The Impact And Mechanism Study Of Host Protein Snapin On Rabies Virus Replication

Rabies is a severe zoonotic viral disease caused by the rabies virus（RABV）that affects the central nervous system and is characterized by symptoms such as hydrophobia,aerophobia,and muscle spasms.Globally,up to 60,000 people die from rabies each year,posing a serious threat to human health and public health security.RABV belongs to the genus Lyssavirus in the family Rhabdoviridae,encoding five structural proteins in the order of nucleoprotein（N）,phosphoprotein（P）,matrix protein（M）,glycoprotein（G）,and RNA-dependent RNA polymerase（L）from the 3’to 5’end.In the lifecycle of RABV,the P protein acts as an auxiliary factor of the polymerase,jointly responsible for the transcription and replication of viral RNA with the N and L proteins.The M protein is crucial for the formation of viral particles,and in the absence of the M protein,virus assembly and budding are severely affected.The G protein is closely related to the pathogenicity of rabies and is associated with the neurotropism of the virus.Each of the five structural proteins plays a role in the infection process of the virus.After infecting the host,RABV briefly replicates in muscle or other local tissues,and then reaches the Central Nervous System（CNS）through motor endplates and axonal transport.Successful infection of the CNS by RABV mainly involves evading host immune responses and protecting infected neurons from apoptosis or premature axonal damage.To elucidate how RABV escapes host immune responses to complete replication and spread,this study will investigate from the perspective of host protein-virus interactions.Our previous research found that RABV infection leads to upregulation of SNAP-associated protein（Snapin）.The host protein Snapin is a multifunctional protein associated with synapses,serving as an important component of the SNARE complex,involved in maintaining presynaptic homeostasis,and playing a crucial role in retrograde axonal transport.Neurons deficient in Snapin exhibit reduced vitality,neurodegeneration,and developmental abnormalities in the CNS,resulting in impaired retrograde axonal transport.As a neurotropic virus,RABV infects the CNS primarily through retrograde axonal transport and synaptic transmission.Does the host protein Snapin play a significant role in RABV infection of the CNS?To elucidate the important role of the host protein Snapin in the replication process of RABV.A recombinant eukaryotic plasmid overexpressing murine Snapin was constructed,and small interfering RNA targeting murine Snapin gene was designed in this study.The results revealed that overexpression of Snapin significantly promoted RABV replication,while knockdown of Snapin markedly inhibited RABV replication,and the regulation of RABV by Snapin was consistent between virulent and avirulent strains.To further elucidate the mechanism by which the host protein Snapin regulates RABV replication,this study first investigated whether Snapin interacts with RABV structural proteins.Immunoprecipitation（Co-IP）and confocal microscopy results demonstrated that the host protein Snapin interacts with the RABV P protein.Subsequently,truncation mutants of Snapin and P protein were constructed based on functional domains to further analyze the key structural domains involved in the interaction between Snapin and RABV P protein.Both Co-IP and confocal microscopy results showed that the 37-78aa region of Snapin interacts with the RABV P protein and is a critical domain for promoting RABV replication;the C-terminal region（172-297aa）of the RABV P protein is a key structural domain involved in the interaction with the host protein Snapin.Given the antagonizing role of the C-terminal region of the RABV P protein played in the interferon signaling pathway,this study investigated whether the host protein Snapin exerts its function by regulating the interferon pathway through the C-terminal region of the P protein.Real-time quantitative PCR（q PCR）results showed that overexpression of Snapin did not significantly affect the expression of interferon downstream signaling factors（ISG15,IFN-β,STAT1,etc.）during RABV infection.Furthermore,transcriptomic sequencing analysis of cell samples infected with the RABV after overexpression or knockdown of Snapin revealed that the intersecting genes mainly targeted the Nuclear Factor kappa-B（NF-κB）signaling pathway.Western blot results demonstrated that overexpression or knockdown of Snapin alone did not affect the NF-κB signaling pathway,but Snapin could significantly activate the NF-κB signaling pathway when co-transfected with RABV P protein or during virus infection.The use of the NF-κB inhibitor JSH-23 further confirmed that the host protein Snapin regulates RABV replication by affecting the NF-κB signaling pathway through the interaction with the RABV P protein.Subsequently,the adaptor molecule Glycogen Synthase Kinase 3β（GSK3β）,targeting the NF-κB signaling pathway,was successfully screened by using combined Co-IP and mass spectrometry analysis.The Co-IP results showed that GSK3βinteracts with the host protein Snapin and RABV P protein,and there is an endogenous interaction between GSK3βand Snapin.To further elucidate the molecular interactions between Snapin,RABV P,and GSK3β.GSK3βand Snapin gene-deficient cell lines were constructed.Co-IP results revealed that the deletion of GSK3βdid not affect the interaction between Snapin and RABV P protein,while the kowndown of Snapin weakened the interaction between GSK3βand RABV P protein.These results suggest that the host protein Snapin plays a crucial role in mediating the interaction between RABV P and the adaptor molecule GSK3β.Previous studies have shown that phosphorylation of threonine 216 of GSK3β（p Tyr216）can activate the NF-κB signaling pathway.Western blot results in this study demonstrated that during RABV infection,overexpression of Snapin significantly activated the phosphorylation levels of GSK3βTyr216 and NF-κB p65,while knockdown of Snapin showed the opposite trend.Further Western blot results confirmed that the Snapin/RABV P complex induces the expression of downstream neuron-related factors by activating GSK3βTyr216phosphorylation and then NF-κB p65 phosphorylation,thereby promoting RABV replication.To validate the impact of Snapin on the pathogenicity of RABV in vivo,this study utilized the RABV reverse genetics system to insert the Snapin gene into the RABV CVS11 genome between the G and L genes,successfully rescuing the recombinant rabies virus expressing Snapin,rCVS11-Snapin.The results of in vitro experiments showed that compared to the parental virus CVS11,rCVS11-Snapin exhibited stronger proliferation ability in N2a cells.BALB/c mice were intracranially infected with rCVS11-Snapin and CVS11（10¹ FFU/each）,respectively,and clinical symptoms were observed and survival rates were recorded.The results indicated that clinical symptoms showed earlier in mice in the rCVS11-Snapin group than those in the CVS11 group;the mortality rate was 80%in the CVS11-infected group and 100%in the rCVS11-Snapin-infected group.q PCR,TCID₅₀,and tissue immunofluorescence results demonstrated that compared to CVS11,rCVS11-Snapin exhibited stronger replication ability in the brains of mice.Subsequently,recombinant adeno-associated virus AAV-PHP.e B-sh Snapin carrying Snapin sh RNA and control virus AAV-PHP.e B-sh NC were constructed and intracranially injected into C57BL/6 mice at a dose of 2×10¹⁰ vg/25μL each.q PCR results showed a significant decrease in Snapin m RNA levels in the brains of mice four weeks after injection of AAV-PHP.e B-sh Snapin,indicating the successful construction of a Snapin gene knockdown model.Following this,the control mice and Snapin gene knockdown mice were intracranially infected with CVS11（10¹ FFU/each）,respectively.The results revealed that compared to the control group,the onset of disease in mice was delayed after Snapin gene knockdown;the viral load in the brain was reduced,and the survival rate of mice was increased.In summary,this study explored the impact of the host protein Snapin on RABV replication from the perspective of virus-host interactions.It was demonstrated that the host protein Snapin modulates RABV replication by interacting with the RABV P protein,which in turn affects the GSK3β/NF-κB signaling pathway.This research provides novel insights into the molecular mechanisms of RABV infection and pathogenesis and offers a new direction for screening novel therapeutic drugs.