RESUMO
BACKGROUND: Protease (PR) inhibitors (PIs) were designed against subtype B virus of human immunodeficiency virus type 1 (HIV-1), but believed to retain its activity against most of the other subtypes. CRF01_AE PR (AE-PR) contains background mutations that are presumed to alter the drug susceptibility of PR. In addition, amino acid variations found in HIV-1 Gag potentially affect the drug susceptibility or catalytic efficiency of PR. METHODS: We studied the impact of naturally occurring amino acid substitutions found in AE-PR and CRF01_AE Gag (AE-Gag) on the drug susceptibility of PR to 9 currently available PIs, using the pNL4-3-derived luciferase reporter virus containing AE-Gag and/or AE-PR genes derived from drug treatment-naïve, HIV-1-infected Thai patients. RESULTS: Sequencing analysis revealed that several mutations were detected in deduced amino acid sequences of AE-PR and AE-Gag genes, as compared to these genes of pNL4-3. Drug susceptibility tests revealed that AE-PR showed a variety of susceptibilities to 9 PIs compared with pNL4-3 PR. In addition, AE-Gag significantly reduced the drug susceptibility of AE-PR and pNL4-3 PR. CONCLUSION: Our results suggest that amino acid variations in AE-PR and AE-Gag play roles in determining the drug susceptibility of CRF01_AE viruses to PIs.
Assuntos
Inibidores da Protease de HIV/farmacologia , Protease de HIV/química , Protease de HIV/genética , HIV-1/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Sequência de Aminoácidos , Farmacorresistência Viral/genética , Regulação Viral da Expressão Gênica/fisiologia , Infecções por HIV/epidemiologia , Infecções por HIV/virologia , HIV-1/genética , Humanos , Dados de Sequência Molecular , Mutação , Tailândia/epidemiologiaRESUMO
Human immunodeficiency virus type 1 (HIV-1) env genes were cloned from blood samples of HIV-1-infected Thai patients, and 35 infectious CRF01_AE envelope glycoprotein (Env)-recombinant viruses were established. In this report, we examined the neutralization susceptibility of these viruses to human monoclonal antibodies, 2G12, IgG1 b12, 2F5 and 4E10, pooled patient plasma, coreceptor antagonists and fusion inhibitor, T-20. The neutralization susceptibility of CRF01_AE Env-recombinant viruses to 2F5, 4E10, patient plasma, coreceptor antagonists and T-20 varied, while most viruses showed low susceptibility to 2G12 and IgG1 b12. Several dual-tropic viruses showed lower susceptibility to 2F5 and 4E10 than CXCR4- or CCR5-tropic viruses. Neutralization susceptibility of the CRF01_AE Env-recombinant virus to pooled patient plasma was negatively correlated with the length of the V1/V2 region or the number of potential N-linked glycosylation sites in conserved regions of gp120. No correlation was found between the coreceptor usage and neutralization susceptibility of the virus to T-20, whereas several dual-tropic viruses showed higher susceptibility to coreceptor antagonists than CXCR4- or CCR5-tropic viruses. We propose that these CRF01_AE Env-recombinant viruses are useful to further study the molecular mechanism of the susceptibility of CRF01_AE Env to neutralizing antibodies and viral entry inhibitors.
Assuntos
Infecções por HIV/virologia , HIV-1/crescimento & desenvolvimento , HIV-1/genética , Recombinação Genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Anticorpos Monoclonais/imunologia , Enfuvirtida , Anticorpos Anti-HIV/imunologia , Proteína gp41 do Envelope de HIV/farmacologia , Inibidores da Fusão de HIV/farmacologia , HIV-1/isolamento & purificação , Humanos , Testes de Neutralização , Fragmentos de Peptídeos/farmacologia , Receptores de HIV/antagonistas & inibidores , TailândiaRESUMO
CRF01_AE is a major subtype of human immunodeficiency virus type 1 (HIV-1) circulating in Southeast Asia, including Thailand. HIV-1 env genes were amplified by polymerase chain reaction from blood samples of HIV-1-infected patients residing in Thailand in 2006, and cloned into the pNL4-3-derived reporter viral construct. Generated envelope protein (Env)-recombinant virus was examined for its infectivity, and then 35 infectious CRF01_AE Env-recombinant viruses were selected. Sequencing analysis revealed that the interclone variation of the deduced amino acid sequences was higher in CRF01_AE env genes isolated in 2006 than in those isolated in the early 1990s, suggesting that env gene variation has been increasing gradually among CRF01_AE viruses prevalent in Thailand. We also examined the characteristics of the deduced amino acid sequences of 35 CRF01_AE env genes. Our results may provide useful information to help in better understanding the genotype of env genes of CRF01_AE viruses currently circulating in Thailand.
Assuntos
Infecções por HIV/virologia , HIV-1/genética , HIV-1/isolamento & purificação , Polimorfismo Genético , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Sequência de Aminoácidos , Sangue/virologia , Genótipo , HIV-1/classificação , Humanos , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase/métodos , Recombinação Genética , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , TailândiaRESUMO
We performed the screening to find the novel host factors affecting human immunodeficiency virus type-1 (HIV-1) replication using the siRNA mini-library consisted with 257 siRNAs directed against cellular genes. J111 cells, a human acute monocytic leukemia cell line, were transfected with individual siRNA, followed by either infected or transfected with the HIV-1 molecular clone with luciferase reporter gene in 96-well plate format. The results showed that six siRNAs significantly enhanced the HIV-1 replication in J111 cells, indicating that the target cellular genes of those siRNAs may negatively regulate HIV-1 replication in normal cell culture condition. We also discuss the possible mechanisms by which those cellular proteins regulate viral replication.
