Alphaviruses are a globally distributed group of enveloped, positive-sense RNA animal viruses. The 5’ two-thirds of the RNA genome is translated into a single, large polypeptide of ∼2500 amino acids. This polypeptide is autoproteolytically cleaved into four subunits, termed nonstructural proteins (nsP) 1-4, which form the replication complex. The nsP1 is the enzyme responsible for capping the viral mRNA and a membrane associated protein. The nsP2 has RNA helicase, triphosphatase activities and is an autoprotease responsible for cleaving the nonstructural polyprotein. The nsP4 is an RNA-dependent, RNA polymerase. The function of nsP3 remains unknown, however, it is the only viral protein phosphorylated by unknown cellular kinases. The nsP3 contains a macro or X domain that binds ADP-ribose and is highly conserved with homologues in rubella virus, hepatitis E virus and coronaviruses, including severe acute respiratory syndrome (SARS) virus. The protease domain of viral nsP2 protein cleaves the nsP1234 polyprotein replication complex into functional proteins. During replication of alphavirus genome, the positive strand of RNA genome is transcribed into a minus strand by the cleavage intermediate nsP123 and nsP4. Cleavage between nsP2/3 causes a template switch from minus strand to positive strand. Although the nonstructural protein 2/3 is essential for RNA replication, its function in viral replication is still not well understood. Here we report the crystal structure of Sindbis virus precursor form of nsP2/3 at 2.94Å resolution. The structure of Sindbis nsP2/3 protein consists of four distinct domains, which are the protease domain, methyltransferase like domain, macro domain and zinc binding domain. Surprisingly, nsP3 contains a novel folding with a previously uncharacterized zinc coordination site. In addition, the cleavage site between nsP2 and nsP3 is buried the two proteins and not accessible for proteolysis. Furthermore, the loop connecting the macro and zinc-binding domains makes direct contact with and causes a structure shift in the nsp2 protease domain. We have observed considerable differences between the pre-cleavage and post-cleavage forms of nsp2/3. The structure suggests that a conformational change in nsP2/3 is required in order for the nsP2 protease to gain access to the nsP2/3 cleavage site.
|School:||Rutgers The State University of New Jersey - New Brunswick and University of Medicine and Dentistry of New Jersey|
|Department:||Graduate School - New Brunswick|
|School Location:||United States -- New Jersey|
|Source:||MAI 49/03M, Masters Abstracts International|
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