Applications of cryo-electron microscopy in the studies of virus and host interactions

Viruses are a group of contagious microbes that have compact structures, containing a nucleic acid core and a protein shell. The replication of viruses requires assistance from hosts which can be almost any cellular organism. Viral infections are often associated with diseases and have been a major threat to the human race. To cope with viral diseases, we need to understand viruses, including their structures, life cycle, pathogenesis and interactions with their hosts. The first structure of a human virus was determined by the Rossmann lab in 1985 using X-ray crystallography.Thanks to the recent advances in both hardware and software, cryo-electron microscopy (cryo-EM) has emerged as a powerful tool to study virus structures. Cryo-EM allows structural determination for a wide range of specimens to high resolution comparable to what can be achieved by X-ray crystallography. Currently two techniques of cryo-EM are commonly used in structural virology: single particles analysis (SPA) and electron tomography (ET). Single particle analysis has been used to determine the structures of viruses complexed with host factors in three studies that are to be discussed with more details in chapters 2-4. The structure of B19 parvovirus complexed with Fabs of a neutralizing human antibody was determined to 3.2 Å resolution. This structure showed that amino acids from three neighboring VP2 proteins form a quaternary structure epitope. In addition, the structure of human rhinovirus-C (RV-C) complexed with its cellular receptor, CDHR3, was determined to 3.9 Å resolution. Despite the low occupancy of the receptors, a “powerful” localized 3D classification procedure helped to select viral particles that had more bound receptors. Furthermore, structures were determined to 10 Å resolution of bacteriophage ΦX174 bound to lipopolysaccharide (LPS) bilayers, before and after genome ejection. These structures showed a series of conformational changes that occurred when a phage penetrated the bacterial membranes. These studies are good examples of applying cryo-EM to investigate virus-host interactions.However, single particle analysis requires samples to be isolated, homogenous and monodispersed. On the contrary, tomography allows in situ studies and is applicable to samples with more flexibility and more heterogeneity. In the case of ΦX174, the structural changes that are involved in the assembly of the H-tube during infection remains a huge mystery. To provide an environment that is more similar to the surface of a bacterial cell, LPS-containing liposomes were mixed with ΦX174 viruses. It was then observed that the ΦX174 particles bound to these liposomes in a very compact manner which was impossible interpret with single particle analysis. Using cryo-ET, 3D volumes of liposome-ΦX174 complexes were reconstructed and structural details were visualized by sub-tomogram classification and averaging.The emergence of cryo-EM has not only made high-resolution structural studies possible but also broadened the scope of samples with which virologists could work. Moreover, studies on flexible and heterogeneous complexes between viruses and host factors are now possible using either single particle analysis or electron tomography. These techniques will help us to understand virus-host relationships and finally, to develop effective anti-viral therapies.