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211 In Vitro Inhibition of Human Immunodeficiency Virus Type 1 ( HIV-1 ) by FOY and Glutathione Reductase
R. Ansovini*1, N. Vanden2, and M. Q. Arens2
1Perugia, Italy and 2Washington Univ. Med. Sch., St. Louis, MO, USA
Background: The optimal yield of correctly folded protein within the endoplasmic reticulum is dependent on the ratio of reduced glutathione (GSH) to oxidized glutathione disulfide (GSSG). Drugs that affect the ratio of GSH to GSSG may affect the efficient folding of proteins made within that compartment which may ultimately have significant impacts on all protein process of the compartment. The ratio of GSH to GSSG would also affect the proper folding of "foreign" proteins made within a cellular compartment, such as proteins from a viral infection. Manipulation of this ratio may adversely affect viral protein folding and thus production of infectious virions. The experiments described herein with HIV-1 are a first step toward investigation of this possibility.
Methods: A synthetic trypsin inhibitor (Camostat, FOY-305) and the enzyme glutathione-reductase (GSSG-red) were tested alone and in combination for their ability to inhibit the growth of HIV-1 in a phenotypic assay employing a recombinant cell (MAGI-CCR5) assay system. In this assay, a beta-galactosidase gene in the recombinant cells is activated in the presence of HIV-1 tat and the resulting blue focus units (BFU) in the cell sheet identify cells infected with HIV: The test virus isolate was HIV-1 resistant to Indinavir (IDV-r genotype M46L/L63M/V82T/184).
Results: FOY, when used individually, had no affect on HIV- IDV-r in our 2-step protease inhibitor assay. GSSG-red alone did not have a significant and reproducible affect. The combination of FOY*GSSG-red inhibited BFU formation by up 56% when both were present at a concentration of 0.01 mum and 48(94% when both were present at 0.05 mum. At the same time, we used a semiquantitative PCR assay to look at the presence of HIV proviral DNA in cells infected with a Nevirapine-resistant strain of virus, NVP-r. The assay employs a nested set of primers to detect the pol gene and produces a product of 129 bp in the second round. The indications from this experiment are that the drug combination (at 0.05 mum) inhibited proviral DNA synthesis to some extent compared with synthesis in cells treated only with Efavirenz or with Efavirenz plus the FOY and GSSG-r combination at 0.01 mum.
Conclusions: The individual drugs, FOY or GSSG-red, when tested alone did not consistently inhibit HIV BFU formation in our phenotypic assay system. The combination of 2 drugs inhibited BFU formation by up to 10-fold and proviral DNA synthesis at 0.05 mum. The data indicate that the drug combination is able to significantly stop the HIV life cycle in the MAGI-CCR5 assay by drug resistant viruses HIV-1 IDV-r.
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