Insights into Minor Group Rhinovirus Uncoating: The X-ray Structure of the HRV2 Empty Capsid

Upon attachment to their respective receptor, human rhinoviruses (HRVs) are internalized into the host cell via different pathways but undergo similar structural changes. This ultimately results in the delivery of the viral RNA into the cytoplasm for replication. To improve our understanding of the conformational modifications associated with the release of the viral genome, we have determined the X-ray structure at 3.0 Å resolution of the end-stage of HRV2 uncoating, the empty capsid. The structure shows important conformational changes in the capsid protomer. In particular, a hinge movement around the hydrophobic pocket of VP1 allows a coordinated shift of VP2 and VP3. This overall displacement forces a reorganization of the inter-protomer interfaces, resulting in a particle expansion and in the opening of new channels in the capsid core. These new breaches in the capsid, opening one at the base of the canyon and the second at the particle two-fold axes, might act as gates for the externalization of the VP1 N-terminus and the extrusion of the viral RNA, respectively. The structural comparison between native and empty HRV2 particles unveils a number of pH-sensitive amino acid residues, conserved in rhinoviruses, which participate in the structural rearrangements involved in the uncoating process

Co-culture of primary rat hepatocytes with rat liver epithelial cells enhances interleukin-6-induced acute-phase protein response

Three different primary rat hepatocyte culture methods were compared for their ability to allow the secretion of fibrinogen and albumin under basal and IL-6-stimulated conditions. These culture methods comprised the co-culture of hepatocytes with rat liver epithelial cells (CC-RLEC), a collagen type I sandwich culture (SW) and a conventional primary hepatocyte monolayer culture (ML). Basal albumin secretion was most stable over time in SW. Fibrinogen secretion was induced by IL-6 in all cell culture models. Compared with ML, CC-RLEC showed an almost three-fold higher fibrinogen secretion under both control and IL-6-stimulated conditions. Induction of fibrinogen release by IL-6 was lowest in SW. Albumin secretion was decreased after IL-6 stimulation in both ML and CC-RLEC. Thus, cells growing under the various primary hepatocyte cell culture techniques react differently to IL-6 stimulation with regard to acute-phase protein secretion. CC-RLEC is the preferred method for studying cytokine-mediated induction of acute-phase proteins, because of the pronounced stimulation of fibrinogen secretion upon IL-6 exposure under these conditions

Human SCARB2-Mediated Entry and Endocytosis of EV71

Enterovirus (EV) 71 infection is known to cause hand-foot-and-mouth disease (HFMD) and in severe cases, induces neurological disorders culminating in fatality. An outbreak of EV71 in South East Asia in 1997 affected over 120,000 people and caused neurological disorders in a few individuals. The control of EV71 infection through public health interventions remains minimal and treatments are only symptomatic. Recently, human scavenger receptor class B, member 2 (SCARB2) has been reported to be a cellular receptor of EV71. We expressed human SCARB2 gene in NIH3T3 cells (3T3-SCARB2) to study the mechanisms of EV71 entry and infection. We demonstrated that human SCARB2 serves as a cellular receptor for EV71 entry. Disruption of expression of SCARB2 using siRNAs can interfere EV71 infection and subsequent inhibit the expression of viral capsid proteins in RD and 3T3-SCARB2 but not Vero cells. SiRNAs specific to clathrin or dynamin or chemical inhibitor of clathrin-mediated endocytosis were all capable of interfering with the entry of EV71 into 3T3-SCARB2 cells. On the other hand, caveolin specific siRNA or inhibitors of caveolae-mediated endocytosis had no effect, confirming that only clathrin-mediated pathway was involved in EV71 infection. Endocytosis of EV71 was also found to be pH-dependent requiring endosomal acidification and also required intact membrane cholesterol. In summary, the mechanism of EV71 entry through SCARB2 as the receptor for attachment, and its cellular entry is through a clathrin-mediated and pH-dependent endocytic pathway. This study on the receptor and endocytic mechanisms of EV71 infection is useful for the development of effective medications and prophylactic treatment against the enterovirus