RÉSUMÉ
Retroviral integration is mediated by intasome nucleoprotein complexes wherein a pair of viral DNA ends are bridged together by a multimer of integrase (IN). Atomic-resolution structures of HIV-1 intasomes provide detailed insights into the mechanism of integration and inhibition by clinical IN inhibitors. However, previously described HIV-1 intasomes are highly heterogeneous and have the tendency to form stacks, which is a limiting factor in determining high-resolution cryo-EM maps. We have assembled HIV-1 intasomes in the presence of excess IN C-terminal domain protein, which was readily incorporated into the intasomes. The purified intasomes were largely homogeneous and exhibited minimal stacking tendencies. The cryo-EM map resolution was further improved to 2.01 Å, which will greatly facilitate structural studies of IN inhibitor action and drug resistance mechanisms. The C-terminal 18 residues of HIV-1 IN, which are critical for virus replication and integration in vitro, have not been well resolved in previous intasome structures, and its function remains unclear. We show that the C-terminal tail participates in intasome assembly, resides within the intasome core, and forms a small alpha helix (residues 271-276). Mutations that disrupt alpha helix integrity impede IN activity in vitro and disrupt HIV-1 infection at the step of viral DNA integration.
Sujet(s)
Cryomicroscopie électronique , Intégrase du VIH , VIH-1 (Virus de l'Immunodéficience Humaine de type 1) , Intégration virale , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/génétique , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/physiologie , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/enzymologie , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/composition chimique , Intégrase du VIH/métabolisme , Intégrase du VIH/composition chimique , Intégrase du VIH/génétique , Humains , Domaines protéiques , Modèles moléculaires , ADN viral/génétique , ADN viral/métabolismeRÉSUMÉ
Retroviral DNA integration is mediated by nucleoprotein complexes (intasomes) in which a pair of viral DNA ends are bridged by a multimer of integrase (IN). Most of the high-resolution structures of HIV-1 intasomes are based on an HIV-1 IN with an Sso7d protein domain fused to the N-terminus. Sso7d-IN aggregates much less than wild-type IN and has been critical for structural studies of HIV-1 intasomes. Unexpectedly, these structures revealed that the common core architecture that mediates catalysis could be assembled in various ways, giving rise to both tetrameric and dodecameric intasomes, together with other less well-characterized species. This differs from related retroviruses that assemble unique multimeric intasomes, although the number of protomers in the intasome varies between viruses. The question of whether the additional Sso7d domain contributes to the heterogeneity of HIV-1 intasomes is therefore raised. We have addressed this by biochemical and structural studies of intasomes assembled with wild-type HIV-1 IN. Negative stain and cryo-EM reveal a similar range of multimeric intasome species as with Sso7d-IN with the same common core architecture. Stacks of intasomes resulting from domain swapping are also seen with both wild-type and Sso7d-IN intasomes. The propensity to assemble multimeric intasome species is, therefore, an intrinsic property of HIV-1 IN and is not conferred by the presence of the Sso7d domain. The recently solved intasome structures of different retroviral species, which have been reported to be tetrameric, octameric, dodecameric, and hexadecameric, highlight how a common intasome core architecture can be assembled in different ways for catalysis.
Sujet(s)
Intégrase du VIH , VIH-1 (Virus de l'Immunodéficience Humaine de type 1) , Intégration virale , Humains , ADN viral/composition chimique , Intégrase du VIH/composition chimique , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/enzymologie , Modèles moléculaires , Nucléoprotéines/composition chimique , Multimérisation de protéinesRÉSUMÉ
Integrase (IN) performs dual essential roles during HIV-1 replication. During ingress, IN functions within an oligomeric "intasome" assembly to catalyze viral DNA integration into host chromatin. During late stages of infection, tetrameric IN binds viral RNA and orchestrates the condensation of ribonucleoprotein complexes into the capsid core. The molecular architectures of HIV-1 IN assemblies that mediate these distinct events remain unknown. Furthermore, the tetramer is an important antiviral target for allosteric IN inhibitors. Here, we determined cryo-EM structures of wildtype HIV-1 IN tetramers and intasome hexadecamers. Our structures unveil a remarkable plasticity that leverages IN C-terminal domains and abutting linkers to assemble functionally distinct oligomeric forms. Alteration of a newly recognized conserved interface revealed that both IN functions track with tetramerization in vitro and during HIV-1 infection. Collectively, our findings reveal how IN plasticity orchestrates its diverse molecular functions, suggest a working model for IN-viral RNA binding, and provide atomic blueprints for allosteric IN inhibitor development.
RÉSUMÉ
HIV-1 integrase (IN) is an important molecular target for the development of anti-AIDS drugs. A recently FDA-approved second-generation integrase strand transfer inhibitor (INSTI) cabotegravir (CAB, 2021) is being marketed for use in long-duration antiviral formulations. However, missed doses during extended therapy can potentially result in persistent low levels of CAB that could select for resistant mutant forms of IN, leading to virological failure. We report a series of N-substituted bicyclic carbamoyl pyridones (BiCAPs) that are simplified analogs of CAB. Several of these potently inhibit wild-type HIV-1 in single-round infection assays in cultured cells and retain high inhibitory potencies against a panel of viral constructs carrying resistant mutant forms of IN. Our lead compound, 7c, proved to be more potent than CAB against the therapeutically important resistant double mutants E138K/Q148K (>12-fold relative to CAB) and G140S/Q148R (>36-fold relative to CAB). A significant number of the BiCAPs also potently inhibit the drug-resistant IN mutant R263K, which has proven to be problematic for the FDA-approved second-generation INSTIs.
Sujet(s)
Inhibiteurs de l'intégrase du VIH , Intégrase du VIH , Raltégravir de potassium/pharmacologie , Inhibiteurs de l'intégrase du VIH/pharmacologie , Pyridones/pharmacologie , Intégrase du VIH/génétiqueRÉSUMÉ
HIV-1 infection depends on the integration of viral DNA into host chromatin. Integration is mediated by the viral enzyme integrase and is blocked by integrase strand transfer inhibitors (INSTIs), first-line antiretroviral therapeutics widely used in the clinic. Resistance to even the best INSTIs is a problem, and the mechanisms of resistance are poorly understood. Here, we analyze combinations of the mutations E138K, G140A/S, and Q148H/K/R, which confer resistance to INSTIs. The investigational drug 4d more effectively inhibited the mutants compared with the approved drug Dolutegravir (DTG). We present 11 new cryo-EM structures of drug-resistant HIV-1 intasomes bound to DTG or 4d, with better than 3-Å resolution. These structures, complemented with free energy simulations, virology, and enzymology, explain the mechanisms of DTG resistance involving E138K + G140A/S + Q148H/K/R and show why 4d maintains potency better than DTG. These data establish a foundation for further development of INSTIs that potently inhibit resistant forms in integrase.
Sujet(s)
Inhibiteurs de l'intégrase du VIH , Intégrase du VIH , Inhibiteurs de l'intégrase du VIH/pharmacologie , Inhibiteurs de l'intégrase du VIH/composition chimique , Oxazines/pharmacologie , Mutation , Intégrase du VIH/génétique , Intégrase du VIH/composition chimique , Intégrase du VIH/métabolismeRÉSUMÉ
HIV-1 DNA is preferentially integrated into chromosomal hot spots by the preintegration complex (PIC). To understand the mechanism, we measured the DNA integration activity of PICs-extracted from infected cells-and intasomes, biochemically assembled PIC substructures using a number of relevant target substrates. We observed that PIC-mediated integration into human chromatin is preferred compared to genomic DNA. Surprisingly, nucleosomes lacking histone modifications were not preferred integration compared to the analogous naked DNA. Nucleosomes containing the trimethylated histone 3 lysine 36 (H3K36me3), an epigenetic mark linked to active transcription, significantly stimulated integration, but the levels remained lower than the naked DNA. Notably, H3K36me3-modified nucleosomes with linker DNA optimally supported integration mediated by the PIC but not by the intasome. Interestingly, optimal intasome-mediated integration required the cellular cofactor LEDGF. Unexpectedly, LEDGF minimally affected PIC-mediated integration into naked DNA but blocked integration into nucleosomes. The block for the PIC-mediated integration was significantly relieved by H3K36me3 modification. Mapping the integration sites in the preferred substrates revealed that specific features of the nucleosome-bound DNA are preferred for integration, whereas integration into naked DNA was random. Finally, biochemical and genetic studies demonstrate that DNA condensation by the H1 protein dramatically reduces integration, providing further evidence that features inherent to the open chromatin are preferred for HIV-1 integration. Collectively, these results identify the optimal target substrate for HIV-1 integration, report a mechanistic link between H3K36me3 and integration preference, and importantly, reveal distinct mechanisms utilized by the PIC for integration compared to the intasomes. IMPORTANCE HIV-1 infection is dependent on integration of the viral DNA into the host chromosomes. The preintegration complex (PIC) containing the viral DNA, the virally encoded integrase (IN) enzyme, and other viral/host factors carries out HIV-1 integration. HIV-1 integration is not dependent on the target DNA sequence, and yet the viral DNA is selectively inserted into specific "hot spots" of human chromosomes. A growing body of literature indicates that structural features of the human chromatin are important for integration targeting. However, the mechanisms that guide the PIC and enable insertion of the PIC-associated viral DNA into specific hot spots of the human chromosomes are not fully understood. In this study, we describe a biochemical mechanism for the preference of the HIV-1 DNA integration into open chromatin. Furthermore, our study defines a direct role for the histone epigenetic mark H3K36me3 in HIV-1 integration preference and identify an optimal substrate for HIV-1 PIC-mediated viral DNA integration.
Sujet(s)
Chromosomes humains , VIH-1 (Virus de l'Immunodéficience Humaine de type 1) , Code histone , Histone , Nucléosomes , Intégration virale , Chromatine/métabolisme , Chromosomes humains/virologie , ADN viral/génétique , ADN viral/métabolisme , Infections à VIH/virologie , Intégrase du VIH/génétique , Intégrase du VIH/métabolisme , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/génétique , Histone/composition chimique , Histone/métabolisme , Humains , Lysine/génétique , Méthylation , Nucléosomes/génétique , Nucléosomes/métabolisme , Nucléosomes/virologie , Intégration virale/génétiqueRÉSUMÉ
The retroviral protein Integrase (IN) catalyzes concerted integration of viral DNA into host chromatin to establish a permanent infection in the target cell. We learned a great deal about the mechanism of catalytic integration through structure/function studies over the previous four decades of IN research. As one of three essential retroviral enzymes, IN has also been targeted by antiretroviral drugs to treat HIV-infected individuals. Inhibitors blocking the catalytic integration reaction are now state-of-the-art drugs within the antiretroviral therapy toolkit. HIV-1 IN also performs intriguing non-catalytic functions that are relevant to the late stages of the viral replication cycle, yet this aspect remains poorly understood. There are also novel allosteric inhibitors targeting non-enzymatic functions of IN that induce a block in the late stages of the viral replication cycle. In this chapter, we will discuss the function, structure, and inhibition of retroviral IN proteins, highlighting remaining challenges and outstanding questions.
Sujet(s)
Inhibiteurs de l'intégrase du VIH , VIH-1 (Virus de l'Immunodéficience Humaine de type 1) , Antirétroviraux , ADN viral , Inhibiteurs de l'intégrase du VIH/pharmacologie , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/génétique , Humains , Retroviridae/génétiqueRÉSUMÉ
Three prime repair exonuclease 1 (TREX1) is the most abundant 3'â5' exonuclease in mammalian cells. It has been suggested that TREX1 degrades HIV-1 DNA to enable the virus to evade the innate immune system. However, the exact role of TREX1 during early steps of HIV-1 infection is not clearly understood. In this study, we report that HIV-1 infection is associated with upregulation, perinuclear accumulation, and nuclear localization of TREX1. However, TREX1 overexpression did not affect reverse transcription or nuclear entry of the virus. Surprisingly, HIV-1 DNA integration was increased in TREX1-overexpressing cells, suggesting a role of the exonuclease in the post-nuclear entry step of infection. Accordingly, preintegration complexes (PICs) extracted from TREX1-overexpressing cells retained higher levels of DNA integration activity. TREX1 depletion resulted in reduced levels of proviral integration, and PICs formed in TREX1-depleted cells retained lower DNA integration activity. Addition of purified TREX1 to PICs also enhanced DNA integration activity, suggesting that TREX1 promotes HIV-1 integration by stimulating PIC activity. To understand the mechanism, we measured TREX1 exonuclease activity on substrates containing viral DNA ends. These studies revealed that TREX1 preferentially degrades the unprocessed viral DNA, but the integration-competent 3'-processed viral DNA remains resistant to degradation. Finally, we observed that TREX1 addition stimulates the activity of HIV-1 intasomes assembled with the unprocessed viral DNA but not that of intasomes containing the 3'-processed viral DNA. These biochemical analyses provide a mechanism by which TREX1 directly promotes HIV-1 integration. Collectively, our study demonstrates that HIV-1 infection upregulates TREX1 to facilitate viral DNA integration. IMPORTANCE Productive HIV-1 infection is dependent on a number of cellular factors. Therefore, a clear understanding of how the virus exploits the cellular machinery will identify new targets for inhibiting HIV-1 infection. The three prime repair exonuclease 1 (TREX1) is the most active cellular exonuclease in mammalian cells. It has been reported that TREX1 prevents accumulation of HIV-1 DNA and enables the virus to evade the host innate immune response. Here, we show that HIV-1 infection results in the upregulation, perinuclear accumulation, and nuclear localization of TREX1. We also provide evidence that TREX1 promotes HIV-1 integration by preferentially degrading viral DNAs that are incompatible with chromosomal insertion. These observations identify a novel role of TREX1 in a post-nuclear entry step of HIV-1 infection.
Sujet(s)
ADN viral/métabolisme , Exodeoxyribonucleases/métabolisme , Infections à VIH/virologie , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/physiologie , Immunité innée/immunologie , Phosphoprotéines/métabolisme , Intégration virale , Réplication virale , Noyau de la cellule , ADN viral/génétique , Exodeoxyribonucleases/génétique , Cellules HEK293 , Infections à VIH/génétique , Cellules HeLa , Humains , Phosphoprotéines/génétiqueRÉSUMÉ
A simple and cost-effective protocol is presented for expression of perdeuterated, Ile/Leu/Val 1H/13C methyl protonated proteins from 100 ml cultures in M9 ++ /D2O medium induced at high (OD600 ~ 10) cell density in shaker flasks. This protocol, which is an extension of our previous protocols for expression of 2H/15N/13C and 1H/13C labeled proteins, yields comparable quantities of protein from 100 ml cell culture to those obtained using a conventional 1 L culture with M9/D2O medium, while using three-fold less α-ketoisovaleric (1,2,3,4-13C4; 3,4',4',4'-d4) and α-ketobutyric (13C4; 3,3-d2) acid precursors.
Sujet(s)
Acides aminés/métabolisme , Biochimie/méthodes , Bioréacteurs/microbiologie , Analyse coût-bénéfice , Deutérium/métabolisme , Protéines Escherichia coli/métabolisme , Escherichia coli/croissance et développement , Protons , Expression des gènes , Isoleucine/métabolisme , Leucine/métabolisme , Valine/métabolismeRÉSUMÉ
The low solubility and aggregation properties of HIV-1 integrase (IN) are major obstacles for biochemical and structural studies. The lens epithelium-derived growth factor (LEDGF) is a cellular factor that binds IN and tethers preintegration complexes to chromatin before integration. The LEDGF also stimulates HIV-1 IN DNA strand transfer activity and improves its solubility in vitro. We show that these properties are conferred by a short peptide spanning residues 178 to 197 of the LEDGF that encompasses its AT-hook DNA-binding elements. The peptide stimulates HIV-1 IN activity both in trans and in cis. Fusion of the peptide to either the N- or C-terminus of IN results in maximal stimulation of concerted integration activity and greatly improves the solubility of the protein and nucleoprotein complexes of IN with viral DNA ends (intasomes). High-resolution structures of HIV-1 intasomes are required to understand the mechanism of IN strand transfer inhibitors (INSTIs), which are front-line drugs for the treatment of HIV-1, and how the virus can develop resistance to INSTIs. We have previously determined the structure of the HIV-1 strand transfer complex intasome. The improved biophysical properties of intasomes assembled with LEDGF peptide fusion IN have enabled us to determine the structure of the cleaved synaptic complex intasome, which is the direct target of INSTIs.
Sujet(s)
Protéines adaptatrices de la transduction du signal/métabolisme , ADN viral/composition chimique , Intégrase du VIH/métabolisme , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/physiologie , Fragments peptidiques/métabolisme , Conformation des protéines , Facteurs de transcription/métabolisme , Intégration virale , Protéines adaptatrices de la transduction du signal/composition chimique , Protéines adaptatrices de la transduction du signal/génétique , ADN viral/génétique , ADN viral/métabolisme , Intégrase du VIH/génétique , Humains , Fragments peptidiques/génétique , Facteurs de transcription/composition chimique , Facteurs de transcription/génétiqueRÉSUMÉ
The HIV intasome is a large nucleoprotein assembly that mediates the integration of a DNA copy of the viral genome into host chromatin. Intasomes are targeted by the latest generation of antiretroviral drugs, integrase strand-transfer inhibitors (INSTIs). Challenges associated with lentiviral intasome biochemistry have hindered high-resolution structural studies of how INSTIs bind to their native drug target. Here, we present high-resolution cryo-electron microscopy structures of HIV intasomes bound to the latest generation of INSTIs. These structures highlight how small changes in the integrase active site can have notable implications for drug binding and design and provide mechanistic insights into why a leading INSTI retains efficacy against a broad spectrum of drug-resistant variants. The data have implications for expanding effective treatments available for HIV-infected individuals.
Sujet(s)
Résistance virale aux médicaments , Inhibiteurs de l'intégrase du VIH/composition chimique , Intégrase du VIH/composition chimique , VIH (Virus de l'Immunodéficience Humaine)/effets des médicaments et des substances chimiques , Composés hétérocycliques avec 4 noyaux ou plus/composition chimique , Complexes multiprotéiques/composition chimique , Nucléoprotéines/composition chimique , Amides , Cryomicroscopie électronique , Conception de médicament , VIH (Virus de l'Immunodéficience Humaine)/composition chimique , Composés hétérocycliques 3 noyaux , Humains , Complexes multiprotéiques/génétique , Naphtyridines/composition chimique , Nucléoprotéines/génétique , Pipérazines , Pyridones , Intégration virale/effets des médicaments et des substances chimiquesRÉSUMÉ
Protein expression in E. coli grown in shaker flasks is a routine and pivotal tool in many research laboratories. To maximize protein yields, cells are normally induced in the middle of the linear growth phase, typically at an OD600 of ≤ 1 for cells grown in Luria-Bertani (LB) medium at 37 °C. We recently showed that the E. coli linear growth phase can be extended to higher cell density when cells are cultured under less than optimal conditions such as in minimal medium and/or at lower temperatures. Maximizing the yield of protein per unit volume of culture is important for reducing the costs, especially when isotopically labeling is required. Here, we present a modified minimal medium and a simple protocol that can increase the protein yield up to fourfold in a pH-stabilized LB medium and up to sevenfold in a modified M9+ medium (M9++). When M9++ medium coupled with the high density (OD600 ~ 6) cell growth protocol are used to express uniformly 15N- or 15N/13C-labeled proteins, the amount of 15NH4Cl and 13C6-glucose for a given cell mass is reduced by 50% and ~ 65%, respectively, relative to the traditional low density (OD600 ~ 1) cell growth protocol with M9 medium; the inclusion of 0.1% LB in the minimal medium permits a reduction in the concentration of both the trace element solution and MgCl2, which can cause precipitation. Mass data indicate that inclusion of 0.1% LB does not significantly affect the isotope enrichment level.
Sujet(s)
Protéines Escherichia coli/métabolisme , Marquage isotopique/méthodes , Milieux de culture/composition chimique , Escherichia coli/croissance et développementRÉSUMÉ
Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is the iterative process by which nucleic acids that can bind with high affinity and specificity (termed aptamers) to specific protein targets are selected. Using a SELEX protocol adapted for Xeno-Nucleic Acid (XNA) as a suitable substrate for aptamer generation, 2'-fluoroarabinonucleic acid (FANA) was used to select several related aptamers to HIV-1 integrase (IN). IN bound FANA aptamers with equilibrium dissociation constants (KD,app) of â¼50-100 pM in a buffer with 200 mM NaCl and 6 mM MgCl2. Comparisons to published HIV-1 IN RNA and DNA aptamers as well as IN genomic binding partners indicated that FANA aptamers bound more than 2 orders of magnitude more tightly to IN. Using a combination of RNA folding algorithms and covariation analysis, all strong binding aptamers demonstrated a common four-way junction structure, despite significant sequence variation. IN aptamers were selected from the same starting library as FA1, a FANA aptamer that binds with pM affinity to HIV-1 Reverse Transcriptase (RT). It contains a 20-nucleotide 5' DNA sequence followed by 59 FANA nucleotides. IN-1.1 (one of the selected aptamers) potently inhibited IN activity and intasome formation in vitro. Replacing the FANA nucleotides of IN-1.1 with 2'-fluororibonucleic acid (F-RNA), which has the same chemical formula but with a ribose rather than arabinose sugar conformation, dramatically reduced binding, suggesting that FANA adopts unique structural conformations that promote binding to HIV-1 IN.
Sujet(s)
Aptamères nucléotidiques/composition chimique , Arabinonucléotides/composition chimique , Intégrase du VIH/composition chimique , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/enzymologie , ARN/composition chimique , Séquence nucléotidique , Technique SELEX , Alignement de séquencesRÉSUMÉ
Integration of a DNA copy of the viral genome into host DNA is an essential step in the replication cycle of HIV-1 and other retroviruses and is an important therapeutic target for drugs. DNA integration is catalyzed by the viral integrase protein and proceeds through a series of stable nucleoprotein complexes of integrase, viral DNA ends and target DNA. These nucleoprotein complexes are collectively called intasomes. Retroviral intasomes undergo a series of transitions between initial formation and catalysis of the DNA cutting and joining steps of DNA integration. Intasomes, rather than free integrase protein, are the target of currently approved drugs that target HIV-1 DNA integration. High-resolution structures of HIV-1 intasomes are needed to understand their detailed mechanism of action and how HIV-1 may escape by developing resistance. Here, we focus on our current knowledge of the structure and function of HIV-1 intasomes, with reference to related systems as required to put this knowledge in context.
Sujet(s)
ADN viral/métabolisme , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/physiologie , Nucléoprotéines/métabolisme , Intégration virale/physiologie , Animaux , ADN viral/génétique , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/composition chimique , Humains , Nucléoprotéines/composition chimique , Nucléoprotéines/génétique , Relation structure-activitéRÉSUMÉ
Like all retroviruses, HIV-1 irreversibly inserts a viral DNA (vDNA) copy of its RNA genome into host target DNA (tDNA). The intasome, a higher-order nucleoprotein complex composed of viral integrase (IN) and the ends of linear vDNA, mediates integration. Productive integration into host chromatin results in the formation of the strand transfer complex (STC) containing catalytically joined vDNA and tDNA. HIV-1 intasomes have been refractory to high-resolution structural studies. We used a soluble IN fusion protein to facilitate structural studies, through which we present a high-resolution cryo-electron microscopy (cryo-EM) structure of the core tetrameric HIV-1 STC and a higher-order form that adopts carboxyl-terminal domain rearrangements. The distinct STC structures highlight how HIV-1 can use the common retroviral intasome core architecture to accommodate different IN domain modules for assembly.
Sujet(s)
VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/composition chimique , Intégration virale , Cryomicroscopie électronique , Cristallographie aux rayons X , ADN viral/composition chimique , ADN viral/ultrastructure , Intégrase du VIH/composition chimique , Intégrase du VIH/ultrastructure , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/physiologie , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/ultrastructure , Humains , Modèles moléculaires , Nucléoprotéines/composition chimique , Nucléoprotéines/ultrastructure , Domaines protéiques , ARN viral/composition chimique , ARN viral/ultrastructureRÉSUMÉ
We present a simple, convenient and robust protocol for expressing perdeuterated proteins in E. coli BL21(DE3) cells in shaker flasks that reduces D2O usage tenfold and d7-glucose usage by 30 %. Using a modified M9 medium and optimized growth conditions, we were able to grow cells in linear log phase to an OD600 of up to 10. Inducing the cells with isopropyl ß-D-1-thiogalactopyranoside at an OD600 of 10, instead of less than 1, enabled us to increase the cell mass tenfold per unit volume of cell culture. We show that protein expression levels per cell are the same when induced at an OD600 between 1 and 10 under these growth conditions. Thus, our new protocol can increase protein yield per unit volume of cell culture tenfold. Adaptation of E. coli from H2O-based to D2O-based medium is also key for ensuring high levels of protein expression in D2O. We find that a simple three-step adaptation approach-Luria-Bertani (LB) medium in H2O to LB in D2O to modified-M9 medium in D2O is both simple and reliable. The method increases the yield of perdeuterated proteins by up to tenfold using commonly available air shakers without any requirement for specialized fermentation equipment.
Sujet(s)
Techniques de culture cellulaire en batch , Escherichia coli/génétique , Escherichia coli/métabolisme , Expression des gènes , Protéines recombinantes/génétique , Protéines recombinantes/métabolisme , Protéines recombinantes/composition chimique , Protéines recombinantes/isolement et purificationRÉSUMÉ
Retroviral DNA integration is mediated by nucleoprotein complexes (intasomes) comprising a pair of viral DNA ends synapsed by a tetramer of integrase. Current integrase inhibitors act on intasomes rather than free integrase protein. Structural and functional studies of intasomes are essential to understand their mechanism of action and how the virus can escape by mutation. To date, prototype foamy virus (PFV) is the only retrovirus for which high-resolution structures of intasomes have been determined. In the PFV intasome structure, only the core domains of the outer subunits are ordered; the N-terminal domain, C-terminal domain, and N-terminal extension domain are disordered. Are these "missing domains" required for function or are they dispensable? We have devised a strategy to assemble "hetero-intasomes" in which the outer domains are not present as a tool to assess the functional role of the missing domains for catalysis of integration. We find that the disordered domains of outer subunits are not required for intasome assembly or catalytic activity as catalytic core domains can substitute for the outer subunits in the case of both PFV and HIV-1 intasomes.
Sujet(s)
ADN viral/métabolisme , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/physiologie , Integrases/métabolisme , Infections à Retroviridae/métabolisme , Spumavirus/physiologie , Protéines virales/métabolisme , Intégration virale , Domaine catalytique , Infections à VIH/métabolisme , Humains , Integrases/composition chimique , Nucléoprotéines/métabolisme , Multimérisation de protéines , Structure tertiaire des protéinesRÉSUMÉ
AIDS has been transformed from a death sentence to a manageable disease for many patients with access to combination antiviral therapy. It is informative to look back on some of the key advances that have led to this transformation. The arsenal of tools currently available to clinicians now includes inhibitors of the viral reverse transcriptase, protease and integrase enzymes. The author discusses some of the key advances that have led to this transformation with an emphasis on the role of basic science in developing integrase inhibitors. Many of the stepping-stones could not easily have been foreseen to lead to medical advances. Treatments for diseases that are yet to emerge will likely depend on the progress made in basic science today.
RÉSUMÉ
The DNA cutting and joining reactions of HIV-1 integration are catalyzed by integrase (IN), a viral protein that functions as a tetramer bridging the two viral DNA ends (intasome). Two major obstacles for biochemical and structural studies of HIV-1 intasomes are 1) the low efficiency of assembly with oligonucleotide DNA substrates, and 2) the non-specific aggregation of both intasomes and free IN in the reaction mixture. By fusing IN with a small non-specific DNA binding protein, Sulfolobus solfataricus chromosomal protein Sso7d (PDB: 1BNZ), we have engineered a highly soluble and hyperactive IN. Unlike wild-type IN, it efficiently catalyzes intasome assembly and concerted integration with oligonucleotide DNA substrates. The fusion IN protein also functions to integrate viral reverse transcripts during HIV-infection. The hyperactive HIV-1 IN may assist in facilitating future biochemical and structural studies of HIV-1 intasomes. Understanding the mechanistic basis of the Sso7d-IN fusion protein could provide insight into the factors that have hindered biophysical studies of wild-type HIV-1 IN and intasomes.