Biomedical subjects

Lisa Z Scheifele

Publications and source records attributed to Lisa Z Scheifele.

5 recordsLinked to original sources

PubMed2006

Transposon insertion site profiling chip (TIP-chip).

Mobile elements are important components of our genomes, with diverse and significant effects on phenotype. Not only can transposons inactivate genes by direct disruption and shuffle the genome through recombination, they can also alter gene expression subtly or powerfully. Currently active transposons are highly polymorphic in host populations, including, among hundreds of others, L1 and Alu elements in humans and Ty1 elements in yeast. For this reason, we wished to develop a simple genome-wide method for identifying all transposons in any given sample. We have designed a transposon insertion site profiling chip (TIP-chip), a microarray intended for use as a high-throughput technique for mapping transposon insertions. By selectively amplifying transposon flanking regions and hybridizing them to the array, we can locate all transposons present in a sample. We have tested the TIP-chip extensively to map Ty1 retrotransposon insertions in yeast and have achieved excellent results in two laboratory strains as well as in evolved Ty1 high-copy strains. We are able to identify all of the theoretically detectable transposons in the FY2 lab strain, with essentially no false positives. In addition, we mapped many new transposon copies in the high-copy Ty1 strain and determined its Ty1 insertion pattern.

Animals↗

PubMed2006

Importin-beta family members mediate alpharetrovirus gag nuclear entry via interactions with matrix and nucleocapsid.

The retroviral Gag polyprotein orchestrates the assembly and release of virus particles from infected cells. We previously reported that nuclear transport of the Rous sarcoma virus (RSV) Gag protein is intrinsic to the virus assembly pathway. To identify cis- and trans-acting factors governing nucleocytoplasmic trafficking, we developed novel vectors to express regions of Gag in Saccharomyces cerevisiae. The localization of Gag proteins was examined in the wild type and in mutant strains deficient in members of the importin-beta family. We confirmed the Crm1p dependence of the previously identified Gag p10 nuclear export signal. The known nuclear localization signal (NLS) in MA (matrix) was also functional in S. cerevisiae, and additionally we discovered a novel NLS within the NC (nucleocapsid) domain of Gag. MA utilizes Kap120p and Mtr10p import receptors while nuclear entry of NC involves the classical importin-alpha/beta (Kap60p/95p) pathway. NC also possesses nuclear targeting activity in avian cells and contains the primary signal for the import of the Gag polyprotein. Thus, the nucleocytoplasmic dynamics of RSV Gag depend upon the counterbalance of Crm1p-mediated export with two independent NLSs, each interacting with distinct nuclear import factors.

Avian Sarcoma Viruses↗

PubMed2005

Detailed mapping of the nuclear export signal in the Rous sarcoma virus Gag protein.

The Rous sarcoma virus (RSV) Gag polyprotein undergoes transient nuclear trafficking as an intrinsic part of the virus assembly pathway. Nuclear export of Gag is crucial for the efficient production of viral particles and is accomplished through the action of a leptomycin B (LMB)-dependent nuclear export signal (NES) in the p10 domain (L. Z. Scheifele, R. A. Garbitt, J. D. Rhoads, and L. J. Parent, Proc. Natl. Acad. Sci. USA 99:3944-3949, 2002). We have now mapped the nuclear export activity to the C-terminal portion of the p10 sequence and identified the four hydrophobic amino acids within this region that comprise a leucine-rich NES. Alteration of these hydrophobic residues resulted in the accumulation of Gag proteins within the nucleus and a budding defect greater than that obtained with LMB treatment of cells expressing the wild-type Gag protein (Scheifele et al., Proc. Natl. Acad. Sci. USA 99:3944-3949, 2002). In addition, export of Gag from the nucleus was found to be a rate-limiting step in virus-like particle production. Consistent with a role for the NES sequence in viral replication, this cluster of hydrophobic residues in p10 is conserved across a wide range of avian retroviruses. Furthermore, naturally occurring substitutions within this region in related viruses maintained nuclear export activity and remained sensitive to the activity of LMB. Using gain-of-function approaches, we found that the hydrophobic motif in p10 was sufficient to promote the nuclear export of a heterologous protein and was positionally independent within the Gag polyprotein. Finally, the export pathway was further defined by the ability of specific nucleoporin inhibitors to prevent the egress of Gag from the nucleus, thereby identifying additional cellular mediators of RSV replication.

Active Transport, Cell Nucleus↗

PubMed2003

Specificity of plasma membrane targeting by the rous sarcoma virus gag protein.

Budding of C-type retroviruses begins when the viral Gag polyprotein is directed to the plasma membrane by an N-terminal membrane-binding (M) domain. While dispersed basic amino acids within the M domain are critical for stable membrane association and consequent particle assembly, additional residues or motifs may be required for specific plasma membrane targeting and binding. We have identified an assembly-defective Rous sarcoma virus (RSV) Gag mutant that retains significant membrane affinity despite having a deletion of the fourth alpha-helix of the M domain. Examination of the mutant protein's subcellular distribution revealed that it was not localized to the plasma membrane but instead was mistargeted to intracytoplasmic membranes. Specific plasma membrane targeting was restored by the addition of myristate plus a single basic residue, by multiple basic residues, or by the heterologous hydrophobic membrane-binding domain from the cellular Fyn protein. These results suggest that the fourth alpha-helix of the RSV M domain promotes specific targeting of Gag to the plasma membrane, either through a direct interaction with plasma membrane phospholipids or a membrane-associated cellular factor or by maintaining the conformation of Gag to expose specific plasma membrane targeting sequences.

Active Transport, Cell Nucleus↗

PubMed2002

Nuclear entry and CRM1-dependent nuclear export of the Rous sarcoma virus Gag polyprotein.

The retroviral Gag polyprotein directs budding from the plasma membrane of infected cells. Until now, it was believed that Gag proteins of type C retroviruses, including the prototypic oncoretrovirus Rous sarcoma virus, were synthesized on cytosolic ribosomes and targeted directly to the plasma membrane. Here we reveal a previously unknown step in the subcellular trafficking of the Gag protein, that of transient nuclear localization. We have identified a targeting signal within the N-terminal matrix domain that facilitates active nuclear import of the Gag polyprotein. We also found that Gag is transported out of the nucleus through the CRM1 nuclear export pathway, based on observations that treatment of virus-expressing cells with leptomycin B resulted in the redistribution of Gag proteins from the cytoplasm to the nucleus. Internal deletion of the C-terminal portion of the Gag p10 region resulted in the nuclear sequestration of Gag and markedly diminished budding, suggesting that the nuclear export signal might reside within p10. Finally, we observed that a previously described matrix mutant, Myr1E, was insensitive to the effects of leptomycin B, apparently bypassing the nuclear compartment during virus assembly. Myr1E has a defect in genomic RNA packaging, implying that nuclear localization of Gag might be involved in viral RNA interactions. Taken together, these findings provide evidence that nuclear entry and egress of the Gag polyprotein are intrinsic components of the Rous sarcoma virus assembly pathway.

Active Transport, Cell Nucleus↗