In order for a virus to establish a chronic infection, it must maintain a dynamic equilibrium to persist against the host's immune response without causing acute disease. To accomplish this, the ...virus must participate in a multitude of complex interactions with the host. Among other virus types, many positive strand RNA viruses are known to establish persistent infections in hosts ranging from plants and livestock to humans. Compared to their eukaryotic hosts, RNA viruses have a very limited genomic capacity. With this restricted coding capacity, how an RNA virus acquires the vast functional capabilities needed for chronic infection becomes an intriguing question. Using the hepatitis C virus (HCV) as a model, we have proposed one route an RNA virus could expand its functional capacity by using a protein containing an intrinsically disordered region (IDR). The structural flexibility of a disordered region allows a protein to establish multiple interactions within the same region. This structural flexibility must be regulated though to provide specificity of function. We, and others, propose phosphorylation can regulate the conformation of an IDR. We hypothesize a 'phosphorylation code' exists for the HCV intrinsically disordered protein, NS5A, that regulates its myriad interactions with both host and viral proteins, allowing this protein to serve an essential role in the virus¡¦ ability to dynamically interact with the host and establish a chronic infection. In the following studies, we have established the ability to purify the intrinsically disordered domain (IDD) of the HCV protein NS5A to a concentration and purity suitable for biophysical characterization. We have determined that the NS5A IDD has a propensity for α-helical formation. Using phosphorylation by PKA as a model, we have determined that a single phosphorylation event alters the global conformation of the NS5A IDD. This conformational change is likely inducing a more stable structure with increased α-helical content. To determine the impact of phosphorylation-induced conformational changes on the function of the NS5A protein, we have explored NS5A IDD binding to SH3 domains from several different proteins. While SH3 domains are structurally similar, we found that not only does the NS5A IDD binding to different SH3 domains differ, but PKA phosphorylation impacts the interaction of each of the SH3 domains uniquely. This study provides insight into a fundamental way binding of SH3 domains to polyproline motifs could be regulated. This exploration of the effects of PKA phosphorylation on the conformation and function of the NS5A IDD has contributed to the overall understanding of the role and regulation of intrinsically disordered proteins and provides an example of how an RNA virus with limited genetic capacity could expand its functional proteome.
