Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2002-03-29
2003-11-25
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S005000, C530S350000, C530S388210
Reexamination Certificate
active
06653103
ABSTRACT:
BACKGROUND OF THE INVENTION
Trafficking of macromolecules between the nucleus and the cytoplasm occurs through nuclear pore complexes (NPCs), large proteinaceous structures (>50 different proteins) that perforate the nuclear envelope. Many small molecules (<40 kDa) can diffuse through NPCs, but large molecules must be transported across NPCs via carrier-mediated and signal-dependent processes. Much of the import and export of molecules across NPCs involves the interaction of transport receptors with their cargoes, the RanGTPase, and components of the NPC (1, 2).
Transport receptors, termed importins and exportins (or karyopherins), bind their appropriate cargoes directly or via specialized adaptor proteins (3). Once these complexes have formed, movement through the NPCs proceeds by a process involving sequential interactions of the receptor-cargo complexes with docking sites on the nuclear pore proteins (nucleoporins). A number of nucleoporins, particularly those containing phenylalanine-glycine (FG) repeat motifs, have been shown to interact directly with transport receptors (4). RanGTPase, which binds to transport receptors, plays a critical role in transport by promoting the association of cargo with export receptors as well as the dissociation of cargo from import receptors. Hydrolysis of RanGTP in the cytoplasm and regeneration of RanGTP in the nucleus sustains a gradient of RanGTP across the nuclear envelope, resulting in delivery of the transport cargoes to the appropriate cell compartments (5, 6).
Carrier-mediated movement across NPCs can be blocked in a variety of ways. Inactivation of RanGTPase leads to a block of most nucleocytoplasmic transport (7). Also, interference with the interactions between receptor-cargo complexes and nucleoporins inhibits nuclear transport. The lectin wheat germ agglutinin, which binds to O-glycosylated nucleoporins, blocks both import and export across NPCs (8), and antibodies to Nup98 or Nup153, two FG repeat-containing components of the NPC, block the export of small nuclear RNAs (snRNAs) and mRNA (9, 10). Likewise, the isolated nucleoporin binding domains of the transport factors importin &bgr; and TAP inhibit the export of mRNA and snRNAs (11, 12). This domain of importin &bgr; is also an efficient inhibitor of protein import.
Infection of eukaryotic cells by viruses can affect the nucleocytoplasmic transport of host-cell proteins and RNAs (13-15). Previously, we and others have demonstrated that the matrix (M) protein of vesicular stomatitis virus (VSV) is a potent inhibitor of nuclear transport (15, 16, and U.S. Pat. No. 5,888,727). M protein, a structural component of VSV virions, blocks the nuclear export of snRNAs and spliced mRNAs as well as the nuclear import of small nuclear ribonucleoproteins (snRNPs) (15 and U.S. Pat. No. 5,888,727). However, it was not known whether a fragment of the VSV M protein could retain its nucleocytoplasmic transport inhibition activity. It was not known either whether M proteins of other viral species had similar nucleocytoplasmic transport inhibition activities.
BRIEF SUMMARY OF THE INVENTION
In one embodiment, the invention is summarized in that a fragment of the VSV M protein and M proteins of other viral species have been demonstrated to have nucleocytoplasmic transport inhibition activity. A methionine conserved in the VSV M protein and M proteins of other viral species mentioned above has been shown to be important for transport inhibition activity of these proteins. Fragments of VSV M proteins that can enter into the nucleus of a cell have also been identified.
In one aspect, the present invention is an isolated M protein-like polypeptide having an amino acid sequence that is at least 26% similar to amino acids 47-229 of SEQ ID NO:1 (VSV M protein amino acid sequence), wherein the amino acid sequence contains methionine at a position corresponding to position 51 of SEQ ID NO:1.
In another aspect, the present invention is an isolated M protein gene-like polynucleotide having a nucleotide sequence that encodes a polypeptide at least 26% similar to amino acids 47-229 of SEQ ID NO:1, wherein the amino acid sequence contains methionine at a position corresponding to position 51 of SEQ ID NO:1. The polypeptide-encoding nucleotide sequence can be operably linked to a promoter to control the expression of the polypeptide.
In another aspect, the present invention is a vector or a host cell that contains the M protein gene-like polynucleotide described above.
In another aspect, the present invention is a method for inhibiting transport of RNA, proteins or RNA-protein complexes between nucleus and cytoplasm of a cell. The method involves exposing the cell to sufficient quantity of a VSV M protein-like polypeptide having an amino acid sequence that is at least 26% similar to amino acids 47-229 of SEQ ID NO:1, wherein the amino acid sequence contains methionine at a position corresponding to position 51 of SEQ ID NO:1.
In another aspect, the present invention is a method of inhibiting import of proteins or RNA-protein complexes from cytoplasm into nucleus of a cell. The method involves exposing the cell to sufficient quantity of a VSV M protein-like polypeptide having an amino acid sequence that is at least 26% similar to amino acids 47-229 of SEQ ID NO:1, wherein the amino acid sequence contains methionine at a position corresponding to position 51 of SEQ ID NO:1.
In another aspect, the present invention is a method of inhibiting export of nucleic acids from nucleus to cytoplasm of a cell. The method involves exposing the cell to sufficient quantity of a VSV M protein-like polypeptide having an amino acid sequence that is at least 26% similar to amino acids 47-229 of SEQ ID NO:1, wherein the amino acid sequence contains methionine at a position corresponding to position 51 of SEQ ID NO:1.
In a related aspect, the present invention is a method for inhibiting transport of RNA, proteins or RNA-protein complexes between nucleus and cytoplasm of a cell. The method involves first analyzing an amino acid sequence that is at least 26% similar to amino acids 47-229 of SEQ ID NO:1 to identify a smaller fragment that retains the ability to inhibit nucleocytoplasmic transport, wherein sized fragments of the amino acid sequence are compared to determine which segments of the amino acid sequence can be deleted without loss of transport inhibition function. The next step of the method involves exposing a cell to sufficient quantity of a VSV M protein-like polypeptide which contains the smaller fragment such that transport of RNA, proteins or RNA-protein complexes across the nuclear envelope is inhibited.
In another related aspect, the present invention is a method for screening for an agent that can alter the activity of an M protein. The first step of the method involves introducing into the nucleus of a cell a VSV M protein-like polypeptide having an amino acid sequence that is at least 26% similar to amino acids 47-229 of SEQ ID NO:1, wherein the amino acid sequence contains methionine at a position corresponding to position 51 of SEQ ID NO:1 and wherein the polypeptide inhibits the nucleocytoplasmic transport of the cell. The method next involves exposing the cell to a test agent and determining the nucleocytoplasmic transport rate of a molecule before and after exposing the cell to the agent, wherein the molecule is selected from a RNA, a protein and a RNA-protein complex.
In another aspect, the present invention is a method for identifying a nuclear import element. The first step of the method involves exposing a cell to sufficient quantity of an M protein-like polypeptide which comprises an amino acid sequence that is at least 26% similar to amino acids 47-229 of SEQ ID NO:1, wherein the amino acid sequence contains methionine at a position corresponding to position 51 of SEQ ID NO:1, so that import of proteins from cytoplasm into the nucleus is inhibited. The method next involves selecting a protein molecule that is imported in the presence of the M protein-like polypeptide and examining the imported mo
Dahlberg James E.
Glodowski Doreen R.
Her Lu-Shiun
Petersen Jeannine M.
Park Hankyel T.
Quarles & Brady LLP
Wisconsin Alumni Research Foundation
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