Macromolecular assembly-driven processing of the 2/3 cleavage site in the alphavirus replicase polyprotein.
Semliki Forest virus (SFV) is a member of the Alphavirus genus, which produces its replicase proteins in the form of a nonstructural (ns) polyprotein precursor P1234. The maturation of the replicase occurs in a temporally controlled manner by protease activity of nsP2. The template preference and enzymatic capabilities of the alphaviral replication complex have a very important connection with its composition, which is irreversibly altered by proteolysis. The final cleavage of the 2/3 site in the ns polyprotein apparently leads to significant rearrangements within the replication complex and thus denotes the "point of no return" for viral replication progression. Numerous studies have devised rules for when and how ns protease acts, but how the alphaviral 2/3 site is recognized remained largely unexplained. In contrast to the other two cleavage sites within the ns polyprotein, the 2/3 site evidently lacks primary sequence elements in the vicinity of the scissile bond sufficient for specific protease recognition. In this study, we sought to investigate the molecular details of the regulation of the 2/3 site processing in the SFV ns polyprotein. We present evidence that correct macromolecular assembly, presumably strengthened by exosite interactions rather than the functionality of the individual nsP2 protease, is the driving force for specific substrate targeting. We conclude that structural elements within the macrodomain of nsP3 are used for precise positioning of a substrate recognition sequence at the catalytic center of the protease and that this process is coordinated by the exact N-terminal end of nsP2, thus representing a unique regulation mechanism used by alphaviruses.
Institute of Technology, University of Tartu, Nooruse 1, Tartu 50411, Estonia. aleksei.lulla@ut.ee
Aleksei
A
Lulla
Valeria
V
Lulla
Andres
A
Merits
D018354:Alphavirus Infections / Q000821:virology; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D008969:Molecular Sequence Data; D020815:Polyproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D012672:Semliki forest virus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D016415:Sequence Alignment; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
D020815:Polyproteins; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase