry, as well as in assessing the efficiency of neutralizing antibodies using the NanoBiT technology. The HiBiT-tag binds in high affinity with LgBiT (Large BiT an 18 kDa fusion protein and complementary subunit of HiBiT peptide), and the resultant complex elicits high intensity luminescence in the presence of substrate. The VLPs produced were morphologically and functionally identical to the native virus, and the HiBiT-tag permitted their quick application in viral binding, entry, and antibody neutralization assays. "Thus, we report a simple setting for generating HiBiT-NiV VLPs which can be utilized in a BSL-2 laboratory, to concurrently quantify features of NiV assembly, binding and entry. This also offers an alternate-safe and effective platform for viral based antibody neutralization assays
PubMed: 38868026
DOI: 10.1016/j.heliyon.2024.e31905 -
The Journal of Biological Chemistry Jun 2024Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a lipid-enveloped virus that acquires its lipid bilayer from the host cell it infects. SARS-CoV-2 can...
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a lipid-enveloped virus that acquires its lipid bilayer from the host cell it infects. SARS-CoV-2 can spread from cell to cell or from patient to patient by undergoing assembly and budding to form new virions. The assembly and budding of SARS-CoV-2 is mediated by several structural proteins known as envelope (E), membrane (M), nucleoprotein (N) and spike (S), which can form virus-like particles (VLPs) when co-expressed in mammalian cells. Assembly and budding of SARS-CoV-2 from the host ER-Golgi intermediate compartment is a critical step in the virus acquiring its lipid bilayer. To date, little information is available on how SARS-CoV-2 assembles and forms new viral particles from host membranes. In this study, we used several lipid binding assays and found the N protein can strongly associate with anionic lipids including phosphoinositides and phosphatidylserine. Moreover, we show lipid binding occurs in the N protein C-terminal domain, which is supported by extensive in silico analysis. We demonstrate anionic lipid binding occurs for both the free and N oligomeric forms, suggesting N can associate with membranes in the nucleocapsid form. Based on these results, we present a lipid-dependent model based on in vitro, cellular and in silico data for the recruitment of N to assembly sites in the lifecycle of SARS-CoV-2.
PubMed: 38866325
DOI: 10.1016/j.jbc.2024.107456 -
Revista Do Instituto de Medicina... 2024The group-specific antigen (gag) plays a crucial role in the assembly, release, and maturation of HIV. This study aimed to analyze the partial sequence of the HIV gag...
The group-specific antigen (gag) plays a crucial role in the assembly, release, and maturation of HIV. This study aimed to analyze the partial sequence of the HIV gag gene to classify HIV subtypes, identify recombination sites, and detect protease inhibitor (PI) resistance-associated mutations (RAMs). The cohort included 100 people living with HIV (PLH) who had experienced antiretroviral treatment failure with reverse transcriptase/protease inhibitors. Proviral HIV-DNA was successfully sequenced in 96 out of 100 samples for gag regions, specifically matrix (p17) and capsid (p24). Moreover, from these 96 sequences, 82 (85.42%) were classified as subtype B, six (6.25%) as subtype F1, one (1.04%) as subtype C, and seven (7.29%) exhibited a mosaic pattern between subtypes B and F1 (B/F1), with breakpoints at p24 protein. Insertions and deletions of amino acid at p17 were observed in 51 samples (53.13%). The prevalence of PI RAM in the partial gag gene was observed in 78 out of 96 PLH (81.25%). Among these cases, the most common mutations were R76K (53.13%), Y79F (31.25%), and H219Q (14.58%) at non-cleavage sites, as well as V128I (10.42%) and Y132F (11.46%) at cleavage sites. While B/F1 recombination was identified in the p24, the p17 coding region showed higher diversity, where insertions, deletions, and PI RAM, were observed at high prevalence. In PLH with virological failure, the analysis of the partial gag gene could contribute to more accurate predictions in genotypic resistance to PIs. This can aid guide more effective HIV treatment strategies.
Topics: Humans; HIV-1; HIV Infections; Genetic Variation; Male; gag Gene Products, Human Immunodeficiency Virus; Female; Adult; Drug Resistance, Multiple, Viral; Mutation; Genotype; Anti-HIV Agents; Middle Aged; Phylogeny; DNA, Viral
PubMed: 38865573
DOI: 10.1590/S1678-9946202466035