OBJECTIVE To establish a model of monkeypox virus(MPXV)infection in suckling rabbits and analyze its pathological changes.METHODS Ten-day-old suckling rabbits were divided into two groups:the MPXV group(intranasally inoculated with 100 μL of MPXV at 106 PFU·mL-1)and the normal control group(intranasally inoculated with 100 μL of PBS solution),with 10 rabbits in each group.After infection,such indicators as body weight,survival rate,and mental state of the suckling rabbits were monitored daily for 15 days.On days 3,9,and 15 post-infection,samples of the heart,liver,spleen,lung,kidney,brain,small intestine,and peripheral blood were collected from each group for RT-qPCR to detect viral the load.Histopathological analysis using HE staining was performed on samples collected on day 9 post-infection.RESULTS Suckling rabbits infected with MPXV exhibited weight loss,reduced survival rates,and such symptoms as lethargy and chilliness,which were similar to the clinical manifes-tations observed in humans infected with MPXV.Viral load results indicated the presence of the virus in the heart,liver,spleen,lung,kidney,brain,small intestine,and peripheral blood,with varying degrees of pathological changes in each tissue and organ.CONCLUSION Intranasal inoculation can help estab-lish a model of MPXV infection in suckling rabbits.

Uch37 is a de-ubiquitinating enzyme that is activated by Rpn13 and involved in the proteasomal degradation of proteins. The full-length Uch37 was shown to exhibit low iso-peptidase activity and is thought to be auto-inhibited. Structural comparisons revealed that within a homo-dimer of Uch37, each of the catalytic domains was blocking the other's ubiquitin (Ub)-binding site. This blockage likely prevented Ub from entering the active site of Uch37 and might form the basis of auto-inhibition. To understand the mode of auto-inhibition clearly and shed light on the activation mechanism of Uch37 by Rpn13, we investigated the Uch37-Rpn13 complex using a combination of mutagenesis, biochemical, NMR, and small-angle X-ray scattering (SAXS) techniques. Our results also proved that Uch37 oligomerized in solution and had very low activity against the fluorogenic substrate ubiquitin-7-amino-4-methylcoumarin (Ub-AMC) of de-ubiquitinating enzymes. Uch37Δ(Hb,Hc,KEKE), a truncation removal of the C-terminal extension region (residues 256-329) converted oligomeric Uch37 into a monomeric form that exhibited iso-peptidase activity comparable to that of a truncation-containing the Uch37 catalytic domain only. We also demonstrated that Rpn13C (Rpn13 residues 270-407) could disrupt the oligomerization of Uch37 by sequestering Uch37 and forming a Uch37-Rpn13 complex. Uch37 was activated in such a complex, exhibiting 12-fold-higher activity than Uch37 alone. Time-resolved SAXS (TR-SAXS) and FRET experiments supported the proposed mode of auto-inhibition and the activation mechanism of Uch37 by Rpn13. Rpn13 activated Uch37 by forming a 1:1 stoichiometric complex in which the active site of Uch37 was accessible to Ub.
Binding Sites ; Catalytic Domain ; Chromatography, Gel ; Crystallography, X-Ray ; Humans ; Membrane Glycoproteins ; chemistry ; genetics ; metabolism ; Nuclear Magnetic Resonance, Biomolecular ; Protein Binding ; Protein Conformation ; Protein Multimerization ; Scattering, Small Angle ; Ubiquitin Thiolesterase ; chemistry ; genetics ; metabolism ; Ultracentrifugation

Algorithms ; Crystallography, X-Ray ; Disulfides ; chemistry ; Models, Molecular ; Protein Conformation ; Proteins ; chemistry