RESUMO
Membrane fusion is the first essential step in HIV-1 replication. The gp41 subunit of HIV-1 envelope protein (Env), a class I fusion protein, achieves membrane fusion by forming a structure called a six-helix bundle composed of N- and C-terminal heptad repeats. We have recently shown that the distal portion of the α9 helix in the C-terminal heptad repeat of X4-tropic HXB2 Env plays a critical role in the late-stage membrane fusion and viral infection. Here, we used R5-tropic JRFL Env and constructed six alanine insertion mutants, 641+A to 646+A, in the further distal portion of α9 where several glutamine residues are conserved (the number corresponds to the position of the inserted alanine in JRFL Env). 644+A showed the most severe defect in syncytia formation. Decreased fusion pore formation activity, revealed by a dual split protein assay, was observed in all mutants except 641+A. Sequence analysis and substitution of inserted 644A with Gln revealed that the glutamine residue at position 644 that forms complex hydrogen-bond networks with other polar residues on the surface of the six-helix bundle is critical for cell-cell fusion. We also developed a split NanoLuc® (Nluc) reporter-based assay specific to the virus-cell membrane fusion step to analyze several of the mutants. Interestingly syncytia-competent mutants failed to display Nluc activities. In addition to defective fusion activity, a reduction of Env incorporation into virions may further contribute to differences in cell-cell and virus-cell fusions.
Assuntos
Bioensaio , Proteína gp41 do Envelope de HIV/metabolismo , HIV-1/metabolismo , Fusão de Membrana , Mutagênese Insercional , Internalização do Vírus , Alanina/genética , Alanina/metabolismo , Linhagem Celular , Proteína gp41 do Envelope de HIV/genética , HIV-1/genética , Humanos , Estrutura Secundária de ProteínaRESUMO
BACKGROUND: The native pre-fusion structure of gp120/gp41 complex of human immunodeficiency virus type 1 was recently revealed. In the model, the helices of gp41 (α6, α7, α8, and α9) form a four-helix collar underneath trimeric gp120. Gp41 is a class I fusion protein and mediates membrane fusion by forming a post-fusion structure called the six-helix bundle (6HB). The comparison of the pre- and post-fusion structures revealed the large conformational changes in gp41 during the antiparallel packing of the N- and C-terminal heptad repeats (NHRs and CHRs) in membrane fusion. Several mutagenesis studies of gp41 performed in the past were interpreted based on 6HB, the only available structure at that time. To obtain an insight about the current pre-fusion structural model and conformational changes during membrane fusion, alanine insertion mutagenesis of the NHR, CHR and connecting loop regions of HXB2 gp41 was performed. The effects of mutations on biosynthesis and membrane fusion were analyzed by immunoblotting and fusion assays, respectively. The extent of membrane fusion was evaluated by split luciferase-based pore formation and syncytia formation assays, respectively. RESULTS: Consistent with the current structural model, drastic negative effects of mutations on biosynthesis and membrane fusion were observed for NHR, loop, and proximal regions of CHR (up to amino acid position 643). The insertions in α9 after it leaves the four-helix collar were tolerable for biosynthesis. These CHR mutants showed varying effects on membrane fusion. Insertion at position 644 or 645 resulted in poor pore and syncytia formation. Efficient pore and syncytia formation almost similar to that of the wild type was observed for insertion at position 647, 648 or 649. However, recovery of virus infectivity was only observed for the insertions beyond position 648. CONCLUSIONS: The mutagenesis data for HXB2 gp41 is in agreement with the recent pre-fusion structure model. The virus infection data suggested that fusion pores sufficiently large enough for the release of the virus genome complex are formed after the completion of 6HB beyond position 648.
Assuntos
Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/metabolismo , Infecções por HIV/virologia , HIV-1/fisiologia , Domínios e Motivos de Interação entre Proteínas , Sequências Repetitivas de Ácido Nucleico , Alanina/genética , Sequência de Aminoácidos , Proteína gp41 do Envelope de HIV/genética , Humanos , Modelos Biológicos , Modelos Moleculares , Mutagênese Insercional , Biossíntese de Proteínas , Conformação Proteica , Internalização do Vírus , Replicação ViralRESUMO
The mature human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) comprises the non-covalently associated gp120 and gp41 subunits generated from the gp160 precursor. Recent structural analyses have provided quaternary structural models for gp120/gp41 trimers, including the variable loops (V1-V5) of gp120. In these models, the V3 loop is located under V1/V2 at the apical center of the Env trimer, and the V4 and V5 loops project outward from the trimeric protomers. In addition, the V4 and V5 loops are predicted to have less movement upon receptor binding during membrane fusion events. We performed insertional mutagenesis using a GFP variant, GFPOPT, placed into the variable loops of HXB2 gp120. This allowed us to evaluate the current structural models and to simultaneously generate a GFP-tagged HIV-1 Env, which was useful for image analyses. All GFP-inserted mutants showed similar levels of whole-cell expression, although certain mutants, particularly V3 mutants, showed lower levels of cell surface expression. Functional evaluation of their fusogenicities in cell-cell and virus-like particle-cell fusion assays revealed that V3 was the most sensitive to the insertion and that the V1/V2 loops were less sensitive than V3. The V4 and V5 loops were the most tolerant to insertion, and certain tag proteins other than GFPOPT could also be inserted without functional consequences. Our results support the current structural models and provide a GFPOPT-tagged Env construct for imaging studies.
Assuntos
Proteínas de Fluorescência Verde/genética , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/metabolismo , HIV/metabolismo , Sequência de Aminoácidos , Western Blotting , Ensaio de Imunoadsorção Enzimática , Técnica Indireta de Fluorescência para Anticorpo , Células HEK293 , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/genética , Humanos , Mutagênese InsercionalRESUMO
Middle East respiratory syndrome (MERS) is an emerging infectious disease associated with a relatively high mortality rate of approximately 40%. MERS is caused by MERS coronavirus (MERS-CoV) infection, and no specific drugs or vaccines are currently available to prevent MERS-CoV infection. MERS-CoV is an enveloped virus, and its envelope protein (S protein) mediates membrane fusion at the plasma membrane or endosomal membrane. Multiple proteolysis by host proteases, such as furin, transmembrane protease serine 2 (TMPRSS2), and cathepsins, causes the S protein to become fusion competent. TMPRSS2, which is localized to the plasma membrane, is a serine protease responsible for the proteolysis of S in the post-receptor-binding stage. Here, we developed a cell-based fusion assay for S in a TMPRSS2-dependent manner using cell lines expressing Renilla luciferase (RL)-based split reporter proteins. S was stably expressed in the effector cells, and the corresponding receptor for S, CD26, was stably coexpressed with TMPRSS2 in the target cells. Membrane fusion between these effector and target cells was quantitatively measured by determining the RL activity. The assay was optimized for a 384-well format, and nafamostat, a serine protease inhibitor, was identified as a potent inhibitor of S-mediated membrane fusion in a screening of about 1,000 drugs approved for use by the U.S. Food and Drug Administration. Nafamostat also blocked MERS-CoV infection in vitro Our assay has the potential to facilitate the discovery of new inhibitors of membrane fusion of MERS-CoV as well as other viruses that rely on the activity of TMPRSS2.
Assuntos
Membrana Celular/efeitos dos fármacos , Guanidinas/farmacologia , Membranas Intracelulares/efeitos dos fármacos , Fusão de Membrana/efeitos dos fármacos , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Inibidores de Proteínas Virais de Fusão/farmacologia , Benzamidinas , Catepsinas/genética , Catepsinas/metabolismo , Fusão Celular , Membrana Celular/metabolismo , Membrana Celular/virologia , Dipeptidil Peptidase 4/genética , Dipeptidil Peptidase 4/metabolismo , Furina/genética , Furina/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Células HEK293 , Ensaios de Triagem em Larga Escala , Interações Hospedeiro-Patógeno , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/virologia , Luciferases/genética , Luciferases/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/crescimento & desenvolvimento , Proteólise , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
Herpes simplex virus (HSV) entry and cell-cell fusion require the envelope proteins gD, gH/gL and gB. We propose that receptor-activated conformational changes to gD activate gH/gL, which then triggers gB (the fusogen) into an active form. To study this dynamic process, we have adapted a dual split protein assay originally developed to study the kinetics of human immunodeficiency virus (HIV) mediated fusion. This assay uses a chimera of split forms of renilla luciferase (RL) and green fluorescent protein (GFP). Effector cells are co-transfected with the glycoproteins and one of the split reporters. Receptor-bearing target cells are transfected with the second reporter. Co-culture results in fusion and restoration of RL, which can convert a membrane permeable substrate into a luminescent product, thereby enabling one to monitor initiation and extent of fusion in live cells in real time. Restoration of GFP can also be studied by fluorescence microscopy. Two sets of split reporters have been developed: the original one allows one to measure fusion kinetics over hours whereas the more recent version was designed to enhance the sensitivity of RL activity allowing one to monitor both initiation and rates of fusion in minutes. Here, we provide a detailed, step-by-step protocol for the optimization of the assay (which we call the SLA for split luciferase assay) using the HSV system. We also show several examples of the power of this assay to examine both the initiation and kinetics of cell-cell fusion by wild type forms of gD, gB, gH/gL of both serotypes of HSV as well as the effect of mutations and antibodies that alter the kinetics of fusion. The SLA can be applied to other viral systems that carry out membrane fusion.
Assuntos
Fusão Celular , Microscopia de Fluorescência/métodos , Proteínas Virais de Fusão/química , Células Cultivadas , Genes Reporter , Interações Hospedeiro-Patógeno , Cinética , Luciferases/análise , Modelos Biológicos , Mutação , Simplexvirus/fisiologia , Proteínas Virais de Fusão/análise , Proteínas Virais de Fusão/genéticaRESUMO
Herpes simplex virus (HSV) entry and cell-cell fusion require glycoproteins gD, gH/gL, and gB. We propose that receptor-activated changes to gD cause it to activate gH/gL, which then triggers gB into an active form. We employed a dual split-protein (DSP) assay to monitor the kinetics of HSV glycoprotein-induced cell-cell fusion. This assay measures content mixing between two cells, i.e., fusion, within the same cell population in real time (minutes to hours). Titration experiments suggest that both gD and gH/gL act in a catalytic fashion to trigger gB. In fact, fusion rates are governed by the amount of gB on the cell surface. We then used the DSP assay to focus on mutants in two functional regions (FRs) of gB, FR1 and FR3. FR1 contains the fusion loops (FL1 and FL2), and FR3 encompasses the crown at the trimer top. All FL mutants initiated fusion very slowly, if at all. However, the fusion rates caused by some FL2 mutants increased over time, so that total fusion by 8 h looked much like that of the WT. Two distinct kinetic patterns, "slow and fast," emerged for mutants in the crown of gB (FR3), again showing differences in initiation and ongoing fusion. Of note are the fusion kinetics of the gB syn mutant (LL871/872AA). Although this mutant was originally included as an ongoing high-rate-of-fusion control, its initiation of fusion is so rapid that it appears to be on a "hair trigger." Thus, the DSP assay affords a unique way to examine the dynamics of HSV glycoprotein-induced cell fusion.
Assuntos
Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Animais , Fusão Celular , Linhagem Celular Tumoral , Análise Mutacional de DNA , Camundongos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismoRESUMO
Recent rapid developments in Asian and African countries bring an opportunity of cross-species transmission of pathogens through unprecedented contacts between people and wild animals. Furthermore, increase of global exchanges of people and products facilitates a rapid spread of infectious diseases worldwide. China has an enormous population with diverse ethnic groups within its wide territory; furthermore, it is experiencing very rapid urbanization. These conditions make China a potential epicenter of emerging infectious diseases. One good example is the SARS incidence in 2003. Therefore, it is essential to include China in a network of research groups of infectious diseases. Here we summarize the ongoing collaborations between the Institute of Medical Science, the University of Tokyo, and its Chinese counterparts.
Assuntos
Doenças Transmissíveis , Comportamento Cooperativo , Pesquisa , Universidades , Animais , China , Controle de Doenças Transmissíveis , Humanos , Comunicação Interdisciplinar , TóquioRESUMO
To help understand the dynamic nature of membrane fusion induced by the human immunodeficiency virus-1 (HIV-1) envelope protein, we developed a new cell-based real-time assay system employing a pair of novel reporter proteins. The reporter proteins consist of a pair of split Renilla luciferase (spRL) fused to split green fluorescent protein (spGFP). The spGFP modules were chosen not only to compensate weak self-association of spRL but also to provide visual reporter signals during membrane fusion. Use of this reporter together with a membrane permeable substrate for Renilla luciferase achieved a simple real-time monitoring of membrane fusion using live cells. We analyzed the HIV-1 envelope mutants whose membrane-spanning domains were replaced with that of glycophorin A or vesicular stomatitis virus G-protein. These mutants showed a slower kinetics of membrane fusion. The analysis of membrane fusion in the presence of fusion inhibitors, soluble CD4 and C34, revealed that these replacements prolonged the period during which the mutants were sensitive to the inhibitors, as compared with the wild type. These results suggest that the mutations within the membrane-spanning domains exerted an allosteric effect on the HIV-1 envelope protein, probably affecting the receptor-induced conformational changes of the ectodomain of the protein.
Assuntos
Proteína gp120 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/química , Fusão de Membrana , Proteínas Recombinantes de Fusão/química , Complexo CD3/metabolismo , Antígenos CD4/metabolismo , Glicoforinas/genética , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas do Envelope Viral/genética , Replicação ViralRESUMO
BACKGROUND: The gp41 subunit of the HIV-1 envelope glycoprotein (Env) has been widely regarded as a type I transmembrane protein with a single membrane-spanning domain (MSD). An alternative topology model suggested multiple MSDs. The major discrepancy between the two models is that the cytoplasmic Kennedy sequence in the single MSD model is assigned as the extracellular loop accessible to neutralizing antibodies in the other model. We examined the membrane topology of the gp41 subunit in both prokaryotic and mammalian systems. We attached topological markers to the C-termini of serially truncated gp41. In the prokaryotic system, we utilized a green fluorescent protein (GFP) that is only active in the cytoplasm. The tag protein (HaloTag) and a membrane-impermeable ligand specific to HaloTag was used in the mammalian system. RESULTS: In the absence of membrane fusion, both the prokaryotic and mammalian systems (293FT cells) supported the single MSD model. In the presence of membrane fusion in mammalian cells (293CD4 cells), the data obtained seem to support the multiple MSD model. However, the region predicted to be a potential MSD is the highly hydrophilic Kennedy sequence and is least likely to become a MSD based on several algorithms. Further analysis revealed the induction of membrane permeability during membrane fusion, allowing the membrane-impermeable ligand and antibodies to cross the membrane. Therefore, we cannot completely rule out the possible artifacts. Addition of membrane fusion inhibitors or alterations of the MSD sequence decreased the induction of membrane permeability. CONCLUSIONS: It is likely that a single MSD model for HIV-1 gp41 holds true even in the presence of membrane fusion. The degree of the augmentation of membrane permeability we observed was dependent on the membrane fusion and sequence of the MSD.
Assuntos
Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/metabolismo , Infecções por HIV/virologia , HIV-1/metabolismo , Linhagem Celular , Membrana Celular/virologia , Proteína gp41 do Envelope de HIV/genética , HIV-1/química , HIV-1/genética , Humanos , Fusão de Membrana , Estrutura Terciária de ProteínaRESUMO
BACKGROUND: The sequences of membrane-spanning domains (MSDs) on the gp41 subunit are highly conserved among many isolates of HIV-1. The GXXXG motif, a potential helix-helix interaction motif, and an arginine residue (rare in hydrophobic MSDs) are especially well conserved. These two conserved elements are expected to locate on the opposite sides of the MSD, if the MSD takes a α-helical secondary structure. A scanning alanine-insertion mutagenesis was performed to elucidate the structure-function relationship of gp41 MSD. RESULTS: A circular dichroism analysis of a synthetic gp41 MSD peptide determined that the secondary structure of the gp41 MSD was α-helical. We then performed a scanning alanine-insertion mutagenesis of the entire gp41 MSD, progressively shifting the relative positions of MSD segments around the helix axis. Altering the position of Gly694, the last residue of the GXXXG motif, relative to Arg696 (the number indicates the position of the amino acid residues in HXB2 Env) around the axis resulted in defective fusion. These mutants showed impaired processing of the gp160 precursor into gp120 and gp41. Furthermore, these Env mutants manifested inefficient intracellular transport in the endoplasmic reticulum and Golgi regions. Indeed, a transplantation of the gp41 MSD portion into the transmembrane domain of another membrane protein, Tac, altered its intracellular distribution. Our data suggest that the intact MSD α-helix is critical in the intracellular trafficking of HIV-1 Env. CONCLUSIONS: The relative position between the highly conserved GXXXG motif and an arginine residue around the gp41 MSD α-helix is critical for intracellular trafficking of HIV-1 Env. The gp41 MSD region not only modulates membrane fusion but also controls biosynthesis of HIV-1 Env.
Assuntos
Proteína gp160 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/química , Infecções por HIV/virologia , HIV-1/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Técnicas de Cocultura , Proteína gp41 do Envelope de HIV/genética , Humanos , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/química , Estrutura Secundária de Proteína/fisiologia , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico , Relação Estrutura-AtividadeRESUMO
Flaviviruses bear class II fusion proteins as their envelope (E) proteins. Here, we describe the development of an in vitro quantitative mosquito-cell-based membrane-fusion assay for the E protein using dual split proteins (DSPs). The assay does not involve the use of live viruses and allows the analysis of a membrane-fusion step independent of other events in the viral lifecycle, such as endocytosis. The progress of membrane fusion can be monitored continuously by measuring the activities of Renilla luciferase derived from the reassociation of DSPs during cell fusion. We optimized the assay to screen an FDA-approved drug library for a potential membrane fusion inhibitor using the E protein of Zika virus. Screening results identified atovaquone, which was previously described as an antimalarial agent. Atovaquone potently blocked the in vitro Zika virus infection of mammalian cells with an IC90 of 2.1 µM. Furthermore, four distinct serotypes of dengue virus were also inhibited by atovaquone with IC90 values of 1.6-2.5 µM, which is a range below the average blood concentration of atovaquone after its oral administration in humans. These findings make atovaquone a likely candidate drug to treat illnesses caused by Zika as well as dengue viruses. Additionally, the DSP assay is useful to study the mechanism of membrane fusion in Flaviviruses.
Assuntos
Antivirais/farmacologia , Vírus da Dengue/efeitos dos fármacos , Dengue/virologia , Fusão de Membrana/efeitos dos fármacos , Proteínas do Envelope Viral/metabolismo , Infecção por Zika virus/virologia , Zika virus/efeitos dos fármacos , Animais , Linhagem Celular , Culicidae , Dengue/tratamento farmacológico , Vírus da Dengue/fisiologia , Citometria de Fluxo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Testes de Sensibilidade Microbiana , Internalização do Vírus/efeitos dos fármacos , Zika virus/fisiologia , Infecção por Zika virus/tratamento farmacológicoRESUMO
Although infection by SARS-CoV-2, the causative agent of coronavirus pneumonia disease (COVID-19), is spreading rapidly worldwide, no drug has been shown to be sufficiently effective for treating COVID-19. We previously found that nafamostat mesylate, an existing drug used for disseminated intravascular coagulation (DIC), effectively blocked Middle East respiratory syndrome coronavirus (MERS-CoV) S protein-mediated cell fusion by targeting transmembrane serine protease 2 (TMPRSS2), and inhibited MERS-CoV infection of human lung epithelium-derived Calu-3 cells. Here we established a quantitative fusion assay dependent on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S protein, angiotensin I converting enzyme 2 (ACE2) and TMPRSS2, and found that nafamostat mesylate potently inhibited the fusion while camostat mesylate was about 10-fold less active. Furthermore, nafamostat mesylate blocked SARS-CoV-2 infection of Calu-3 cells with an effective concentration (EC)50 around 10 nM, which is below its average blood concentration after intravenous administration through continuous infusion. On the other hand, a significantly higher dose (EC50 around 30 mM) was required for VeroE6/TMPRSS2 cells, where the TMPRSS2-independent but cathepsin-dependent endosomal infection pathway likely predominates. Together, our study shows that nafamostat mesylate potently inhibits SARS-CoV-2 S protein-mediated fusion in a cell fusion assay system and also inhibits SARS-CoV-2 infection in vitro in a cell-type-dependent manner. These findings, together with accumulated clinical data regarding nafamostat's safety, make it a likely candidate drug to treat COVID-19.
Assuntos
Anticoagulantes/farmacologia , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Guanidinas/farmacologia , Pneumonia Viral/tratamento farmacológico , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Internalização do Vírus/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2 , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Benzamidinas , Betacoronavirus/metabolismo , COVID-19 , Linhagem Celular , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Ésteres , Gabexato/análogos & derivados , Gabexato/farmacologia , Células HEK293 , Humanos , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , SARS-CoV-2 , Serina Endopeptidases/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Células VeroRESUMO
The expression and solubilization of insoluble proteins have been facilitated by the introduction of protein tags. In our analyses of viral protein R (Vpr) of human immunodeficiency virus 1 (HIV-1), however, several conventional tag proteins enhanced its expression but failed to solubilize it. Therefore, we decided to explore whether proteins derived from Thermus thermophilus HB8 (T. th.), a highly heat-stable bacterium, could be used as tag proteins to enhance the solubilization of Vpr. Based on the data accumulated during the recent structural genomics project of T. th., we selected 15 T. th. proteins with high expression levels and solubilities. From this group, we identified a T. th. tag protein that expressed Vpr in a soluble form. Furthermore, two T. th. tag proteins, including the identified one, were found to solubilize the extremely insoluble membrane-spanning domain of the envelope protein of HIV-1. When green fluorescent protein (GFP) was used as a passenger protein of T. th. tags, the brightness and stability of GFP were similar to those of untagged GFP, suggesting that the T. th. tags do not negatively affect the function of the passenger protein. Thus, data of structural genomics can be applied to generate a customized versatile protein tag for protein analyses.
Assuntos
Thermus thermophilus/química , Proteínas Virais/análise , Genômica/métodos , Proteínas de Fluorescência Verde , HIV-1/química , Proteínas do Vírus da Imunodeficiência Humana/análise , Humanos , Técnicas de Sonda MolecularRESUMO
OBJECTIVE: Tat-dependent transcriptional elongation is crucial for the replication of HIV-1 and depends on positive transcription elongation factor b complex (P-TEFb), composed of cyclin dependent kinase 9 (CDK9) and cyclin T. Hexamethylene bisacetamide-induced protein 1 (HEXIM1) inhibits P-TEFb in cooperation with 7SK RNA, but direct evidence that this inhibition limits the replication of HIV-1 has been lacking. In the present study we examined whether the expression of FLAG-tagged HEXIM1 (HEXIM1-f) affected lentiviral replication in human T cell lines. METHODS: HEXIM1-f was introduced to five human T cell lines, relevant host for HIV-1, by murine leukemia virus vector and cells expressing HEXIM1-f were collected by fluorescence activated cell sorter. The lentiviral replication kinetics in HEXIM1-f-expressing cells was compared with that in green fluorescent protein (GFP)-expressing cells. RESULTS: HIV-1 and simian immunodeficiency virus replicated less efficiently in HEXIM1-f-expressing cells than in GFP-expressing cells of the five T cell lines tested. The viral revertants were not immediately selected in culture. In contrast, the replication of vaccinia virus, adenovirus, and herpes simplex virus type 1 was not limited. The quantitative PCR analyses revealed that the early phase of viral life cycle was not blocked by HEXIM1. On the other hand, Tat-dependent transcription in HEXIM1-f-expressing cells was substantially repressed as compared with that in GFP-expressing cells. CONCLUSION: These data indicate that HEXIM1 is a host factor that negatively regulates lentiviral replication specifically. Elucidating the regulatory mechanism of HEXIM1 might lead to ways to control lentiviral replication.
Assuntos
Lentivirus/fisiologia , Proteínas de Ligação a RNA/fisiologia , Replicação Viral/fisiologia , Western Blotting/métodos , Linhagem Celular , DNA Complementar/genética , Vetores Genéticos , HIV-1/fisiologia , Humanos , Plasmídeos , Proteínas de Ligação a RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Vírus da Imunodeficiência Símia/fisiologia , Linfócitos T/metabolismo , Linfócitos T/virologia , Fatores de Transcrição , TransfecçãoRESUMO
HIV-1 is an etiological agent of AIDS. One of the targets of the current anti-HIV-1 combination chemotherapy, called highly active antiretroviral therapy (HAART), is HIV-1 protease (PR), which is responsible for the processing of viral structural proteins and, therefore, essential for virus replication. Here, we describe an in vitro transcription/translation-based method of phenotyping HIV-1 PR. In this system, both substrate and PR for the assay can be prepared by in vitro transcription/translation. Protease activity is estimated by the cleavage of a substrate, as measured by enzyme-linked immunosorbent assay (ELISA). This assay is safe, rapid, and requires no special facility to be carried out. Our rapid phenotyping method of HIV-1 PR may help evaluate drug resistance, useful when choosing an appropriate therapeutic regiment, and could potentially facilitate the discovery of new drugs effective against HIV-1 PR.
Assuntos
Protease de HIV/biossíntese , HIV-1/enzimologia , Biologia Molecular/métodos , Biossíntese de Proteínas , Farmacorresistência Viral/genética , Ensaio de Imunoadsorção Enzimática , Inibidores de Integrase de HIV/farmacologia , Protease de HIV/genética , HIV-1/efeitos dos fármacos , Técnicas In Vitro , Transcrição GênicaRESUMO
Characterizing cellular factors involved in the life cycle of human immunodeficiency virus type 1 (HIV-1) is an initial step toward controlling replication of HIV-1. Actin polymerization mediated by the Arp2/3 complex has been found to play a critical role in some pathogens' intracellular motility. We have asked whether this complex also contributes to the viral life cycles including that of HIV-1. We have used both the acidic domains from actin-related protein (Arp) 2/3 complex-binding proteins such as the Wiscott-Aldrich syndrome protein (N-WASP) or cortactin, and siRNA directing toward Arp2 to inhibit viral infection. HIV-1, simian immunodeficiency virus (SIV), and intracellular mature vaccinia virus (IMV) were sensitive to inhibition of the Arp2/3 complex, whereas MLV, HSV-1, and adenovirus were not. Interestingly, pseudotyping HIV-1 with vesicular stomatitis virus G protein (VSV-G) overcame this inhibition. Constitutive inhibition of the Arp2/3 complex in the T-cell line H9 also blocked replication of HIV-1. These data suggested the existence of an Arp2/3 complex-dependent event during the early phase of the life cycles of both primate lentiviruses and IMV. Inhibiting the HIV-1's ability to activate Arp2/3 complex could be a potential chemotherapeutic intervention for acquired immunodeficiency syndrome (AIDS).
Assuntos
Proteínas do Citoesqueleto/antagonistas & inibidores , Espaço Intracelular/virologia , Lentivirus de Primatas/fisiologia , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Vaccinia virus/fisiologia , Síndrome da Imunodeficiência Adquirida/terapia , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Proteína 2 Relacionada a Actina , Proteína 3 Relacionada a Actina , Adenoviridae/fisiologia , Animais , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Proteínas do Citoesqueleto/metabolismo , HIV-1/genética , HIV-1/metabolismo , HIV-1/fisiologia , Humanos , Fusão de Membrana , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Ligação Proteica , Vírus da Imunodeficiência Símia/fisiologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Replicação Viral , Proteína Neuronal da Síndrome de Wiskott-AldrichRESUMO
Here we report that sparsomycin, a streptococcal metabolite, enhances the replication of HIV-1 in multiple human T cell lines at a concentration of 400 nM. In addition to wild-type HIV-1, sparsomycin also accelerated the replication of low-fitness, drug-resistant mutants carrying either D30N or L90M within HIV-1 protease, which are frequently found mutations in HIV-1-infected patients on highly active antiretroviral therapy (HAART). Of particular interest was that replication enhancement appeared profound when HIV-1 such as the L90M-carrying mutant displayed relatively slower replication kinetics. The presence of sparsomycin did not immediately select the fast-replicating HIV-1 mutants in culture. In addition, sparsomycin did not alter the 50% inhibitory concentration (IC50) of antiretroviral drugs directed against HIV-1 including nucleoside reverse transcriptase inhibitors (lamivudine and stavudine), non-nucleoside reverse transcriptase inhibitor (nevirapine) and protease inhibitors (nelfinavir, amprenavir and indinavir). The IC50s of both zidovudine and lopinavir against multidrug resistant HIV-1 in the presence of sparsomycin were similar to those in the absence of sparsomycin. The frameshift reporter assay and Western blot analysis revealed that the replication-boosting effect was partly due to the sparsomycin's ability to increase the -1 frameshift efficiency required to produce the Gag-Pol transcript. In conclusion, the use of sparsomycin should be able to facilitate the drug resistance profiling of the clinical isolates and the study on the low-fitness viruses.
Assuntos
Farmacorresistência Viral/efeitos dos fármacos , HIV-1/efeitos dos fármacos , Esparsomicina/farmacologia , Replicação Viral/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , HIV-1/genética , Humanos , Concentração Inibidora 50 , Modelos Biológicos , Mutação , Streptococcaceae/metabolismo , TransfecçãoRESUMO
The membrane-spanning domain (MSD) of HIV-1 envelope protein (Env) has an additional glycine residue within a well-conserved putative transmembrane helix-helix interaction motif, GXXXG, and forms a G(690)G(691)XXG(694) sequence (G, glycine; X, any residues; the numbering indicates the position within the Env of an infectious molecular clone, HXB2). Different from vesicular stomatitis virus G (VSV-G), the glycine residues of the GXXXG motif of HIV-1 showed higher tolerance against mutations, and a simultaneous substitution of G690 and G694 with leucine residues only modestly decreased fusion activity and replication capacity of HIV-1. When G691 was further substituted with alanine, phenylalanine or leucine residue while G690 and G694 were substituted with leucine residues, the efficiency of membrane fusion decreased, with the decrease greatest occurring with the leucine substitution, a less severe decrease with phenylalanine, and the least severe decrease with alanine. Substitution with leucine residue also decreased the incorporation of Env onto virions, and the mutant showed the most delayed replication profile. Thus the presence of the extra glycine residue, G691, may increase the tolerance of the other two glycine residues against mutations than VSV-G. The fact that a more severe defect was observed for the leucine residue than the phenylalanine residue suggested that the function of Env depended on the steric nature rather than on the simple volume of the side chain of the amino acid residue at position 691. Based on this result, we propose a hypothetical model of the association among MSDs of gp41, in which G(691) locates itself near the helix-helix interface.
Assuntos
Glicina/genética , Proteína gp41 do Envelope de HIV/genética , HIV-1/patogenicidade , Mutação , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/genética , Sequência de Aminoácidos , Animais , Células COS , Fusão Celular , Chlorocebus aethiops , Citometria de Fluxo , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/fisiologia , HIV-1/genética , HIV-1/fisiologia , Humanos , Células Jurkat , Fusão de Membrana , Dados de Sequência Molecular , Proteínas Virais de Fusão/metabolismo , Replicação Viral/genéticaRESUMO
Human immunodeficiency virus type 1 (HIV-1) is a causative agent of acquired immunodeficiency syndrome (AIDS) in humans. In the last decade, the functions of HIV-1-encoded genes have been intensively studied. These studies have contributed to the development of the effective anti-AIDS drugs directing against the HIV-1-encoded enzymes, namely reverse transcriptase and protease. However, even the combination of these drugs is not sufficient enough to stop the progression of AIDS partly due to the emergence of drug-resistant HIV-1 mutants as well as the severe side effects. Understanding the molecular mechanisms by which cellular factors support the efficient replication of HIV-1 should contribute to develop means to control the progression of AIDS. This field is now expanding rapidly. Here we review the host factors involved in the replication of HIV-1 and highlight some findings that have a substantial impact on the retroviral research.
Assuntos
Infecções por HIV/fisiopatologia , HIV-1/fisiologia , Replicação Viral/fisiologia , Infecções por HIV/virologia , Humanos , Ligação Proteica , Proteínas Virais/fisiologiaRESUMO
Human immunodeficiency virus type I (HIV-1), a causative agent of AIDS, is affecting today more than 35 millions of people worldwide. The advance of anti-HIV chemotherapy has made AIDS a chronic non-fatal disease in resourceful countries. Long-awaited anti-HIV-1 vaccine is still not with us yet; however, great progress in structural analyses of the envelope protein of HIV-1 in recent years starts to shed light on rational intervention targeted at the envelope protein, as will be reviewed in this article.