RESUMO
Human retroviruses, including human T cell leukemia virus type 1 (HTLV-1) and HIV type 1 (HIV-1), encode an antisense gene in the negative strand of the provirus. Besides coding for proteins, the messenger RNAs (mRNAs) of retroviral antisense genes have also been found to regulate transcription directly. Thus, it has been proposed that retroviruses likely localize their antisense mRNAs to the nucleus in order to regulate nuclear events; however, this opposes the coding function of retroviral antisense mRNAs that requires a cytoplasmic localization for protein translation. Here, we provide direct evidence that retroviral antisense mRNAs are localized predominantly in the nuclei of infected cells. The retroviral 3' LTR induces inefficient polyadenylation and nuclear retention of antisense mRNA. We further reveal that retroviral antisense RNAs retained in the nucleus associate with chromatin and have transcriptional regulatory function. While HTLV-1 antisense mRNA is recruited to the promoter of C-C chemokine receptor type 4 (CCR4) and enhances transcription from it to support the proliferation of HTLV-1-infected cells, HIV-1 antisense mRNA is recruited to the viral LTR and inhibits sense mRNA expression to maintain the latency of HIV-1 infection. In summary, retroviral antisense mRNAs are retained in nucleus, act like long noncoding RNAs instead of mRNAs, and contribute to viral persistence.
Assuntos
HIV-1/genética , Vírus Linfotrópico T Tipo 1 Humano/genética , Latência Viral/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Expressão Gênica/genética , Regulação Viral da Expressão Gênica/genética , Proteínas do Vírus da Imunodeficiência Humana/genética , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Humanos , Cultura Primária de Células , Regiões Promotoras Genéticas/genética , Provírus/genética , RNA Antissenso/genética , RNA Antissenso/metabolismo , RNA Mensageiro/metabolismo , RNA Viral/genética , Proteínas dos Retroviridae/genética , Proteínas dos Retroviridae/metabolismo , Sequências Repetidas Terminais/genética , Transcrição Gênica/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Proteínas Virais/metabolismo , Replicação Viral/genéticaRESUMO
Viruses causing chronic infection artfully manipulate infected cells to enable viral persistence in vivo under the pressure of immunity. Human T-cell leukemia virus type 1 (HTLV-1) establishes persistent infection mainly in CD4+ T cells in vivo and induces leukemia in this subset. HTLV-1-encoded Tax is a critical transactivator of viral replication and a potent oncoprotein, but its significance in pathogenesis remains obscure due to its very low level of expression in vivo. Here, we show that Tax is expressed in a minor fraction of leukemic cells at any given time, and importantly, its expression spontaneously switches between on and off states. Live cell imaging revealed that the average duration of one episode of Tax expression is â¼19 hours. Knockdown of Tax rapidly induced apoptosis in most cells, indicating that Tax is critical for maintaining the population, even if its short-term expression is limited to a small subpopulation. Single-cell analysis and computational simulation suggest that transient Tax expression triggers antiapoptotic machinery, and this effect continues even after Tax expression is diminished; this activation of the antiapoptotic machinery is the critical event for maintaining the population. In addition, Tax is induced by various cytotoxic stresses and also promotes HTLV-1 replication. Thus, it seems that Tax protects infected cells from apoptosis and increases the chance of viral transmission at a critical moment. Keeping the expression of Tax minimal but inducible on demand is, therefore, a fundamental strategy of HTLV-1 to promote persistent infection and leukemogenesis.
Assuntos
Regulação Viral da Expressão Gênica , Produtos do Gene tax/metabolismo , Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/patogenicidade , Leucemia-Linfoma de Células T do Adulto/virologia , Linfócitos T/virologia , Replicação Viral , Produtos do Gene tax/genética , Infecções por HTLV-I/genética , Infecções por HTLV-I/metabolismo , Humanos , Leucemia-Linfoma de Células T do Adulto/genética , Leucemia-Linfoma de Células T do Adulto/metabolismo , Análise de Célula Única , Ativação ViralRESUMO
[This corrects the article DOI: 10.1371/journal.ppat.1006120.].
RESUMO
Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia-lymphoma (ATL) and inflammatory diseases. To enhance cell-to-cell transmission of HTLV-1, the virus increases the number of infected cells in vivo. HTLV-1 bZIP factor (HBZ) is constitutively expressed in HTLV-1 infected cells and ATL cells and promotes T-cell proliferation. However, the detailed mechanism by which it does so remains unknown. Here, we show that HBZ enhances the proliferation of expressing T cells after stimulation via the T-cell receptor. HBZ promotes this proliferation by influencing the expression and function of multiple co-inhibitory receptors. HBZ suppresses the expression of BTLA and LAIR-1 in HBZ expressing T cells and ATL cells. Expression of T cell immunoglobulin and ITIM domain (TIGIT) and Programmed cell death 1 (PD-1) was enhanced, but their suppressive effect on T-cell proliferation was functionally impaired. HBZ inhibits the co-localization of SHP-2 and PD-1 in T cells, thereby leading to impaired inhibition of T-cell proliferation and suppressed dephosphorylation of ZAP-70 and CD3ζ. HBZ does this by interacting with THEMIS, which associates with Grb2 and SHP-2. Thus, HBZ interacts with the SHP containing complex, impedes the suppressive signal from PD-1 and TIGIT, and enhances the proliferation of T cells. Although HBZ was present in both the nucleus and the cytoplasm of T cells, HBZ was localized largely in the nucleus by suppressed expression of THEMIS by shRNA. This indicates that THEMIS is responsible for cytoplasmic localization of HBZ in T cells. Since THEMIS is expressed only in T-lineage cells, HBZ mediated inhibition of the suppressive effects of co-inhibitory receptors accounts for how HTLV-1 induces proliferation only of T cells in vivo. This study reveals that HBZ targets co-inhibitory receptors to cause the proliferation of infected cells.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Proliferação de Células/fisiologia , Infecções por HTLV-I/transmissão , Vírus Linfotrópico T Tipo 1 Humano/patogenicidade , Proteínas/metabolismo , Proteínas dos Retroviridae/metabolismo , Linfócitos T/imunologia , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Complexo CD3/metabolismo , Linhagem Celular Tumoral , Encefalomielite Autoimune Experimental/virologia , Proteína Adaptadora GRB2/metabolismo , Infecções por HTLV-I/virologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Células Jurkat , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptor de Morte Celular Programada 1/biossíntese , Receptor de Morte Celular Programada 1/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Receptores Imunológicos/biossíntese , Receptores Imunológicos/metabolismo , Proteínas dos Retroviridae/genética , Proteína-Tirosina Quinase ZAP-70/metabolismoRESUMO
BACKGROUND: HIV-1 typically develops resistance to any single antiretroviral agent. Combined anti-retroviral therapy to reduce drug-resistance development is necessary to control HIV-1 infection. Here, to assess the utility of a combination of antibody and fusion inhibitor treatments, we investigated the potency of monoclonal antibodies at neutralizing HIV-1 variants that are resistant to fusion inhibitors. RESULTS: Mutations that confer resistance to four fusion inhibitors, enfuvirtide, C34, SC34, and SC34EK, were introduced into the envelope of HIV-1JR-FL, a CCR5-tropic tier 2 strain. Pseudoviruses with these mutations were prepared and used for the assessment of neutralization sensitivity to an array of antibodies. The resulting neutralization data indicate that the potencies of some antibodies, especially of those against the CD4 binding site, V3 loop, and membrane-proximal external region epitopes, were increased by the mutations in gp41 that conferred resistance to the fusion inhibitors. C34-, SC34-, and SC34EK-resistant mutants showed more sensitivity to monoclonal antibodies than enfuvirtide-resistant mutants. An analysis of C34-resistant mutations revealed that the I37K mutation in gp41 HR1 is a key mutation for C34 resistance, low infectivity, neutralization sensitivity, epitope exposure, and slow fusion kinetics. The N126K mutation in the gp41 HR2 domain contributed to C34 resistance and neutralization sensitivity to anti-CD4 binding site antibodies. In the absence of L204I, the effect of N126K was antagonistic to that of I37K. The results of a molecular dynamic simulation of the envelope trimer confirmation suggest that an I37K mutation induces the augmentation of structural fluctuations prominently in the interface between gp41 and gp120. Our observations indicate that the "conformational unmasking" of envelope glycoprotein by an I37K mutation is one of the mechanisms of neutralization sensitivity enhancement. Furthermore, the enhanced neutralization of C34-resistant mutants in vivo was shown by its high rate of neutralization by IgG from HIV patient samples. CONCLUSIONS: Mutations in gp41 that confer fusion inhibitor resistance exert enhanced sensitivity to broad neutralizing antibodies (e.g., VRC01 and 10E8) and other conventional antibodies developed in HIV-1 infected patients. Therefore, next-generation fusion inhibitors and monoclonal antibodies could be a potential combination for future regimens of combined antiretroviral therapy.
RESUMO
3,4-Dihydro-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-imine (PD 404182) and 3,4-dihydro-2H-benzo[4,5]isothiazolo[2,3-a]pyrimidine are the heterocyclic antiretroviral agents against human immunodeficiency virus type 1 (HIV-1) infection. On the basis of similar structure-activity relationships of anti-HIV activities toward the early-stage of viral infection between these unique scaffolds, the transformations under the bioassay conditions were investigated. The distinctive S-N bond in the isothiazolopyrimidine scaffold was immediately cleaved under reductive conditions in the presence of GSH to generate a thiophenol derivative. A similar rapid conversion of PD 404182 into the same thiophenol derivative was observed, suggesting that pyrimidobenzothiazine and isothiazolopyrimidine scaffolds may work as prodrug forms of the common bioactive thiophenol derivatives.
Assuntos
Fármacos Anti-HIV/química , Infecções por HIV/tratamento farmacológico , Pró-Fármacos/química , Pirimidinas/química , Benzotiadiazinas/química , Química Farmacêutica , Desenho de Fármacos , Glutationa/química , HIV-1/efeitos dos fármacos , Humanos , Iminas/química , Espectroscopia de Ressonância Magnética , Nitrogênio/química , Relação Estrutura-Atividade , Enxofre/química , Tiazinas/química , Tiazóis/químicaRESUMO
3,4-Dihydro-2H-benzo[4,5]isothiazolo[2,3-a]pyrimidine is a newly identified antiviral agent against human immunodeficiency virus type 1 (HIV-1) infection, derived from 3,4-dihydro-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-imine (PD 404182). The introduction of the hydrophobic 8-aryl substituent on the benzene substructure improved its anti-HIV activity, resulting in the identification of 6-fold more potent analogs. In addition, it was demonstrated that these isothiazolopyrimidine derivatives exert anti-HIV effects at an early stage of viral infection.
Assuntos
Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , Pirimidinas/química , Pirimidinas/farmacologia , Humanos , Iminas/química , Iminas/farmacologia , Relação Estrutura-Atividade , Tiazinas/química , Tiazinas/farmacologiaRESUMO
Simian retrovirus type 4 (SRV-4), a simian type D retrovirus, naturally infects cynomolgus monkeys, usually without apparent symptoms. However, some infected monkeys presented with an immunosuppressive syndrome resembling that induced by simian immunodeficiency virus infection. Antiretrovirals with inhibitory activity against SRV-4 are considered to be promising agents to combat SRV-4 infection. However, although some antiretrovirals have been reported to have inhibitory activity against SRV-1 and SRV-2, inhibitors with anti-SRV-4 activity have not yet been studied. In this study, we identified antiretroviral agents with anti-SRV-4 activity from a panel of anti-human immunodeficiency virus (HIV) drugs using a robust in vitro luciferase reporter assay. Among these, two HIV reverse transcriptase inhibitors, zidovudine (AZT) and tenofovir disoproxil fumarate (TDF), potently inhibited SRV-4 infection within a submicromolar to nanomolar range, which was similar to or higher than the activities against HIV-1, Moloney murine leukemia virus, and feline immunodeficiency virus. In contrast, nonnucleoside reverse transcriptase inhibitors and protease inhibitors did not exhibit any activities against SRV-4. Although both AZT and TDF effectively inhibited cell-free SRV-4 transmission, they exhibited only partial inhibitory activities against cell-to-cell transmission. Importantly, one HIV integrase strand transfer inhibitor, raltegravir (RAL), potently inhibited single-round infection as well as cell-free and cell-to-cell SRV-4 transmission. These findings indicate that viral expansion routes impact the inhibitory activity of antiretrovirals against SRV-4, while only RAL is effective in suppressing both the initial SRV-4 infection and subsequent SRV-4 replication.
Assuntos
Antirretrovirais/farmacologia , Retrovirus dos Símios/efeitos dos fármacos , Adenina/análogos & derivados , Adenina/farmacologia , Sequência de Aminoácidos , Animais , Genes Reporter , Integrases/genética , Luciferases/análise , Luciferases/genética , Testes de Sensibilidade Microbiana/métodos , Organofosfonatos/farmacologia , Inibidores de Proteases/farmacologia , DNA Polimerase Dirigida por RNA/genética , Inibidores da Transcriptase Reversa/farmacologia , Alinhamento de Sequência , Tenofovir , Zidovudina/farmacologiaRESUMO
T-20EK is a novel fusion inhibitor designed to have enhanced α-helicity over T-20 (enfuvirtide) through engineered electrostatic interactions between glutamic acid (E) and lysine (K) substitutions. T-20EK efficiently suppresses wild-type and T-20-resistant variants. Here, we selected T-20EK-resistant variants. A combination of L33S and N43K substitutions in gp41 were required for high resistance to T-20EK. While these substitutions also caused resistance to T-20, they did not cause cross-resistance to other known fusion inhibitors.
Assuntos
Farmacorresistência Viral , Proteína gp41 do Envelope de HIV/farmacologia , Inibidores da Fusão de HIV/farmacologia , HIV-1/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Linhagem Celular Tumoral , Enfuvirtida , Ácido Glutâmico/metabolismo , HIV-1/genética , Humanos , Lisina/genética , Lisina/metabolismo , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Eletricidade EstáticaRESUMO
The structure-activity relationship of phenylpyrazole derivative 1 was investigated for the development of novel anti-HIV agents. Initial efforts revealed that the diazenyl group can be replaced by an aminomethylene group. In addition, we synthesized various derivatives by the reductive amination of benzaldehydes with 5-aminopyrazoles and carried out parallel structural optimization on the benzyl group and the pyrazole ring. This optimization led to a six-fold more potent derivative 32j than the lead compound 1, and this derivative has a 3',4'-dichloro-(1,1'-biphenyl)-3-yl group.
Assuntos
Fármacos Anti-HIV/química , Derivados de Benzeno/síntese química , Pirazóis/síntese química , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/farmacologia , Derivados de Benzeno/química , Derivados de Benzeno/farmacologia , HIV/efeitos dos fármacos , Concentração Inibidora 50 , Pirazóis/química , Pirazóis/farmacologia , Relação Estrutura-AtividadeRESUMO
To investigate the mechanism of action of the potent antiviral compound PD 404182, three novel photoaffinity probes equipped with a biotin or alkyne indicator were designed and synthesized based on previous structure-activity relationship studies. These probes retained the potent anti-HIV activity of the original pyrimidobenzothiazine derivatives. In photoaffinity labeling studies using HIV-1-infected H9 cells (H9IIIB), eight potential proteins were observed to bind PD 404182.
Assuntos
Alcinos/química , Fármacos Anti-HIV/química , Biotina/química , HIV-1/efeitos dos fármacos , Marcadores de Fotoafinidade/química , Proteínas Virais/metabolismo , Alcinos/síntese química , Alcinos/farmacologia , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/farmacologia , Biotina/síntese química , Biotina/farmacologia , Linhagem Celular , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/metabolismo , Humanos , Marcadores de Fotoafinidade/síntese química , Marcadores de Fotoafinidade/farmacologia , Ligação ProteicaRESUMO
3,4-Dihydro-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-imine (PD 404182) is a virucidal heterocyclic compound active against various viruses, including HCV, HIV, and simian immunodeficiency virus. Using facile synthetic approaches that we developed for the synthesis of pyrimido[1,2-c][1,3]benzothiazin-6-imines and related tricyclic derivatives, the parallel structural optimizations of the central 1,3-thiazin-2-imine core, the benzene part, and the cyclic amidine part of PD 404182 were investigated. Replacement of the 6-6-6 pyrimido[1,2-c][1,3]benzothiazin-6-imine framework with 5-6-6 or 6-6-5 derivatives led to a significant loss of anti-HIV activity, and introduction of a hydrophobic group at the 9- or 10-positions improved the potency. In addition, we demonstrated that the PD 404182 derivative exerts anti-HIV effects at an early stage of viral infection.
Assuntos
Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , Iminas/química , Iminas/farmacologia , Tiazinas/química , Tiazinas/farmacologia , Fármacos Anti-HIV/síntese química , Infecções por HIV/tratamento farmacológico , Células HeLa , Humanos , Iminas/síntese química , Testes de Sensibilidade Microbiana , Pirimidinas/síntese química , Pirimidinas/química , Pirimidinas/farmacologia , Relação Estrutura-Atividade , Tiazinas/síntese químicaRESUMO
3,4-Dihydro-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-imine (PD 404182) is an antiretroviral agent with submicromolar inhibitory activity against human immunodeficiency virus-1 (HIV-1) and HIV-2 infection. In the current study, the structure-activity relationships of accessory groups at the 3- and 9-positions of pyrimido[1,2-c][1,3]benzothiazin-6-imine were investigated for the development of more potent anti-HIV agents. Several different derivatives containing a 9-aryl group were designed and synthesized using Suzuki-Miyaura cross-coupling and Ullmann coupling reactions. Modification of the m-methoxyphenyl or benzo[d][1,3]dioxol-5-yl group resulted in improved anti-HIV activity. In addition, the 2,4-diazaspiro[5.5]undec-2-ene-fused benzo[e][1,3]thiazine derivatives were designed and tested for their anti-HIV activities. The most potent 9-(benzo[d][1,3]dioxol-5-yl) derivative was two-threefold more effective against several strains of HIV-1 and HIV-2 than the parent compound, PD 404182.
Assuntos
Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , Iminas/química , Iminas/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Fármacos Anti-HIV/síntese química , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Iminas/síntese química , Testes de Sensibilidade Microbiana , Estrutura Molecular , Pirimidinas/síntese química , Relação Estrutura-AtividadeRESUMO
Human immunodeficiency virus (HIV) gp41 plays a key role in viral fusion; the N- and C-terminal heptad repeats (N-HR and C-HR) of gp41 form a stable 6-helical conformation for fusion. Therefore, HR-derived peptides, such as enfuvirtide (T-20), inhibit HIV-1 fusion by acting as decoys, and have been used for the treatment of HIV-1 infection. However, the efficacy of T-20 is attenuated by resistance mutations in gp41, including V38A and N43D. To suppress the resistant variants, we previously developed electrostatically constrained peptides, SC34 and SC34EK, and showed that both exhibited potent anti-HIV-1 activity against wild-type and T-20-resistant variants. In this study, to clarify the resistance mechanism to this next generation of fusion inhibitors, we selected variants with resistance to SC34 and SC34EK in vitro. The resistant variants had multiple mutations in gp41. All of these mutations individually caused less than 6-fold resistance to SC34 and SC34EK, indicating that there is a significant genetic barrier for high-level resistance. Cross-resistance to SC34 and SC34EK was reduced by a simple difference in the polarity of two intramolecular electrostatic pairs. Furthermore, the selected mutations enhanced the physicochemical interactions with N-HR variants and restored activities of the parental peptide, C34, even to resistant variants. These results demonstrate that our approach of designing gp41-binding inhibitors using electrostatic constraints and information derived from resistance studies produces inhibitors with enhanced activity, high genetic barrier, and distinct resistance profile from T-20 and other inhibitors. Hence, this is a promising approach for the design of future generation peptide fusion inhibitors.
Assuntos
Inibidores da Fusão de HIV/farmacologia , Sequência de Aminoácidos , Antivirais/química , Antivirais/farmacologia , Linhagem Celular , Desenho de Fármacos , Farmacorresistência Viral , Variação Genética , Humanos , Cinética , Dados de Sequência Molecular , Mutação , Peptídeos/química , Fenótipo , Homologia de Sequência de Aminoácidos , Eletricidade EstáticaRESUMO
Administration of convalescent plasma or neutralizing monoclonal antibodies (mAbs) is a potent therapeutic option for coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, SARS-CoV-2 variants with mutations in the spike protein have emerged in many countries. To evaluate the efficacy of neutralizing antibodies induced in convalescent patients against emerging variants, we isolate anti-spike mAbs from two convalescent COVID-19 patients infected with prototypic SARS-CoV-2 by single-cell sorting of immunoglobulin-G-positive (IgG+) memory B cells. Anti-spike antibody induction is robust in these patients, and five mAbs have potent neutralizing activities. The efficacy of most neutralizing mAbs and convalescent plasma samples is maintained against B.1.1.7 and mink cluster 5 variants but is significantly decreased against variants B.1.351 from South Africa and P.1 from Brazil. However, mAbs with a high affinity for the receptor-binding domain remain effective against these neutralization-resistant variants. Rapid spread of these variants significantly impacts antibody-based therapies and vaccine strategies against SARS-CoV-2.
Assuntos
Anticorpos Neutralizantes/imunologia , COVID-19/imunologia , COVID-19/terapia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Antivirais/imunologia , COVID-19/virologia , Linhagem Celular , Células HEK293 , Humanos , Imunização Passiva , Masculino , Mutação , Testes de Neutralização , Domínios Proteicos , Glicoproteína da Espícula de Coronavírus/genética , Soroterapia para COVID-19RESUMO
Integrase (IN), an essential enzyme of human immunodeficiency virus (HIV), is an attractive antiretroviral drug target. The antiviral activity and resistance profile in vitro of a novel IN inhibitor, elvitegravir (EVG) (also known as JTK-303/GS-9137), currently being developed for the treatment of HIV-1 infection are described. EVG blocked the integration of HIV-1 cDNA through the inhibition of DNA strand transfer. EVG inhibited the replication of HIV-1, including various subtypes and multiple-drug-resistant clinical isolates, and HIV-2 strains with a 50% effective concentration in the subnanomolar to nanomolar range. EVG-resistant variants were selected in two independent inductions, and a total of 8 amino acid substitutions in the catalytic core domain of IN were observed. Among the observed IN mutations, T66I and E92Q substitutions mainly contributed to EVG resistance. These two primary resistance mutations are located in the active site, and other secondary mutations identified are proximal to these primary mutations. The EVG-selected IN mutations, some of which represent novel IN inhibitor resistance mutations, conferred reduced susceptibility to other IN inhibitors, suggesting that a common mechanism is involved in resistance and potential cross-resistance. The replication capacity of EVG-resistant variants was significantly reduced relative to both wild-type virus and other IN inhibitor-resistant variants selected by L-870,810. EVG and L-870,810 both inhibited the replication of murine leukemia virus and simian immunodeficiency virus, suggesting that IN inhibitors bind to a conformationally conserved region of various retroviral IN enzymes and are an ideal drug for a range of retroviral infections.
Assuntos
Fármacos Anti-HIV/farmacologia , Farmacorresistência Viral/genética , HIV-1/efeitos dos fármacos , HIV-2/efeitos dos fármacos , Inibidores de Integrase/farmacologia , Quinolonas/farmacologia , Substituição de Aminoácidos/genética , Sítios de Ligação , Humanos , Vírus da Leucemia Murina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Replicação Viral/genéticaRESUMO
Cell-to-cell spread of HIV permits ongoing viral replication in the presence of antiretroviral therapy and is suggested to be a major contributor to sexual transmission by mucosal routes. Fusion inhibitors that prevent viral entry have been developed, but their clinical applications have been limited by weak antiviral activity, short half-life, and the low genetic barrier to development of resistance. We examined the inhibitory activities of a series of single-chain variable fragments (scFvs) targeting the V3 and CD4i epitopes against both cell-free and cell-to-cell HIV infection. We found that all anti-V3 scFvs, including two newly constructed scFvs, showed broad neutralization activity against a panel of subtype B viruses compared with the corresponding IgGs. All scFvs neutralized cell-free infection by HIV-1JR-FL WT and fusion inhibitor-resistant mutants. In addition, all anti-V3 scFvs and some CD4i scFvs significantly inhibited cell fusion, while their IgG counterparts did not. Furthermore, scFvs-fusion inhibitors combinations, such as C34 and SC34, showed synergistic inhibition of cell fusion by both HIV-1JR-FL WT and fusion inhibitor-resistant mutants. The most prominent combinational effect was observed for 916B2 CD4i scFv with SC34. The delayed fusion kinetics of fusion inhibitor-resistant mutants partly explain their synergistic inhibition by such combinations. Our data demonstrate the advantages of using scFvs over their parent IgGs for inhibiting both cell-free and cell-to-cell infection. High synergistic inhibition of cell fusion by using scFvs-fusion inhibitors combinations suggests the possibility of intensification therapy adding this combination to current anti-HIV treatment regimens.
RESUMO
The infection routes of HIV-1 can affect several viral properties, including dissemination, pathogenesis, and immune evasion. In this study, we evaluated the inhibitory activity of a wide variety of anti-HIV drugs, focusing on the impact that different infection pathways have on their efficacy. Compared to cell-free infection, inhibitory activities were reduced in cell-to-cell productive transmission for all drugs tested. We detected weak reporter-expressing target cells after cell-to-cell transmission in the presence of integrase strand transfer inhibitors (INSTIs). Further analysis revealed that this expression was mainly due to unintegrated circular HIV (cHIV) DNAs, consisting of 1-LTR and 2-LTR circles. When in vitro-constructed cHIV DNAs were introduced into cells, the production of infectious and intercellular transmittable virions was observed, suggesting that cHIV DNA could be a source of infectious virus. These results highlight some advantages of the cell-to-cell infection mode for viral expansion, particularly in the presence of anti-retroviral drugs.
Assuntos
Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Internalização do Vírus/efeitos dos fármacos , Liberação de Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Linhagem Celular , HumanosRESUMO
T-20 (enfuvirtide) resistance is caused by the N43D primary resistance mutation at its presumed binding site at the N-terminal heptad repeat (N-HR) of gp41, accompanied by the S138A secondary mutation at the C-terminal HR of gp41 (C-HR). We have discovered that modifying T-20 to include S138A (T-20S138A) allows it to efficiently block wild-type and T20-resistant viruses, by a mechanism that involves improved binding of T-20S138A to the N-HR that contains the N43D primary mutation. To determine how HIV-1 in turn escapes T-20S138A we used a dose escalation method to select T-20S138A-resistant HIV-1 starting with either wild-type (HIV-1WT) or T-20-resistant (HIV-1N43D/S138A) virus. We found that when starting with WT background, I37N and L44M emerged in the N-HR of gp41, and N126K in the C-HR. However, when starting with HIV-1N43D/S138A, L33S and I69L emerged in N-HR, and E137K in C-HR. T-20S138A-resistant recombinant HIV-1 showed cross-resistance to other T-20 derivatives, but not to C34 derivatives, suggesting that T-20S138A suppressed HIV-1 replication by a similar mechanism to T-20. Furthermore, E137K enhanced viral replication kinetics and restored binding affinity with N-HR containing N43D, indicating that it acts as a secondary, compensatory mutation. We therefore introduced E137K into T-20S138A (T-20E137K/S138A) and revealed that T-20E137K/S138A moderately suppressed replication of T-20S138A-resistant HIV-1. T-20E137K/S138A retained activity to HIV-1 without L33S, which seems to be a key mutation for T-20 derivatives. Our data demonstrate that secondary mutations can be consistently used for the design of peptide inhibitors that block replication of HIV resistant to fusion inhibitors.
Assuntos
Substituição de Aminoácidos , Fármacos Anti-HIV/farmacologia , Desenho de Fármacos , Farmacorresistência Viral/genética , Proteína gp41 do Envelope de HIV/farmacologia , HIV-1/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Sequência de Aminoácidos , Fármacos Anti-HIV/química , Farmacorresistência Viral/efeitos dos fármacos , Enfuvirtida , Células HEK293 , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/genética , HIV-1/genética , HIV-1/fisiologia , Humanos , Cinética , Dados de Sequência Molecular , Mutação , Fragmentos de Peptídeos/química , Replicação Viral/efeitos dos fármacosRESUMO
CXCR4 is a coreceptor of HIV-1 infection in host cells. Through a photocrosslinking study to identify receptors involved in internalization of oligoarginine cell-penetrating peptides (CPPs), we found that CXCR4 serves as a receptor that stimulates macropinocytic uptake of the arginine 12-mer peptide (R12) but not of the 8-mer. We also found that stimulating CXCR4 with its intrinsic ligands, stromal cell-derived factor 1α and HIV-1 envelope glycoprotein 120, induced macropinocytosis. R12 had activity to prevent viral infection for HIV-1(IIIB), a subtype of HIV-1 that uses CXCR4 as a coreceptor for entry into susceptible cells, whereas the addition of a macropinocytosis inhibitor, dimethylamiloride, resulted in enhancement of viral infection. The present study shows that CXCR4 triggers macropinocytosis, which may have implications for the cellular uptake of oligoarginine CPPs and internalization of HIV.