Efficient production of Hantaan and Puumala pseudovirions for viral tropism and neutralization studies.

Puumala (PUUV) and Hantaan (HTNV) viruses are hantaviruses within the family Bunyaviridae and associated with Hemorrhagic Fever with Renal Syndrome (HFRS) in humans. Little is known about how these viruses interact with host cells, though pathogenic hantaviruses interact with α(v)ß(3) integrin. To study host cell interactions and rapidly test the ability of antibodies to prevent infection, we produced HTNV and PUUV pseudovirions on a vesicular stomatitis virus (VSV) core. Similar to replication-competent hantaviruses, infection was low-pH-dependent. Despite broad cell tropism, several human T cell lines were poorly permissive to hantavirus pseudovirions, compared to VSV, indicating a relative block to infection at the level of entry. Stable expression of α(v)ß(3) integrin in SupT1 cells did not restore infectivity. Finally, the pseudovirion system provided a rapid, quantitative, and specific method to screen for neutralizing antibodies in immune sera.

Molecular characterization of the Ran-binding zinc finger domain of Nup153.

The nuclear pore complex is the gateway for selective traffic between the nucleus and cytoplasm. To learn how building blocks of the pore can create specific docking sites for transport receptors and regulatory factors, we have studied a zinc finger module present in multiple copies within the nuclear pores of higher eukaryotes. All four zinc fingers of human Nup153 were found to bind the small GTPase Ran with dissociation constants ranging between 5 and 40 mum. In addition a fragment of Nup153 encompassing the four tandem zinc fingers was found to bind Ran with similar affinity. NMR structural studies revealed that a representative Nup153 zinc finger adopts the same zinc ribbon structure as the previously characterized Npl4 NZF module. Ran binding was mediated by a three-amino acid motif (Leu(13)/Val(14)/Asn(25)) located within the two zinc coordination loops. Nup153 ZnFs bound GDP and GTP forms of Ran with similar affinities, indicating that this interaction is not influenced by a nucleotide-dependent conformational switch. Taken together, these studies elucidate the Ran-binding interface on Nup153 and, more broadly, provide insight into the versatility of this zinc finger binding module.

Studying nuclear disassembly in vitro using Xenopus egg extract.

Xenopus egg extract provides an extremely powerful approach in the study of cell cycle regulated aspects of nuclear form and function. Each egg contains enough membrane and protein components to support multiple rounds of cell division. Remarkably, incubation of egg extract with DNA in the presence of an energy regeneration system is sufficient to induce formation of a nuclear envelope around DNA. In addition, these in vitro nuclei contain functional nuclear pore complexes, which form de novo and are capable of supporting nucleocytoplasmic transport. Mitotic entry can be induced by the addition of recombinant cyclin to an interphase extract. This initiates signaling that leads to disassembly of the nuclei. Thus, this cell-free system can be used to decipher events involved in mitotic remodeling of the nuclear envelope such as changes in nuclear pore permeability, dispersal of membrane, and disassembly of the lamina. Both general mechanisms and individual players required for orchestrating these events can be identified via biochemical manipulation of the egg extract. Here, we describe a procedure for the assembly and disassembly of in vitro nuclei, including the production of Xenopus egg extract and sperm chromatin DNA.