From Science to Fiction – Connecting In Vivo and In Vitro Results in Polyprotein Processing of Coronaviruses

Polyprotein processing is a common strategy in many positive sense single-stranded RNA ((+)ssRNA) viruses. This highly regulated process is crucial for viral progeny and ensures the release of functional replicase proteins in the correct location and at the right time. Coronaviruses (CoVs) have one of the largest genomes on average among (+)ssRNA viruses requiring a unique replication-transcription complex (RTC) with proofreading function that prevents error catastrophe. Two thirds of the CoV genome encode for the non-structural proteins (nsps) that drive replication. These are directly synthesized by RNA genome translation after infection as two large polyproteins pp1a and pp1ab. A regulated polyprotein proteolytic auto-processing is essential for viral growth and always has been an interesting target for therapeutics. Here, we present an overview of polyprotein processing and RTC research in CoVs in vitro and in vivo over the last 30 years. We highlight cutting-edge methodologies such as super resolution microscopy or structural mass spectrometry approaches and demonstrate how these have contributed to polyprotein research, e.g. by providing comprehensive structural models. We illustrate exciting examples of polyprotein processing in other viruses that could be transferred to CoVs, too. Additionally, we identify critical knowledge gaps in polyprotein processing and RTC assembly, proposing future perspectives to address these limitations.