of the application) Retrovirus vectors have received considerable attention as gene delivery vehicles for potential use in human therapy of single gene disorders. Recombinant murine retroviruses have been developed as gene transfer vehicles to deliver functional gene products into cells that contain nonfunctional or deficient product. Recently, my laboratory isolated an infectious molecular clone of a serogroup 2 simian retrovirus (SRV; D2/RHE/OR/V1) found within Asian macaques. This retrovirus exhibits broad tropism within the nonhuman primate. Sequence analysis of this molecular clone has allowed us to define the genetic structure of the envelope (env) glycoprotein gene and to develop a structural model of simian retrovirus-cell interaction. The overall goal of Specific Aim 1 is to develop the simian retrovirus as a gene delivery vehicle. This vector will represent the first primate-directed retrovirus vector specifically designed as a potential gene delivery vehicle. We will assess the ability of the SRV gene transfer vehicle to infect and express transgenes in established nonhuman primate and human cell lines, and then utilize this vector as a gene delivery vehicle for the expression of reporter genes in nonhuman primates. Specifically, we will a) identify the simian retrovirus psi packaging signal, and b) construct SRV packaging cell lines and gene transfer vehicles and test the retroviral recombinants in established monkey and human cell lines, with ultimate in vivo testing in rhesus macaques. Nucleocytoplasmic transport of macromolecules, including nuclear export of RNA, is mediated by specific proteins that harbor explicit export signals. The SRV genome contains a cis-acting element (constitutive transport element; CTE) that binds to cellular protein(s), facilitating the nuclear export of unspliced SRV RNA. The intergenic region identified in the simian retrovirus, Mason-Pfizer monkey virus (MPMV), enables rev-independent human immunodeficiency virus-1 (HIV-1) replication, and can promote the nuclear export of unspliced HIV mRNA in the absence of rev and rev-responsive element (RRE). We therefore hypothesize that the intergenic spacer region between the env gene and 3'-long terminal repeat (LTR) contains a potential CTE that facilitates nuclear export of SRV RNA. The research described in Specific Aim 2 will help us identify important determinants of simian retrovinus trafficking that may provide further refinement in vector development. Specifically, we will a) define the precise sequence of the SRV CTE, using RNA mobility shift and RNase digestion/footprinting analyses, b) demonstrate the functional importance of the CTE sequence in nucleocytoplasmic export of SRV RNA, using site-directed mutagenesis, cell fractionation, and in situ hybridization procedures, and c) identify the gene(s) encoding cellular protein factor(s) involved in nucleocytoplasmic export of SRV RNA, using the yeast three-hybrid selection system.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21DK053462-02
Application #
2882808
Study Section
Special Emphasis Panel (ZDK1-GRB-7 (O1))
Program Officer
Mckeon, Catherine T
Project Start
1998-03-25
Project End
2001-02-28
Budget Start
1999-03-01
Budget End
2001-02-28
Support Year
2
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Oregon Regional Primate Research Center
Department
Type
DUNS #
City
Beaverton
State
OR
Country
United States
Zip Code
97006
Wen, Yi; Feng, Jing; Sachs, George (2013) Helicobacter pylori 5'ureB-sRNA, a cis-encoded antisense small RNA, negatively regulates ureAB expression by transcription termination. J Bacteriol 195:444-52
Marcus, Elizabeth A; Sachs, George; Wen, Yi et al. (2012) Role of the Helicobacter pylori sensor kinase ArsS in protein trafficking and acid acclimation. J Bacteriol 194:5545-51
Marcus, E A; Inatomi, N; Nagami, G T et al. (2012) The effects of varying acidity on Helicobacter pylori growth and the bactericidal efficacy of ampicillin. Aliment Pharmacol Ther 36:972-9
Sachs, George; Shin, Jai Moo; Vagin, Olga et al. (2007) The gastric H,K ATPase as a drug target: past, present, and future. J Clin Gastroenterol 41 Suppl 2:S226-42
Munson, Keith; Law, Richard J; Sachs, George (2007) Analysis of the gastric H,K ATPase for ion pathways and inhibitor binding sites. Biochemistry 46:5398-417
Wen, Yi; Feng, Jing; Scott, David R et al. (2007) The HP0165-HP0166 two-component system (ArsRS) regulates acid-induced expression of HP1186 alpha-carbonic anhydrase in Helicobacter pylori by activating the pH-dependent promoter. J Bacteriol 189:2426-34
Zer, Cindy; Sachs, George; Shin, Jai Moo (2007) Identification of genomic targets downstream of p38 mitogen-activated protein kinase pathway mediating tumor necrosis factor-alpha signaling. Physiol Genomics 31:343-51
Wen, Yi; Feng, Jing; Scott, David R et al. (2006) Involvement of the HP0165-HP0166 two-component system in expression of some acidic-pH-upregulated genes of Helicobacter pylori. J Bacteriol 188:1750-61
Sachs, G; Kraut, J A; Wen, Y et al. (2006) Urea transport in bacteria: acid acclimation by gastric Helicobacter spp. J Membr Biol 212:71-82
Shin, Jai Moo; Homerin, Michel; Domagala, Florence et al. (2006) Characterization of the inhibitory activity of tenatoprazole on the gastric H+,K+ -ATPase in vitro and in vivo. Biochem Pharmacol 71:837-49

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