Rotavirus is a clinically significant pathogen and is the causative agent of life-threatening gastroenteritis in many young humans and animals, leading to approximately 500,000 deaths annually, with 85% occurring in developing countries globally. The mature infectious rotavirus particle, also called triple-layered particles (TLPs), is formed by three concentric layers of protein that surround the viral genome composed of eleven segments of doublestranded RNA (dsRNA). One of the essential steps in the rotavirus infection is the viral recognition and the recruitment of the host cell surface receptors, followed by the entry of the virion into the host cell cytoplasm triggering the viral replication process. Therefore, an understanding of the mechanism associated with the initial viral-host cell interactions would undoubtedly impart a critical insight that may lead to the development of an effective therapeutic. The rotavirus cell entry process is a complicated process and is poorly understood. The outermost layer of the virion is composed of two major proteins, the glycoprotein VP7, and the spike protein VP4. The VP4 spike protein plays a significant role in receptor binding and cell penetration. Rotavirus infectivity is significantly enhanced by the proteolytic cleavage of VP4 into its functional fragments VP8* and VP5* that remain associated with the virion. The globular head of the spike VP8*, mediates initial rotavirus interactions with cell membrane glycoconjugates, including those containing sialic acid (Sia). Here, we report the first X-ray crystal structure of the rotavirus carbohydrate-recognizing protein VP8* from porcine strain CRW-8 in complex with the GM1bGc ganglioside (which is the N-glycolyl form of GM1b) (Chapter 3). Our structure not only provides the first critical insights into the molecular basis of the interaction between rotavirus VP8* and a larger ganglioside, but has also allowed us to map the nature of rotavirus interactions with other gangliosides, for example, GM1a and GD1a. The introduction of animal rotavirus strains into the human population is referred as zoonotic transmission. This inherent capability of rotaviruses to reassort and to cross the interspecies barrier, especially between animals and humans, poses a significant challenge to counteract these viruses. Limited attention has been given to these zoonotic events and this is turning into a major threat to human society. Therefore, we directed our studies to these animal rotaviruses that are capable of crossing inter-species barrier. Our saturation transfer difference (STD) NMR and Isothermal Titration Calorimetry (ITC) studies demonstrated that canine K9 shows a preference for the N-glycolyl form of Sia (Neu5Gc, which is not expressed in humans) over its N-acetyl form (Neu5Ac). Interestingly, the K9 VP8* displayed a higher binding affinity for the N-acetyl form of Sia when compared to the binding affinity shown by other animal rotavirus strains (rhesus RRV and porcine CRW-8) for the same ligand. This indicates that canine K9 could bind efficiently to the glycans that are expressed in humans (Chapter 2). We also present the X-ray crystal structure of canine K9 VP8* in its apo form (Chapter 5) as well as in complex with a Neu5Ac derivative (Chapter 2). The K9 VP8* complex structure provides a template for further glycan-based drug design and discovery. The thesis Chapter 4 reports the first crystallographic structure of the VP8* from an animal rotavirus strain that is in its Sia-free form (porcine TFR-41 VP8*). This structure revealed an unexpected discovery of substantial rearrangements when compared to Sia-bound VP8* porcine CRW-8 VP8* (particularly in the Sia-binding site) that have not been seen in any other VP8* structure. Similarly, we have also determined the X-ray crystal structure of canine K9 VP8* (Chapter 5) and rhesus RRV VP8* (Chapter 6) in their Sia-free form, and the comparison with their respective Sia-bound form displayed a similar pattern of structural rearrangement as that seen in TFR-41 VP8* structure. Thus, the plasticity of VP8* dictates its structural adaptability to utilize a wider range of cellular glycans that are identified as their binding partners. Intriguingly, we have also discovered a novel deep hydrophobic cavity that emerges as a consequence of the structural rearrangement observed in VP8*. This cavity is loaded with an endogenous fatty acid (acquired by bacterial expression), and it lies at the site equivalent to the Sia-binding site in VP8*. We assessed the identity of the fatty acid using mass spectrometry, and it supports our X-ray crystal structure determination. Lastly, this novel hydrophobic cavity was exploited to identify promising molecules that could have an ability to bind within this cavity (Chapter 6). The cavity in VP8* shows some comparable features to that found in enteroviruses and thus provided an opportunity to test the viral uncoating inhibitors (pocket binders, most intensely studied antiviral category in enteroviruses and many picornaviruses). Pleconaril (a pocket binder) is a broad-spectrum antiviral agent that is used in picornavirus respiratory infections. We present the first evidence, using 19F NMR technique, that pleconaril interacts with the fatty acid treated VP8* and that the interaction is not observed when VP8* is used alone. In summary, the work contained within this thesis has contributed significantly to the rotavirus research and the findings that relate to the novel hydrophobic pocket has unfolded an entirely new area of research with tremendous potential for the design and discovery of future drug candidates.