Purified retroviral constitutive transport enhancer elements that enhance nucleocytoplasmic transport of mRNA, and methods of making and using the elements
Abstract
A novel retroviral nucleotide sequence comprising a constitutive transport enhancer which functions to transport mRNA transcripts from the nucleus to the cytoplasm of a cell, wherein the mRNA transcript is either differentially spliced, alternatively spliced, incompletely spliced, or unspliced. Also disclosed is a recombinant attenuated HIV containing only structural proteins gag, pol, and env, wherein the expression of the structural proteins is rev-independent and facilitated by the constitutive transport enhancer.
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Inventors:
Assignee:
Appl.No.:
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Hammarskjold; Marie-Louise (Earlysville, VA); Rekosh; David (Earlysville, VA); Bray; Molly (Charlottesville, VA); Hunter; Eric (Birmingham, AL)
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| Current U.S. Class: |
435/236; 424/207.1; 424/208.1; 536/24.1 |
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Intern'l Class: |
C12N 007/04; C12N 015/00; C07H 021/04; A61K 039/21 |
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Field of Search: |
435/5,7.21,172,236,172.3 536/24.1 424/207.1,208.1 |
References Cited [Referenced By]
Other References
Bray et al.,"A Small Element from the Mason-Pfizer Monkey Virus Genome Makes Human Immunodeficiency Virus Type 1 Expression and Replication Rev-Independent", Proc. Natl. Acad. Sci. USA, vol. 91, Feb. 1994, pp. 1256-1260.
Osborne and Silver, 1993, "Nucleocytoplasmic transport in the yeast Saccharomyces cerevisiae", Annu. Rev. Biochem. 62:219-254.
Kadowaki et al., 1992, "A conditional yeast mutant deficient in mRNA transport from nucleus to cytoplasm", Proc, Natl. Acad. Sci. USA 89:2312-2316.
Del Pozzo et al., 1994, "Control of nucleo-cytoplasmic HLA-DRA mRNA partitioning by interaction of a retention signal with compartmentalized proteins", J. Mol. Biol. 240:193-204.
Alonso-Caplen et al., 1992, "Nucleocytoplasmic transport: the influenza virus NS1 protein regulates the transport of spliced NS2 mRNA and its precursor NS1 mRNA", Genes Devel. 6:255-267.
Latchman, D., 1990, "Cell-type-specific splicing factors and the regulation of alternative RNA splicing", New Biol. 2:297-303.
Gomeza et al., 1994, "Cellular distribution and regulation by cAMP of the GABA transporter (GAT-1) mRNA", Molec. Brain Res. 21:150-156.
Primary Examiner:Budens; Robert D..
Assistant Examiner: Parkin; Jeffrey S.
Attorney, Agent or Firm: Hodgson, Russ, Andrews, Woods & Goodyear
Goverment Interests
This invention was made with government support under grant numbers AI-25721 and AI-25784, awarded by the National Cooperative Drug Discovery Group; and grant numbers AI-27290, AI-30399, and AI-33319, awarded by the National Institutes of Allergy and Infectious Disease. The government has certain rights in this invention.
Claims
1. A purified sub-genomic nucleic acid molecule encoding a cis-acting retroviral, mRNA constitutive transport enhancer element comprising the nucleotide sequence of SEQ ID NO:2, or an analogous nucleic acid molecule obtained from simian retrovirus type 1 (SRV-1) or simian retrovirus type 2 (SRV-2), wherein said nucleic acid molecule enhances the nuclear to cytoplasmic transport of a heterologous m-RNA transcript when present in said transcript.
2. The nucleic acid molecule of claim 1, wherein said heterologous mRNA transcript is a rev-dependent HIV transcript containing RNA sequences for at least one gene selected from the group consisting of env, gag, pol, vif, vpr, and vpu, and said nuclear to cytoplasmic transport occurs in the absence of Rev expression.
3. A recombinant, attenuated, rev-deficient proviral human immunodeficiency virus clone containing the cis-acting retroviral constitutive transport enhancer element according to claim 1, wherein said transport enhancer element is present in a correct orientation and transcribed in a cell as part of a mRNA transcript of one or more HIV genes, wherein the genes are selected from the group consisting of env, gag, and pol, and wherein said attenuation results in impaired viral replication.
4. The recombinant, attenuated, rev-deficient proviral human immunodeficiency virus clone of claim 3, wherein the constitutive transport enhancer element is inserted into the nef coding region of the infectious proviral clone.
5. A purified and isolated nucleotide sequence comprising a constitutive transport enhancer wherein the nucleotide sequence consists of SEQ ID NO:2.