Abstract
The NS1 protein of mink enteritis virus (MEV) is a multidomain and multifunctional protein that plays a critical role in viral replication, with predicted nuclease, helicase and transactivation activities. The nuclease and helicase domains of NS1 protein are involved in interaction with viral DNA. Herein, potential amino acids critical for DNA binding in the MEV NS1 were mutated, all of which resulted in a termination of viral production from an infectious MEV clone. Although E121, H129/131, Y212 and K470/472 mutants retained their P38 and 5'UTR transactivation activity, K196/197 and K406 mutations eliminated this. Interestingly, VP2 protein was produced following transfection of F81 cells with pMEV-NS1-196K2G (K196G and K197G) and pMEV-NS1-K406G when pNS1 was co-transfected in trans, indicating that the substitutions did not affect the integrity of the DNA sequence that bound to NS1 protein but inhibited the biological properties of NS1 protein itself. The ability of NS1 protein to interact with SP1 was inhibited by both 196K2G and K406G substitutions, while 196K2G resulted in failure to bind to the DNA-binding sites in the P38 promoter, and the oligomerization of K406G was inhibited. All of these could explain the transcriptional repression.