RESUMO
OBJECTIVE: To compare in vitro replication kinetics and nucleoside analog susceptibilities of a natural feline immunodeficiency virus (FIV) isolate (FIV-Maxam), a molecular clone of FIV (FIV-pPPR), and two (-)-beta-L-2',3'-dideoxy-3'-thiacytidine- (3TC-) resistant mutants of FIV-pPPR. SAMPLE POPULATION: Peripheral blood mononuclear cells (PBMC) from 4 specific-pathogenfree cats. PROCEDURE: Two point mutations corresponding to mutations of human immunodeficiency virus type 1 (HIV-1) were engineered into the highly conserved YMDD motif of the reverse transcriptase- (RT-) encoding region of the FIV-pPPR pol gene. Replication kinetics and nucleoside analog susceptibilities of FIV-Maxam, FIV-pPPR, and the 2 mutant viruses were measured in vitro, using feline PBMC. RESULTS: Replication kinetics and nucleoside analog susceptibilities were similar between FIV-Maxam and FIV-pPPR. However, FIV-Maxam was significantly more susceptible to 3TC. A methionine-to-valine mutation at codon 183 (M183V) of the RT-encoding region of the pol gene of FIV-pPPR conferred high-level phenotypic resistance to 3TC and cross-resistance to the related compound (-)-beta-L-2',3'-dideoxy-5-fluoro-3'-thiacytidine. CONCLUSIONS AND CLINICAL RELEVANCE: Similarities between FIV-Maxam and FIV-pPPR suggest that results of studies performed using FIV-pPPR will have relevance to natural FIV infection in cats. In vitro evaluation of nucleoside analog susceptibilities of FIV-Maxam may help determine concentrations of nucleoside analogs required for effective treatment of FIV-infected cats. IMPACT FOR HUMAN MEDICINE: 3TC resistance of FIV-pPPR M183V was similar in magnitude to that of HIV-1 M184V, a mutant described in infected humans treated with 3TC. Thus, FIV-pPPR M183V may be a useful model for studying the in vivo effects of 3TC resistance on lentivirus pathogenesis.
Assuntos
Gatos , Genes pol/genética , Vírus da Imunodeficiência Felina/efeitos dos fármacos , Vírus da Imunodeficiência Felina/genética , Inibidores da Transcriptase Reversa/farmacologia , Animais , Antígenos Virais/análise , Antígenos Virais/biossíntese , Clonagem Molecular , DNA Viral/química , Resistência Microbiana a Medicamentos/genética , Síndrome de Imunodeficiência Adquirida Felina/tratamento farmacológico , Síndrome de Imunodeficiência Adquirida Felina/virologia , Feminino , Vírus da Imunodeficiência Felina/fisiologia , Cinética , Lamivudina/farmacologia , Masculino , Mutagênese Sítio-Dirigida , Mutação Puntual , Reação em Cadeia da Polimerase/veterinária , DNA Polimerase Dirigida por RNA/efeitos dos fármacos , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , Organismos Livres de Patógenos Específicos , Replicação Viral/fisiologiaRESUMO
High-affinity, high-specificity RNA ligands for reverse transcriptase from feline immunodeficiency virus (FIV) were isolated from an RNA library by the SELEX (Systematic Evolution of Ligands by EXponential enrichment) procedure. The selected RNA ligands bound to FIV reverse transcriptase with dissociation constants in the nanomolar range. One of the ligands was a potent inhibitor of the RNA-dependent DNA polymerase activity of both the recombinant and the virion-derived FIV reverse transcriptase. It also inhibited the reverse transcriptase from an FIV mutant that is resistant to 3'-azido-3'-deoxythymidine (AZT). The inhibition of FIV reverse transcriptase was competitive with respect to template-primer and noncompetitive with respect to deoxyribonucleoside 5'-triphosphates. This ligand was specific for the FIV enzyme and did not inhibit other reverse transcriptases tested (avian myeloblastosis virus, Moloney murine leukemia virus, and human immunodeficiency virus type 1).