ABSTRACT
Previous studies have shown that morphine stimulates simian immunodeficiency virus (SIV) replication in SIV-infected human CEM x174 cells as well as in monkey lymphocytes through a mechanism of delaying the lysis of infected cells (Biochem. Biophys. Res. Commun. 195:1165-1173, 1993). The present study describes the identification of brain-like opioid receptor sequences in RNA transcripts of both CEM x174 cells and monkey lymphocytes. Study of the gene sequence of a lymphocyte opioid receptor encompassing the third transmembrane domain and the third cytoplasmic loop indicates a 96% homology in amino acid composition to the delta opioid receptor in brain cells. Expression of such an opioid receptor sequence in lymphocytic cells is constitutive, since it could be detected in both saline-treated and morphine-treated monkeys as well as in morphine-treated monkeys after detoxification.
Subject(s)
Gene Expression , Lymphocytes/metabolism , Receptors, Opioid, delta/genetics , Amino Acid Sequence , Animals , Base Sequence , Blotting, Southern , Brain/metabolism , Cells, Cultured , DNA Primers , Humans , Macaca mulatta , Male , Molecular Sequence Data , Polymerase Chain Reaction , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Homology, Amino AcidABSTRACT
Infection with the macaque strain of the simian immunodeficiency virus (SIVmac) induces simian immunodeficiency syndrome in rhesus macaques. This report describes the isolation and identification of antigenic variants of SIVmac in one of the infected monkeys (macaque #22803). Eight naive rhesus monkeys were inoculated with a titered viral stock of the molecularly cloned SIVmac239. Standard serological analysis revealed that all but two were seroconverted. Western blot analysis confirmed the seronegativity of macaque #22803. In addition, sera recovered from this monkey were not able to neutralize the parent SIVmac239. However, virus could be isolated from all of the infected animals, including macaque #22803. Sera recovered were reactive to the autologous virus. The results suggest that the virus from macaque #22803 may have undergone extensive antigenic shift in vivo. To test this hypothesis, a portion of the gag gene was amplified by the polymerase chain reaction (PCR), cloned, and sequenced. Sequence analysis revealed amino acid changes that were clustered between amino acids 200-245. Evaluation of the possible selective pressures contributing to the observed viral mutation revealed that in comparison with the other SIVmac239-infected monkeys, macaque #22803 produced an unusually high T cell proliferative response toward mitogen stimulation before infection, and continued to display a persistently high plasma viremia titer after infection.
Subject(s)
Antigenic Variation , Antigens, Viral/biosynthesis , Gene Products, gag/biosynthesis , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Immunodeficiency Virus/immunology , Amino Acid Sequence , Animals , Antibodies, Viral/blood , Antigens, Viral/isolation & purification , Base Sequence , Blotting, Western , Cloning, Molecular , DNA Primers , Enzyme-Linked Immunosorbent Assay , Gene Products, gag/isolation & purification , Genes, gag , Lymphocyte Activation , Lymphocytes/immunology , Lymphocytes/virology , Macaca mulatta , Molecular Sequence Data , Neutralization Tests , Polymerase Chain Reaction , RNA, Viral/analysis , Simian Acquired Immunodeficiency Syndrome/blood , Simian Immunodeficiency Virus/genetics , Simian Immunodeficiency Virus/isolation & purification , T-Lymphocytes/immunology , Time FactorsSubject(s)
Antiviral Agents/pharmacology , Benzamides/pharmacology , Nitroso Compounds/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors , Retroviridae/drug effects , B-Lymphocytes/drug effects , Benzamides/toxicity , Cell Line , Cell Survival/drug effects , Coloring Agents , Humans , Nitroso Compounds/toxicity , RNA-Directed DNA Polymerase , Simian Immunodeficiency Virus/drug effects , T-Lymphocytes/drug effects , Tetrazolium Salts , Thiazoles , Virus Replication/drug effectsABSTRACT
The 3-nitrosobenzamide (NOBA) drug abolishes SIV replication sharply at 20 microM concentration when CEM x 174 cells are preincubated for 1 h with the drug prior to viral infection. Treatment of CEM x 174 cells with 20 microM NOBA resulted in the inhibition of the synthesis of the DNA sequence coding for the gag gene, as determined by the PCR technique. Cell viability was directly proportional to the antiviral action of NOBA. Replication of AZT-resistant SIV 23740 in MMU 23740 cells in vitro was suppressed by NOBA in a concentration-dependent manner without significant effects on cell viability. Reverse transcriptase activity of SIVmac239 was unaffected by NOBA up to 800 microM concentration. Preincubation of two SIV strains with NOBA completely abolished their infectivity in human PHA-PBL cells. Replication of two strains of SIV in PHA-PBL cells was also inhibited by NOBA.
