Molecular Mechanisms Of Poly(A)-binding Proteins Regulating Pseudorabies Virus Replication

Poly(A)-binding proteins(PABPs,PABPCs)are conserved and expressed extensively in eukaryotes,which bind with poly(A)on the 3' terminal and interact with eukaryotic translation initiation factor 4F(eIF4F)on the 5' terminal,leading to the mRNA circularization.When pseudorabies virus(PRV)infects host cells to produce progeny viruses,the viral mRNA also contains poly(A)and cap structures.Importantly,this virus exploits host translation system for viral protein production to promote viral proliferation.However,the role of mRNA circularization in viral protein synthesis during PRV infection remains largely unknown.At the early stage,using mass spectrometry and bioinformatics analysis,we found that PRV infection led to significant differential expression PABPC proteins in host cells.Due to the important role in the process of mRNA stability and protein synthesis,we wanted to explore the function and the underlying mechanism of PABPC1 and PABPC4 proteins during PRV infection.In this study,we first examined the effect on PABPC by PRV infection.As a result,we found that the mRNA level of PABPC1 was upregulated and the mRNA level of PABPC4 was downregulated during PRV infection.Consistently,the protein level of PABPC1 was increased and the protein level of PABPC4 was reduced.These results suggest that PRV infection increases the expression of PABPC1 and decreases the expression of PABPC4.Next,we performed transient overexpression and knockdown assays to determine the effects on PRV proliferation.Consequently,the g B protein levels in PK15 cells overexpressing Flag-PABPC1 or Flag-PABPC4 were increased than in the control groups.We also examined the viral titers and found that the viral titers were increased in the presence of Flag-PABPC4 or Flag-PABPC4.In contrast,the g B protein levels and viral titers were decreased in PABPC1 or PABPC4 knockdown cells.To further explore the mechanisms by which PABPC1 and PABPC4 facilitate viral growth,we identified some viral proteins that might interact with PABPC1 and PABPC4 through mass spectrum(MS)and bioinformatics analysis.Using co-IP assay,we found that viral proteins,US2,UL18,UL46,UL50,and UL54 could interact with PABPC1.Importantly,US2,UL18,and UL46 could also interact with PABPC4.Previous studies have indicated that PABPC1 promotes mRNA circularization by interacting with eIF4 G,leading to mRNA stabilization and translation enhancement.Therefore,we investigated the interaction between viral proteins and eIF4 F.The co-IP result showed that UL46 could interact with eIF4 E,indicating UL46 could help viral mRNA circularization.Next,we focused on the role of UL46 and hypothesized that UL46 could bind with viral mRNA during viral infection.Using RNA-immunoprecipitation(RIP)and deep-sequencing,we found that PABPC1 and PABPC4 could bind with cellular mRNA at 3' end,but bind with viral mRNA both at 5' and3' ends.In addition,UL46 could also bind with viral mRNA both at 5' and 3' ends,indicating UL46 could promote viral mRNA circularization.Intriguingly,we found that US2 and UL18 could target eIF4 G for degradation by autophagy,thus impairing host translation machinery.Finally,we found that US2 or UL18 caused differential expression of proteins in host.Notably,we generated a recombinant pseudorabies virus that can express HA-PABPC4,because we wanted to employ this virus to effectively control coronavirus based on our previous studies that PABPC4 could suppress coronavirus proliferation by targeting the nucleoprotein for autophagic degradation.In conclusion,we found that both PABPC1 and PABPC4 facilitated PRV growth and that viral infection promoted the expression of PABPC1 and dampened the expression of PABPC4.Further results showed that PRV UL46 could interact with PABPC1,PABPC4,and eIF4 E.Additionally,UL46 could bind with viral mRNA at both 5' and 3' ends,indicating UL46 may play an important role in mRNA circularization.However,we found that US2 and UL18 proteins could target and degrade eIF4 G in a lysosomal pathway,thus impairing host translation machinery.Our results provide new insights into the virus-host interaction and add new understanding of the function of viral proteins in PRV pathogenesis.