LxCxE-motif mediated interaction of viral proteins with the retinoblastoma tumor suppressor: a common theme in DNA and RNA virus-host interactions

CHEMES, L.B.; GLAVINA, J; SANCHEZ, IE; DE PRAT-GAY, G.

Buenos Aires

Simposio; 3rd ICGEB WORKSHOP ON HUMAN RNA VIRUSES; 2012

ICGEB y Fundación Instituto Leloir IIBBA-CONICET

The retinoblastoma protein (Rb) controls cell cycle and development in eukaryotes. Rb is frequently mutated in human cancer and is inactivated by many viral proteins. Rb is a "hub" in a large interaction network that functions through the establishment of multiple protein-protein interactions [1]. Rb harbors a conserved protein interaction surface termed the “LxCxE cleft” that binds with high (nanomolar) affinity to the LxCxE linear motif, a short functional sequence found in over 30 host proteins that interact with Rb, including HDAC, cyclin D, the ATP dependent helicase BRG1, the methyltransferase DNMT1 and transcription factors Elf1 and UBF among others. The LxCxE motif is also present in proteins from numerous DNA and RNA viruses, including the E7 oncoprotein from human papillomavirus (HPV) and the NS5B polymerase from hepatitis C virus (HCV). Viruses are thought to use LxCxE-mediated interactions in order to fulfill their replication cycle. Long term sustained cell cycle subversion in infected cells is thought to lead, in some cases, to the development of human cancer. Currently, very little is known about the molecular properties of the LxCxE-Rb interaction, and about the mechanisms through which viral proteins that harbor this linear motif can compete effectively with binding of endogenous Rb targets. Interestingly, the LxCxE motif is found both in disordered regions, where it is readily exposed for binding, as well as in globular domains, where its conformation is restricted and must require large conformational changes that expose the motif for interaction. We have analyzed the interaction between HPV E7 and Rb. The LxCxE motif in E7 is highly conserved and located in the intrinsically disordered E7N domain. Binding of the LxCxE motif is modulated by the neighboring and highly acidic CKII-PEST region, and phosphorylation of serine residues within this region enhances the binding affinity by five-fold. In addition, the interaction is mediated through at least two other secondary, lower-affinity binding sites in E7, which further increases binding affinity to the sub-nanomolar range. The E7-Rb interaction was found to have a strong electrostatic component, which provides with a fast recognition speed. We propose that these molecular features of the E7-Rb interaction may be optimized to compete effectively with endogenous LxCxE-mediated interactions. Current research is aimed at studying the properties of the NS5B-Rb interaction. Opposed to HPV E7, the LxCxE motif in NS5B is located in a structured and solvent-protected region of the protein, next to the active site. Therefore, in this case the interaction must require a large conformational change to expose the motif. We aim to compare the interaction mechanisms of HPV E7 and HCV NS5B, revealing shared and virus-specific interaction features of these two important human pathogens. Future studies are also aimed at studying cellular LxCxEmediated interactions in order to understand the molecular basis of competition between cellular and viral LxCxE targets.