Molecular Mechanism Of Ribosomal Protein S5 In Calicivirus Translation And Replication

Caliciviridae is a non-enveloped,single-stranded positive-RNA virus.The virus family is composed of Norovirus,Sapovirus,Lagovirus,Vesivirus,and Nebovirus,which can infect a variety of hosts,including humans,rabbits,cats and mice,posing a serious threat to the health of the host.Since most caliciviruses cannot be cultured stably in vitro,the molecular biology research of these viruses are seriously hindered.In particular,the molecular mechanism of host regulation of calicivirus translation and replication still needs to be further explored,and there still exists lots of blind spots and difficulties.Our previous studies have found that the expression level of ribosomal protein S5(RPS5)is significantly positively correlated with the translation and replication level of calicivirus protein,suggesting that RPS5may be involved in the translation and replication process of calicivirus protein,but the molecular mechanism is unclear.In this study,Rabbit hemorrhagic disease virus(RHDV)and Feline calicivirus(FCV)were used as research objects,and biochemical experimental techniques such as RNA pulldown,Co-IP and GST pulldown were used to study the role of RPS5 in calicivirus and its mechanism at the cellular level.This paper mainly includes the following three aspects:(1)Construction of RPS5 overexpression and knockout cell lines.First,we constructed p LOV-RPS5-GFP and lenti-RPS5-sg RNA recombinant plasmids,using lentivirus packaging system and CRISPR-Cas9 system,and successfully obtained RPS5 overexpression and knockout cell lines that can stably express in cells are named RK-RPS5-GFP,CRFK-RPS5-GFP,RK-RPS5^-/-sg1,RK-RPS5^-/-sg2,CRFK-RPS5^-/-sg1 and CRFK-RPS5^-/-sg2 cell lines.IFA and Western blotting experiments were identified,and the results showed that RPS5 overexpression cell lines of different sources can express efficiently and stably,and RPS5 was effectively knocked down in RPS5 knockout cell lines.The constructed RPS5overexpression and knockout cell lines provide a research platform for studying the role of the host ribosomal protein RPS5 in calicivirus translation and replication.(2)Interaction between RPS5 and Calicivirus genomic RNA extremities sequences.First,we predicted the structure of RHDV 5'and 3'extremities(Ex)RNA,and found that both 5'and 3'Ex RNA contained stem-loop structures;Subsequently,RHDV 5'and 3'Ex t RSA recombinants plasmids were constructed.Using RNA-pulldown assay and mass spectrometry(MS),several host proteins were discovered which interact with RHDV 5'and 3'Ex RNA in RK-13 cells.To further investigate the role of RPS5 in RHDV replication,si RNAs for RPS5 and RPS5 eukaryotic expression plasmids were used to change the expression level of RPS5 in RK-13 cells and the results showed that the RHDV replication and translation levels were positively correlated with the expression level of RPS5.It was also verified that RPS5 promoted RHDV replication by constructing RPS5 stable overexpression cell lines and RPS5knockdown cell lines.(3)The interaction between RPS5 and calicivirus non-structural proteins.First,we have determined that RPS5 transfers from the nucleus to the cytoplasm at the initial stage of FCV infection through immunoconfocal real-time fluorescence,and nuclear-cytoplasmic separation experiments,and participated in the translation of FCV protein.In the middle and late stages of infection,RPS5 was degraded,but its mechanism needs to be explored.Subsequently,it was determined that RPS5 co-localized with FCV non-structural proteins NS1/2,NS3,NS6,and Rd Rp by IFA experiments.Through Co-IP and GST-Pulldown experiments,we determined that RPS5 interacted with FCV non-structural proteins NS3 and Rd Rp.Therefore,we successfully made FCVNS3 and Rd Rp polyclonal antibodies.Through IFA experiments,we determined the endogenous RPS5 and non-structural proteins NS3 and Rd Rp are co-localized in FCV infected cells.Subsequently,truncated eukaryotic plasmids of the FCV non-structural proteins NS3 and Rd Rp were constructed,and through Co-IP experiments,the functional domains of RPS5 and NS3 and Rd Rp were successfully identified.The IP-MS experiment successfully screened the host ribosomal proteins that interacted with RPS5,and found that RPS29,RPL29,and RPL15specifically associated with RPS5 after virus infection.In summary,we constructed RPS5 overexpression and knockout cell lines of different source cells,which provides a platform for this research.Then,we found that RPS5 interacted with viral RNA terminal sequences and RPS5 interacts with viral non-structural proteins.And then we use the difference of RPS5binding ribosomal protein before and after viral infection to elucidate the molecular mechanism of RPS5promoting viral protein translation at the cellular level.These results provide clues and basis for further study of the molecular mechanism of calicivirus translation and replication.