Astrovirus is a non-enveloped (T = 3), positive-sense RNA virus that is transmitted via the ingestion of contaminated food and water. Generally targeting susceptible populations, astrovirus results in gastroenteritis presenting with nausea, dehydration, and diarrhea. The Astroviridae family encompasses the genera Mamastrovirus and Avastrovirus, which are capable of infecting mammals and avians, respectively. In addition to presenting a human health concern, astrovirus can infect a variety of domesticated animals such as bovines, ducks, turkeys, chickens, minks, and pigs. Avastrovirus is associated with life-threatening symptoms like nephritis and hepatitis in avian populations. Alarmingly, the members of Astroviridae engage in high rate of inter-species homologous recombination ensuing in rapid strain evolution developing into a precipitous and unpredictable expansion of cell tropism, which have been implicated in causing neurological syndromes. In astrovirus, the capsid serves as the outer barrier that encapsulates nucleic acids, protects the virion against environmental stresses, defines the cell tropism, and triggers the host immune response via antigenic display. The capsid protein (CP) is translated as an assembly competent polypeptide (VP90) that is composed of three structural domains: a conserved amino-terminal domain, a central hyper-variable domain, and an acidic carboxy-terminal domain, which is thought to constitute an assembly-related sorting domain. Upon capsid assembly, the CP polypeptide is proteolytically processed by host caspases and extracellular proteases, resulting in the liberation of mature CP proteins that is concomitant with the induction of infectivity via an unknown maturation-dependent mechanism. It is postulated that proteolysis results in the liberation of small peptides from a 10 kD capsid segment that may modulate infectivity as a viral penetration factor. We propose a series of biophysical and structural approaches to dissect the link between capsid maturation and the induction of infectivity in human astrovirus. Capsid assembly as a function of CP domain will be ascertained by over-expressing constructs including CP70-796, CP70-646 and CP70-418 coupled with imaging via negative-staining EM, which will verify which domains provide the cementing force necessary for capsid assembly. An atomic resolution structure of the capsid surface (VP34) will be built using X-ray crystallography coupled with a cryo-EM reconstruction of both the immature and mature capsid to clarify how the capsid lattice reorganizes as a consequence of proteolytic processing. Likewise, the spike complement of the capsid as a function of maturation state will be verified with ultracentrifugation and silver-staining SDS-PAGE. Finally, the presence of liberated viral penetration factors will be ascertained through ESI-MS and the molecular basis of infectivity acquired from maturation will be quantified through liposome permeability and floatation assay. This work will improve our understanding of the astrovirus capsid structure, assembly, and maturation-dependent infectivity, which will aid in the design of therapeutics to combat this emerging pathogen.
In addition to its canonical role in causing gastroenteritis in juveniles, the elderly, and in immunocompromised individuals, astrovirus has been implicated in neurological syndromes such as shaking mink syndrome and viral encephalitis in humans (Blomstrom et al., 2010;Quan et al., 2010). Astrovirus appears to engage in a rapid expansion of cell tropism through the exchange of the spike protrusion domain of the capsid through homologous recombination (Babkin et al., 2012). In this study, we propose a series of experiments to dissect the link between capsid assembly and maturation with the induction of infectivity, which can serve as a basis for rational drug design.
