Noroviruses, in the genus Norovirus, are a significant cause of viral gastroenteritis in humans and animals. For almost 50 years, the lack of a cultivation system for human noroviruses (HuNoVs) was a major barrier to understanding virus biology and the development of effective antiviral strategies. This review presents a historical perspective of the development of a cultivation system for HuNoVs in human intestinal epithelial cell cultures. Successful cultivation was based on the discovery of genetically-encoded host factors required for infection, knowledge of the site of infection in humans, and advances in the cultivation of human intestinal epithelial cells achieved by developmental and stem cell biologists. The human stem cell-derived enteroid cultivation system recapitulates the multicellular, physiologically active human intestinal epithelium, and allows studies of virus-specific replication requirements, evaluation of human host-pathogen interactions, and supports the pre-clinical assessment of methods to prevent and treat HuNoV infections.

A variety of pathogens, including viruses, bacteria and parasites, target cellular lipid droplets for their replication. Rotaviruses (RVs) infect the villous epithelium of the small intestine and are a major cause of acute gastroenteritis in infants and young children worldwide. RVs induce and require lipid droplets for the formation of viroplasms, sites of virus genome replication, and nascent particle assembly. Here we review the role of lipid droplets in RV replication. Inhibitors of fatty acid synthesis or chemicals that interfere with lipid droplet homeostasis decrease the number and size of viroplasms and the yield of infectious virus. We used a genetically engineered RV, delayed in viroplasm assembly, to show an early interaction of RV nonstructural protein NSP2 and the lipid droplet-associated protein phospho-PLIN1. The interaction between NSP2 and phospho-PLIN1 suggests that we have identified part of the mechanism of RV-induced lipid droplet formation. These studies demonstrate that RV is an excellent model to dissect the cellular process of lipid droplet formation and to determine how RV induces and usurps lipid droplet biogenesis to form viroplasm/lipid droplets for virus replication.

Autophagy is a cellular response activated by many pathogens, but the mechanism of activation is largely unknown. Recently we showed for the first time that rotavirus initiates the autophagy pathway through a calcium-mediated mechanism. Expression of the rotavirus-encoded NSP4, a pore-forming protein (viroporin), elicits the release of endoplasmic reticulum (ER) lumenal calcium into the cytoplasm of the infected cell. The increased cytoplasmic calcium activates a calcium signaling pathway involving calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) and 5' adenosine monophosphate-activated protein kinase (AMPK) to trigger autophagy. Rotavirus further manipulates autophagy membrane trafficking to transport viral ER-associated proteins to viroplasms, sites of viral genome replication and immature particle assembly. Transport of viral proteins to viroplasms is required for assembly of infectious virus. Thus, NSP4, a multifunctional viral protein known to regulate infectious particle assembly, also modulates membrane trafficking by orchestrating the activation of autophagy to benefit viral replication.

In a morphogenic step that is exceedingly rare for nonenveloped viruses, immature rotavirus particles assemble in replication centers called viroplasms, and bud through cytoplasmic cellular membranes to acquire the outer capsid proteins for infectious particle assembly. Historically, the intracellular membranes used for particle budding were thought to be endoplasmic reticulum (ER) because the rotavirus nonstructural protein NSP4, which interacts with the immature particles to trigger budding, is synthesized as an ER transmembrane protein. This present study shows that NSP4 exits the ER in COPII vesicles and that the NSP4-containing COPII vesicles are hijacked by the cellular autophagy machinery, which mediates the trafficking of NSP4 to viroplasms. Changing the paradigm for rotavirus maturation, we propose that the cellular membranes required for immature rotavirus particle budding are not an extension of the ER but are COPII-derived autophagy isolation membranes.