Two Types Of Virus-host Interaction Between Chlorovirus And Chlorella Variablis

PBCV-1,the type speices of chloroviruses,is the first strain that has been fully sequenced in phycodnsviruses;Chlorella variabilis,the host of the virus,has also been sequenced for its full-length of genome,thus the chlorovirus-host system is a good model system for studying algal virus-host interactions.In this study,we conducted a series of preliminary discussions for the possibility that PBCV-1 encodes the ubiquitin ligase complex E3 components,vSkp1 and vANK,to hijack ubiquitin-proteasome system of the host,and the enzymatic properties of the copper-zinc superoxide dismutase(tcv Cu,Zn-SOD)that encoded by the virus and may have the function of suppressing its host ROS system.In this study,the protein expression vectors vSkp1 and A607 R of the virus were constructed,and obtain the soluble expression of protein vSkp1.Pulldown experiments were performed between the protein vSkp1 and the supernatant of Chlorella variablis NC64 A with or without virus infection.The results were analyzed by mass spectrometry and obtain 29 proteins,include twenty five of F-Box proteins,two of Cullin proteins,one of Ringbox protein of the host and one of F-Box protein of the virus.The protein with high coverage was selected to optimized and synthesized based on yeast codon,then amplifying the protein fragments and linking them with the activation domain in the yeast two-hybrid system to form a "prey" plasmid;the optimized Skp1 protein gene fragment of virus and host was ligated with the binding domain to form the "bait" plasmid.The bait and prey plasmids were co-transformed into yeast strain Y2 HGold and Y187 to verify protein-protein interactions.The results showed that all five proteins interacted with chSkp1 and vSkp1.In our laboratory,the tcv Cu,Zn-SOD protein expressed by the chlorella virus PBCV-1 has been shown to have the function of eliminating superoxide anion and suppressing host ROS defense system.Using pyrogallol autoxidation method to measure the specific enzyme activity of SOD,and the result is 16050 U/mg.A preliminary study was conducted on its enzymatic properties and it was found that when the mass concentration of the enzyme was 1 ?g/mL,the temperature,pH,and denaturants have little effect on the activity of vSOD;when the mass concentration was 0.1 ?g/mL,the temperature and SDS have a strong influence on the activity of vSOD;when the mass concentration is 0.05 ?g/mL,the temperature,pH,and denaturants have a strong influence on the activity of vSOD.