Gregory W. Moseley’s research while affiliated with Monash University (Australia) and other places

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Publications (91)


Analysis of mechanisms of the rabies virus P protein-nucleocapsid interaction using engineered N-protein peptides and potential applications in antivirals design
  • Article

December 2024

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13 Reads

Antiviral Research

Jingyu Zhan

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Shatabdi Chakraborty

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Construction of BFV icDNA clones. (A). Schematic presentation of a plasmid containing BFV icDNA. pUC57-Kan was used as a backbone, and five DNA fragments covering the complete BFV genome (11.5 kb) were synthesized and assembled using digestion with indicated restriction enzymes (XhoI, BgIII, Smal, NotI, and BsiWI). In fragment A, the 5′ end of the BFV genome was placed under an SP6 promotor, and a unique AvrII site, used for plasmid linearization, was placed immediately downstream of poly(A) sequence of virus genome. (B). Schematic presentation of recombinant BFV genomes, BFV-IC, BFVV1911D, BFVT1325P+V1911D, and BFVT1325P. Amino acid substitutions are shown above, and nucleotide substitutions are below the drawings; for amino acid residues, the numbers indicate their positions in P1234 of BFV-IC. Arrows indicate the sub-genomic promoter of BFV. The numbers at the right of the drawing indicate infectivity of corresponding RNA transcripts (PFU/μg of RNA) in the ICA, N/A—not analyzed.
Comparison of in vitro phenotypes of BFV2193-FI and BFV-IC. Multi-step growth curves of BFV2193-FI and BFV-IC. Vero (A), IFNAR-/- MEF (B), and WT MEF (C) cells were infected at an MOI of 0.1 and supernatants were collected at 0, 3, 6, 9, 12, 24, and 48 h p.i. Viral titers were determined by plaque assay and are represented as PFU/mL. The dotted line represents the limit of detection. Each data point represents the mean ± standard deviation (SD) from two independent experiments performed on triplicates. *P < 0.05 and **P < 0.01 using two-way ANOVA with a Bonferroni post hoc test.
Comparison of in vivo phenotypes of BFV2193-FI and BFV-IC. C57BL/6 mice (n = 5) were infected subcutaneously with 10⁵ PFU of BFV2193-FI or BFV-IC or mock infected with PBS. Mice were assessed for disease score (A) and weight gain (B). Serum (C), quadriceps (D), and ankle (E) were collected on days 1, 3, and 5 p.i. Viral titers in the tissues were determined by plaque assay. The dotted line represents the limit of detection. H&E staining of longitudinal sections of quadriceps from the infected mice was performed at 7 and 10 days p.i. (F). Scale bar = 200 µm. The cell infiltrates were quantified using ImageScope (G). **P < 0.01; ****P < 0.0001 using two-way ANOVA with a Bonferroni post hoc test.
nsP2-T1325P and nsP4-V1911D substitutions attenuate BFV in vitro. IFNAR-/- MEF (A), WT MEF (B), AF319 (C), and C6/36 (D) cells were infected with BFV-IC, BFVT1325P, and BFVT1325P+V1911D at an MOI 0.1. Samples were collected and analyzed by plaque assay. The dotted line represents the limit of detection. Each data point represents the mean ± SD from two independent experiments performed in triplicates. *P < 0.05; **P < 0.01; ***P < 0.001, and ****P < 0.0001 using two-way ANOVA with a Bonferroni post hoc test.
Subcellular localization of BFV nsP3. (A) Schematic presentation of the genome of BFV-P3mCh. Inserted sequence encoding for mCherry is shown in red. The arrow indicates the sub-genomic promoter of BFV. (B) BHK-21 cells were infected with BFV-P3mCh or SFV-P3mCh (as a control) at an MOI of 1.0. Cells were fixed 24 h p.i. Nuclei were counterstained with DAPI, and cells were analyzed for mCherry fluorescence using a Zeiss LSM710 confocal microscope. (C) C6/36 and AF319 cells were infected with the BFV-P3mCh at an MOI of 1.0. Cells were fixed 24 h p.i. Nuclei were counterstained with DAPI, and cells were analyzed for mCherry fluorescence using a Zeiss LSM710 confocal microscope. (D) Vero cells and C6/36 cells were infected with BFV-P3mCh at an MOI of 1.0, fixed 24 h p.i. and treated with dsRNA-specific mouse monoclonal J2 antibody as primary antibody and anti-mouse Alexa Fluor 488 conjugated antibody as the secondary antibody. In addition to mCherry and DAPI fluorescence, cells were analyzed for dsRNA signal using a Nikon N1R + confocal microscope. Scale bar = 10 µm.

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Exploring Barmah Forest virus pathogenesis: molecular tools to investigate non-structural protein 3 nuclear localization and viral genomic determinants of replication
  • Article
  • Full-text available

July 2024

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36 Reads

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2 Citations

Barmah Forest virus (BFV) is a mosquito-borne virus that causes arthralgia with accompanying rash, fever, and myalgia in humans. The virus is mainly found in Australia and has caused outbreaks associated with significant health concerns. As the sole representative of the Barmah Forest complex within the genus Alphavirus, BFV is not closely related genetically to other alphaviruses. Notably, basic knowledge of BFV molecular virology has not been well studied due to a lack of critical investigative tools such as an infectious clone. Here we describe the construction of an infectious BFV cDNA clone based on Genbank sequence and demonstrate that the clone-derived virus has in vitro and in vivo properties similar to naturally occurring virus, BFV field isolate 2193 (BFV2193-FI). A substitution in nsP4, V1911D, which was identified in the Genbank reference sequence, was found to inhibit virus rescue and replication. T1325P substitution in nsP2 selected during virus passaging was shown to be an adaptive mutation, compensating for the inhibitory effect of nsP4-V1911D. The two mutations were associated with changes in viral non-structural polyprotein processing and type I interferon (IFN) induction. Interestingly, a nuclear localization signal, active in mammalian but not mosquito cells, was identified in nsP3. A point mutation abolishing nsP3 nuclear localization attenuated BFV replication. This effect was more prominent in the presence of type I interferon signaling, suggesting nsP3 nuclear localization might be associated with IFN antagonism. Furthermore, abolishing nsP3 nuclear localization reduced virus replication in mice but did not significantly affect disease. IMPORTANCE Barmah Forest virus (BFV) is Australia’s second most prevalent arbovirus, with approximately 1,000 cases reported annually. The clinical symptoms of BFV infection include rash, polyarthritis, arthralgia, and myalgia. As BFV is not closely related to other pathogenic alphaviruses or well-studied model viruses, our understanding of its molecular virology and mechanisms of pathogenesis is limited. There is also a lack of molecular tools essential for corresponding studies. Here we describe the construction of an infectious clone of BFV, variants harboring point mutations, and sequences encoding marker protein. In infected mammalian cells, nsP3 of BFV was located in the nuclei. This finding extends our understanding of the diverse mechanisms used by alphavirus replicase proteins to interact with host cells. Our novel observations highlight the complex synergy through which the viral replication machinery evolves to correct mutation errors within the viral genome.

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Giant Virus Global Proteomics Innovation: Comparative Evaluation of In-Gel and In-Solution Digestion Methods

April 2024

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27 Reads

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1 Citation

Omics: a Journal of Integrative Biology

With their unusually large genome and particle sizes, giant viruses (GVs) defy the conventional definition of viruses. Although most GVs isolated infect unicellular protozoans, such as amoeba, studies in the last decade have established their much wider prevalence infecting most eukaryotic supergroups and some giant viral families with the potential to be human pathogens. Their complexity, almost autonomous life cycle, and enigmatic evolution necessitate the study of GVs. The accurate assessment of GV proteome is a veritable challenge. We have compared the coverage of global protein identification using different methods for GVs isolated in Mumbai, Mimivirus Bombay (MVB), Powai Lake Megavirus (PLMV), and Kurlavirus (KV), along with two previously studied GVs, Acanthamoeba polyphaga Mimivirus (APMV) and Marseillevirus (MV). Our study shows that the simultaneous use of in-gel and in-solution digestion methods can significantly increase the coverage of protein identification in the global proteome analysis of purified GV particles. Combining the two methods of analyses, we identified an additional 72 proteins in APMV and 114 in MV compared with what have been previously reported. Similarly, proteomes of MVB, PLMV, and KV were analyzed, and a total of 242 proteins in MVB, 287 proteins in PLMV, and 174 proteins in KV were identified. Our results suggest that a combined methodology of in-gel and in-solution methods is more efficient and opens up new avenues for innovation in global proteome analysis of GVs. Future planetary health research on GVs can benefit from consideration of a broader range of proteomics methodologies as illustrated by the present study.


Figure 2. P3 nuclear bodies are liquid and closely associated with known nuclear MLOs. (A) P3 NBs undergo fusion: Live-cell time-lapse CLSM imaging of HeLa cells transfected to express GFP-P3 and imaged 16 h p.t.. Images of a nucleus of a transfected cell are shown over a 16 s period with 4 s intervals (extracted from Supplementary Movie 1). (B) HeLa cells transfected to express GFP-P3 were fixed and immunostained with antibodies against protein markers for specific NBs: Cajal bodies (Coilin, NOLC1), paraspeckles (NONO, RBM14), PML-NBs (PML protein); Gem bodies (SMN); and labeled with AlexaFluor-568 secondary antibodies (Alexa-568). Images are representative of ≥ 5 fields of view.
Figure 5. P1 and P3 wt, KRm and D289N show long-range conformational differences. Small Angle X-Ray Scattering data for (A) P1 (black, magenta) and (B) wt P3 (black, brown), P3 KRm (red, orange) and P3 D289N (blue, cyan) and the corresponding p(r) curves for (C) P1 (magenta) and (D) wt P3 (black), P3 KRm (red) and P3 D289N (blue). The p(r) curve for P3 D289N is consistent with a smaller radius of gyration while for P3 KRm it is larger compared to wt P3 (Table S1). The Kratky plots for (E) P1 (magenta) and (F) wt P3 (black), P3 KRm (red) and P3 D289N (blue) rise to a plateau that are representative of proteins that contain significant regions of disorder. EOM modeling distribution of (G) P1 (magenta), (H) wt P3 (black), (I) P3 KRm (red) and (J) P3 D289N (blue) and DENSS models (K) P1, (L) wt P3, (M) P3 KRm and (N) P3 D289N show P1 relative to P3 has a narrow distribution of conformations, whereas the P3 proteins are distributed between open and closed conformers with P3 D289N showing a greater preference for closed and R3 KRm for open.
Figure 7. P1, wt P3 and P3 mutants show in vitro phase separation. (A) CLSM images show that P1, wt P3, P3-D289N and P3-KRm form phase-separated droplets on addition of 10% PEG 6000. P1 and P3-KRm, compared to wt P3, show enhancement of phase separation (higher number of droplets and larger radius of droplets at equivalent protein concentrations), whereas P3-D289N shows a reduction in phase separation at the protein concentrations tested (12.5 to 75 μM). Note that droplet count and size (apparent radii of droplets) distribution is dependent on protein concentration with a tendency to form larger coalesced droplets with a net lower droplet count at high concentrations, indicative of droplet fusion especially for P1 and P3-KRm. Images are representative of three fields of view, scale bar: 20 μm. (B, C) Images were used to estimate (B) droplet count (mean ± SEM, n = 3) and (C) size distribution (radius) with mean ± SEM indicated for droplets, representative from 3 fields of view, determined using the in-built particle analyzer algorithm in ImageJ (Fiji) (see Materials and Methods).
Definition of mechanisms of viral protein multifunctionality reveals key roles of ‘conformer diversity’ and RNA interaction in forming host interfaces

February 2024

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89 Reads

Multifunctionality of viral genes is critical for processes in replication and modulation of infected cells. P gene of rabies virus generates the full-length protein, P1, and the truncated isoform, P3, which gains unique phenotypes lacking in P1, including interactions with multiple cellular membrane-less organelles (MLOs, liquid-liquid phase-separated (LLPS) structures), important to immune evasion. The gain-of-function by P3 proposes that multifunctionality of P isoforms is not merely due to their complement of independent modules, but is regulated by complex interactions of globular and intrinsically disordered regions (IDRs). However, the molecular basis of gain-of-function is unknown. Here we report biophysical and cellular analyses of P1 and P3, identifying a network of intra-protomer interactions involving the globular C-terminal domain and N-terminal IDRs, which differ between the isoforms. Mutagenesis of P3 identified substitutions causing gain- and loss-of-function for MLO interactions, associated with altered interactions of N- and C-terminal regions. Despite reduced MLO association of P1 and P3-loss-of-function mutants compared with wild-type P3, they retain capacity for LLPS in vitro, suggesting that specific inter-molecular interactions enable MLO targeting. P3 and P1 interact similarly with multiple MLO-associated proteins, but RNA binding is only observed for P3, and is enhanced or diminished by gain- and loss-of-function mutations, respectively. These data indicate that differences in interfaces formed between distant regions in P protein isoforms regulate protein-RNA interactions as a principal mechanism in the acquisition of unique functions/MLO interactions by P3, identifying a novel strategy in viral protein multifunctionality.


Uptake of T4 phage by mammalian cells does not trigger a pro-inflammatory immune response
(A) A549 cells and (B) MDCK-I cells incubated with T4 phages for 2 hours. Images were taken with a confocal microscope; the plasma membrane is shown in magenta, T4 phage DNA in green, and the cell nucleus in blue. (C) A549 cells transfected with NF-κB-dependent luciferase reporter plasmid, or (D) IFN-β promoter-dependent luciferase reporter plasmid, followed by 48 hours incubation with 10⁹ T4 phages/mL or a Filter control. Differentiated WT (E) or STING KO (F) BMDM cells were incubated for 18 hours 10⁷ T4 phages/mL, Filter control, Capsid-only or transfected with phage DNA using Lipofectamine 2000. Raw data can be found in S1 Data; each set of data follows the normality law; P values between the different groups were calculated from a one-way ANOVA with multiple comparisons, shown as stars (P < 0.0001 = ****; A: F (3, 12) = 31.06; B: F (3, 12) = 2.812; C: F (4, 25) = 5.7; D: F (4, 25) = 0.8181).
Network analysis of mammalian cells treated with T4 phages
(A) Kinexus KAM-1325 antibody microarray with MDCK-I cells after 8 hours of incubation with T4 phages. (B) Kinexus KAM-2000 antibody microarray with A549 cells after 8 hours of incubation with T4 phages. Figures report major cellular pathways of the main up- and down-regulated leads from the network analysis. Boxes highlighted in red are proteins discussed in this manuscript. The color gradient and arrow width indicate the Log2 fold change values.
Phage application to in vitro mammalian cells leads to enhanced growth and proliferation
(A) Cell cycle stage repartition within the A549 cell population after 8- or 24-hour incubation with phages or Filter control (data are mean with error bars representing 95% CI, n = 3 independent replicates with 100,000 cells analyzed). Not all the cells are included in a cell cycle phase (S4 and S5 Figs). P values of each cell cycle stage between the Filter control and T4 phage were calculated using a two-way ANOVA, shown as stars (F (3, 32) = 2.237). (B) Cell proliferation assay as measured via absorbance (540 nm) using a modified MTT colorimetric assay with A549 cells incubated with phages for 24, 48, or 72 hours (data are mean with error bars representing 95% CI, n = 3 independent replicates). Raw data can be found in S2 Data; P values were calculated using a two-way ANOVA, shown as stars (F (2, 224) = 1,015).
Overview of the effect of exogenous phages on cellular pathways
(A) Innate immune pathways in BMDM and A549 cells. Phage DNA is protected by the phage capsid and is not detected by the TLR9 or cGAS-STING. (B) The effect of phages on MDCK-I and A549 cells after 8 hours. The AKT pathway on the left and the CDK1 pathway on the right show the major cellular changes detected in response to T4 phage.
Mammalian cells internalize bacteriophages and use them as a resource to enhance cellular growth and survival

October 2023

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136 Reads

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17 Citations

There is a growing appreciation that the direct interaction between bacteriophages and the mammalian host can facilitate diverse and unexplored symbioses. Yet the impact these bacteriophages may have on mammalian cellular and immunological processes is poorly understood. Here, we applied highly purified phage T4, free from bacterial by-products and endotoxins to mammalian cells and analyzed the cellular responses using luciferase reporter and antibody microarray assays. Phage preparations were applied in vitro to either A549 lung epithelial cells, MDCK-I kidney cells, or primary mouse bone marrow derived macrophages with the phage-free supernatant serving as a comparative control. Highly purified T4 phages were rapidly internalized by mammalian cells and accumulated within macropinosomes but did not activate the inflammatory DNA response TLR9 or cGAS-STING pathways. Following 8 hours of incubation with T4 phage, whole cell lysates were analyzed via antibody microarray that detected expression and phosphorylation levels of human signaling proteins. T4 phage application led to the activation of AKT-dependent pathways, resulting in an increase in cell metabolism, survival, and actin reorganization, the last being critical for macropinocytosis and potentially regulating a positive feedback loop to drive further phage internalization. T4 phages additionally down-regulated CDK1 and its downstream effectors, leading to an inhibition of cell cycle progression and an increase in cellular growth through a prolonged G1 phase. These interactions demonstrate that highly purified T4 phages do not activate DNA-mediated inflammatory pathways but do trigger protein phosphorylation cascades that promote cellular growth and survival. We conclude that mammalian cells are internalizing bacteriophages as a resource to promote cellular growth and metabolism.


Sub-nucleolar trafficking of Hendra virus matrix protein is regulated by ubiquitination and oligomerisation

August 2023

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31 Reads

Hendra virus (HeV) is a highly pathogenic member of the Henipavirus genus (order Mononegavirales ), the replication cycle of which occurs primarily in the cytoplasm. The HeV matrix protein (HeV M) plays critical roles in viral assembly and budding at the plasma membrane, but also undergoes nuclear/nucleolar trafficking, to accumulate in nucleoli early in infection and, later, localise predominantly at the plasma membrane. Previously we found that HeV M protein targets specific sub-nucleolar compartments (corresponding to the FC-DFC (fibrillar centre (FC)/dense fibrillar component (DFC)) where it interacts with the nucleolar protein Treacle and modulates rRNA biogenesis by subverting the host nucleolar DNA damage response, indicating the importance of specific sub-nucleolar trafficking to infection. However, the mechanisms underlying targeting and movement between sub-nucleolar compartments by viral or cellular proteins remain poorly defined. Here, we assessed the molecular regulation of HeV M protein nucleolar/sub-nucleolar trafficking, finding that in infected cells and in cells expressing HeV M protein alone, M protein localizes into Treacle-enriched FC-DFC at early time points, and that FC-DFC localization is subsequently lost due to relocalization into the surrounding granular component (GC) of the nucleolus. Analysis using mutated M proteins and pharmacological modulation of ubiquitination indicate that this dynamic localization is regulated by ubiquitination and oligomerisation, with ubiquitination required for retention of HeV M in Treacle-enriched sub-nucleolar compartments, and oligomerisation required for egress. To our knowledge, this study provides the first direct insights into the dynamics and mechanisms of viral protein trafficking between sub-nucleolar compartments, important to the interplay between HeV M protein and host cell factors during infection. AUTHOR SUMMARY Henipaviruses, including Hendra (HeV) and Nipah viruses, cause deadly diseases in humans and livestock and are considered priority diseases by the World Health Organization due to their epidemic potential and lack of effective treatments. Understanding how these viruses interact with host cells is essential for developing new therapeutics. Our study examines the matrix (M) protein of henipaviruses and its interaction with the nucleolus, a cell structure that mediates ribosome production, and is a common target for various viruses, although their functions are largely unresolved. Previously, we showed that the HeV M protein targets a sub-nucleolar structure, called the FC-DFC, to modulate ribosome biogenesis. Here, we report that the M protein’s movement between sub-nucleolar compartments is controlled by two processes: ubiquitination, which causes accumulation of the protein in the FC-DFC, and oligomerization, which is associated with exit. Similar mechanisms are also observed in other henipaviruses. Our findings reveal mechanisms regulating the hijacking of host cell functions by henipaviruses and suggest new potential targets for antiviral therapies. This study is the first to investigate how viral proteins move within the nucleolus, offering new insights into interactions that may be significant to multiple viruses.


Biochemical Reconstitution of the Mimiviral Base Excision Repair Pathway

June 2023

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25 Reads

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2 Citations

Journal of Molecular Biology

Viruses are believed to be the obligate intracellular parasites that only carry genes essential for infecting and hijacking the host cell machinery. However, a recently discovered group of viruses belonging to the phylum nucleocytovirocota, also known as the nucleo-cytoplasmic large DNA viruses (NCLDVs), possess a number of genes that code for proteins predicted to be involved in metabolism, and DNA replication, and repair. In the present study, first, using proteomics of viral particles, we show that several proteins required for the completion of the DNA base excision repair (BER) pathway are packaged within the virions of Mimivirus as well as related viruses while they are absent from the virions of Marseillevirus and Kurlavirus that are NCLDVs with smaller genomes. We have thoroughly characterized three putative base excision repair enzymes from Mimivirus, a prototype NCLDV and successfully reconstituted the BER pathway using the purified recombinant proteins. The mimiviral uracil-DNA glycosylase (mvUDG) excises uracil from both ssDNA and dsDNA, a novel finding contrary to earlier studies. The putative AP-endonuclease (mvAPE) specifically cleaves at the abasic site created by the glycosylase while also exhibiting the 3'-5' exonuclease activity. The Mimivirus polymerase X protein (mvPolX) can bind to gapped DNA substrates and perform single nucleotide gap-filling followed by downstream strand displacement. Furthermore, we show that when reconstituted in vitro, mvUDG, mvAPE, and mvPolX function cohesively to repair a uracil-containing DNA predominantly by long patch BER and together, may participate in the BER pathway during the early phase of Mimivirus life-cycle.





Citations (67)


... Mutations disrupting this NLS lead to attenuated BFV replication, suggesting that nsP3 nuclear localization may be associated with interferon antagonism and is important for viral replication and pathogenicity [12]. ...

Reference:

Conserved Nuclear Localization Signal in NS2 Protein of Bombyx Mori Bidensovirus: A Potential Invertebrate ssDNA Virus Trait
Exploring Barmah Forest virus pathogenesis: molecular tools to investigate non-structural protein 3 nuclear localization and viral genomic determinants of replication

... They align with one of the two jelly-roll folds of their top Dali hits, the MCP of PBCV-1 (PDB: 5TIP) and the VP2 subviral particle of infectious bursal disease virus (PDB: 2DF7), respectively ( Fig. 4a and b). Proteomics data show that both gp109 and 225 are packaged inside the mature MaV particle [57]. Whether either of these two SJR proteins acts as the penton or they have minor structural roles in the overall capsid assembly of MaV needs to be investigated experimentally. ...

Giant Virus Global Proteomics Innovation: Comparative Evaluation of In-Gel and In-Solution Digestion Methods
  • Citing Article
  • April 2024

Omics: a Journal of Integrative Biology

... The World Health Organization defines pharmacokinetics as the study of drug absorption, distribution, metabolism, and elimination. Additionally, pharmacists recognize the release of the active ingredient as a step prior to the drug's absorption [124]. ...

Reference:

PUBLICADO
Mammalian cells internalize bacteriophages and use them as a resource to enhance cellular growth and survival

... PolX has also been identified in the African swine fever virus as well as in other dsDNA virus belonging to the socalled giant viruses (Nucleocytoviricota phyla) such as Megavirus, Mimivirus, and certain phycodnaviruses (Chen et al. 2017;Lad et al. 2023). During viral replication within swine macrophages, the ASFV is counterattacked by the cellular defense mechanisms, which leads to an increase in the viral mutation rate. ...

Biochemical Reconstitution of the Mimiviral Base Excision Repair Pathway
  • Citing Article
  • June 2023

Journal of Molecular Biology

... For instance, the RABV P3 protein, which is a shortened version of the pathogenic P protein, showcases exclusive functions that are absent in its longer variations. A study conducted by Sethi et al. [39] examines P3 from the pathogenic RABV strain Nishigahara (Nish) and a modified strain known as Ni-CE. The study delves into the intricacies of intra-protomer interactions, highlighting the connection between the globular C-terminal domain and the intrinsically disordered regions (IDRs) of the N-terminal region. ...

Structural insights into the multifunctionality of rabies virus P3 protein

Proceedings of the National Academy of Sciences

... Viral infections can strategically manipulate host ribosomal RNA (rRNA) metabolism to influence immune responses, promoting both viral replication and evasion of the host immune system [21,22]. Viral infections can strategically manipulate host ribosomal RNA (rRNA) metabolism to influence immune responses, promoting both viral replication and evasion of the host immune system [23,24]. ...

Henipaviruses and lyssaviruses target nucleolar Treacle protein and regulate ribosomal RNA synthesis

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... Images acquired from live and IF stained cells by CLSM were analysed using ImageJ freeware software as previously. 24 ...

Lyssavirus P Protein Isoforms Diverge Significantly in Subcellular Interactions Underlying Mechanisms of Interferon Antagonism

... Reporter assays for IFN induction and IFN signaling have been described previously (59,(82)(83)(84)(85). Briefly, to measure the activity of RIG-I and MDA5 activation of IFNβ induction pathways and effects thereon of expression of BFV nsp3, HEK-293T cells were co-transfec ted in triplicate with 40 ng pRL-TK (which expresses Renilla luciferase (RLuc) under the control of the constitutively active thymidine kinase promoter), 250 ng pGL3-IFNβ (in which firefly luciferase (Fluc) is under the control of the IFN-β promoter) and 250 ng plasmids to express the indicated GFP-fused nsP3 proteins or the control proteins ORF6-StrepII [expressing the SARS-CoV-2 ORF6 protein, a potent inhibitor of RIG-I/ MDA5-activated IFN induction, fused to StrepII tag (82)] or pEGFP-CVS-N [expressing the nucleoprotein (N protein) of rabies virus, which lacks intrinsic antagonist activity to the IFN-induction pathway (86)]. ...

Deactivation of the antiviral state by rabies virus through targeting and accumulation of persistently phosphorylated STAT1

... P forms dimers [24,25], and each protomer (297 aa) consists of a long N-terminal intrinsically disordered region (aa 1-88) and a Cterminal region made of two folded domains, the dimerization domain, MD (aa 89-132), and the C-terminal domain (aa 194-295), CTD, which are connected by a flexible linker (aa 133-193) [18,20,25,26] (Figure 1B,C). As part of its role in viral replication, P works as a hub, in which structural and functional modules interact with their different partners ( Figure 1C): (i) by a module (aa 1-39) located at its N-terminus, and P acts as a chaperone of unassembled RNA-free nucleoprotein (N 0 ) [27-31] ( Figure 1D); (ii) by a module encompassing the last part of the N-terminal region (aa 51-88), and P binds to L [32], and by its CTD, it binds to the NC [26,33,34], tethering the polymerase to its template and ensuring its processivity during RNA synthesis [17] ( Figure 1E,F); (iii) by being dimeric and forming weak multivalent interactions with itself and with N, and P acts as a scaffold protein for the formation of membrane-less viral factory compartments (MLO) by liquid-liquid phase separation [35,36]. In our previous study, we generated a model of P CTD bound to a circular polymeric N-RNA complex, where the binding site for P CTD involved two adjacent N protomers ( Figure 1F) [33]. ...

Molecular Basis of Functional Effects of Phosphorylation of the C-Terminal Domain of the Rabies Virus P Protein

... Another interesting application of our methodology is in the investigation of how the cell responds to toxic or otherwise antagonistic substances. For a simple illustration, we picked demecolcine, a membrane-permeable antineoplastic drug that disrupts the microtubule network within the cell by depolymerizing existing tubules and limiting their subsequent reformation 40 . To visualize the microtubule network in COS-7 cells, we transfected a cell culture to express an α-tubulin-pmEGFP fusion protein. ...

Nanoscale characterization of drug-induced microtubule filament dysfunction using super-resolution microscopy