The Structural Basis Of African Swine Fever Virus PA104R Binding To DNA And Its Inhibition By Stilbene Derivatives

African swine fever(ASF)is an acute,febrile and hemorrhagic animal infectious diseases caused by the African swine fever virus(ASFV),and is one of the animal diseases notifiable to the World Organization for Animal Health(OIE).ASF is also a List A animal disease in China.ASFV mainly infects domestic pigs and wild boars,with a fatality rate of 100%.The ASF epidemic was first reported in Kenya in 1921 and has been spreading worldwide since then.After its appearance in China in August 2018,ASF has caused hundreds of millions of dollars in economic losses,seriously affecting public health and safety as well as the healthy development of the pig industry.Due to its large genome structure and complex immune escape mechanism,there are currently no commercial vaccines,drugs,and reliable treatments that can prevent the spread of ASFV.The heat-unstable nucleoid protein(HU)and integration host factors(IHF)families are composed of histone-like proteins expressed by a variety of bacteria.They are abundantly present in the nucleoid of bacteria and possess the nucleic acid binding ability.The HU/IHF family plays an architectural role in DNA supercoiling and compaction,and therefore participates in bacterial gene replication,DNA recombination and repair,cell division,and gene transcription regulation.As a Nucleocytoplasmic large DNA virus(NCLDV),ASFV has a genome length of up to 170-193 kbp,which encodes more than 200 proteins.pA104R,a histone-like protein located in ASFV nucleoside,is composed of 104 amino acids,and possesses sequence identity and functional similarity with the HU/IHF family members.It has been reported that pA104R acts simultaneously with pP1192R,a type Ⅱ topoisomerase encoded by ASFV,and introduces superhelical structure into the ASFV genome.This superhelical structure narrows the virus nucleic acid volume and helps the virus nucleic acid packaged into the new generation of progeny virus,thus is crucial in the process of virus replication.Based on this,we have a strong interest in how pA104R protein mediates the formation of complex DNA structures and whether pA104R can be used as a target for the screening of small molecule inhibitors.We first analyzed the nucleic acid binding ability of pA104R and found that it binds both double-stranded DNA(dsDNA)and single-stranded DNA(ssDNA)greater than 5 bp with no sequence dependence.Subsequently,the molecular structure of pA104R prior to binding DNA molecules(apo-pA104R)and the complex structure of its binding DNA molecule(pA104R-DNA)were resolved by crystallographic methods.We found that pA104R has a high structural similarity to the HU/1HF family proteins of bacteria,which composed of a a-helical "body" and two prominent β-strand nucleic acid-binding "arms".Despite the high structural similarity between pA104R and the bacterial HU/IHF family proteins,there were significant differences in the binding pattern of DNA molecules between pA104R and the HU/IHF family proteins.The complex structures of several bacterial HU/IHF family protein-binding DNAs all showed that the two nucleic acid-binding arms(β-ribbon arms)of HU/IHF family proteins insert into the minor groove of DNA molecules when binding DNA molecules.In contrast,the nucleic acid-binding arms of pA 104R inserts into the major groove of the DNA molecule.However,this difference in binding mode did not affect the ability of pA104R in folding the DNA molecule.An overall DNA bending angle of 93.80 is observed in crystal packing of the pA104R-DNA complex structure,which provides clues to the mechanism of DNA condensation by pA104R.Furthermore,we found two stilbene derivatives,SD1 and SD4,which targeting the Mycobacterium tuberculosis HU protein can also bind to pA104R and block the binding of pA104R to DNA at the molecular level.We then tested whether SD1 and SD4 can also inhibit replication of ASFV replication in swine macrophages(PAM),and found both SD1 and SD4 displayed strong inhibition of ASFV DNA levels and the titers of infectious progeny virus in the supernatants in a dose-dependent manner.The nucleic acid copy number of the virus decreased to about 22%and 5%of that of the control group 48 hours after administration at 50 μm,and the TCID50 of the virus decreased to about 9%and 3%of that of the control group.These results indicate that the inhibition rate of the two small molecules against the virus at the cellular level can reach more than 90%,suggesting that they have an important application prospect in the clinical treatment or prevention of ASFV infection.In conclusion,we have made important progress in structure and function of the important viral proteins of ASFV and the antiviral strategies based on this.We have elucidated the high-resolution crystal structure of ASFV nucleic acid binding protein pAl 04R before and after binding DNA molecules,and revealed the molecular basis of pA104R mediated DNA formation into advanced structures.In addition,two small molecule compounds were found to inhibit ASFV infection at the cellular level,which provided an important basis for anti-ASFV drug design and disease prevention and control of targeted viral proteins.