Publications by category
Journal articles
Rankovic S, Ramalho R, Aiken C, Rousso I (2018). PF74 Reinforces the HIV-1 Capsid to Impair Reverse Transcription-Induced Uncoating.
Journal of Virology,
92(20).
Abstract:
PF74 Reinforces the HIV-1 Capsid to Impair Reverse Transcription-Induced Uncoating
ABSTRACTThe RNA genome of human immunodeficiency virus type 1 (HIV-1) is enclosed in a cone-shaped capsid shell that disassembles following cell entry via a process known as uncoating. During HIV-1 infection, the capsid is important for reverse transcription and entry of the virus into the target cell nucleus. The small molecule PF74 inhibits HIV-1 infection at early stages by binding to the capsid and perturbing uncoating. However, the mechanism by which PF74 alters capsid stability and reduces viral infection is presently unknown. Here, we show, using atomic force microscopy (AFM), that binding of PF74 to recombinant capsid-like assemblies and to HIV-1 isolated cores stabilizes the capsid in a concentration-dependent manner. At a PF74 concentration of 10 μM, the mechanical stability of the core is increased to a level similar to that of the intrinsically hyperstable capsid mutant E45A. PF74 also prevented the complete disassembly of HIV-1 cores normally observed during 24 h of reverse transcription. Specifically, cores treated with PF74 only partially disassembled: the main body of the capsid remained intact and stiff, and a cap-like structure dissociated from the narrow end of the core. Moreover, the internal coiled structure that was observed to form during reverse transcriptionin vitropersisted throughout the duration of the measurement (∼24 h). Our results provide direct evidence that PF74 directly stabilizes the HIV-1 capsid lattice, thereby permitting reverse transcription while interfering with a late step in uncoating.IMPORTANCEThe capsid-binding small molecule PF74 inhibits HIV-1 infection at early stages and perturbs uncoating. However, the mechanism by which PF74 alters capsid stability and reduces viral infection is presently unknown. We recently introduced time-lapse atomic force microscopy to study the morphology and physical properties of HIV-1 cores during the course of reverse transcription. Here, we apply this AFM methodology to show that PF74 prevented the complete disassembly of HIV-1 cores normally observed during 24 h of reverse transcription. Specifically, cores with PF74 only partially disassembled: the main body of the capsid remained intact and stiff, but a cap-like structure dissociated from the narrow end of the core HIV-1. Our result provides direct evidence that PF74 directly stabilizes the HIV-1 capsid lattice.
Abstract.
Rankovic S, Varadarajan J, Ramalho R, Aiken C, Rousso I (2017). Reverse Transcription Mechanically Initiates HIV-1 Capsid Disassembly.
Journal of Virology,
91(12).
Abstract:
Reverse Transcription Mechanically Initiates HIV-1 Capsid Disassembly
ABSTRACT
. The HIV-1 core consists of the viral genomic RNA and several viral proteins encased within a conical capsid. After cell entry, the core disassembles in a process termed uncoating. Although HIV-1 uncoating has been linked to reverse transcription of the viral genome in target cells, the mechanism by which uncoating is initiated is unknown. Using time-lapse atomic force microscopy, we analyzed the morphology and physical properties of isolated HIV-1 cores during the course of reverse transcription in vitro. We found that, during an early stage of reverse transcription the pressure inside the capsid increases, reaching a maximum after 7 h. High-resolution mechanical mapping reveals the formation of a stiff coiled filamentous structure underneath the capsid surface. Subsequently, this coiled structure disappears, the stiffness of the capsid drops precipitously to a value below that of a pre-reverse transcription core, and the capsid undergoes partial or complete rupture near the narrow end of the conical structure. We propose that the transcription of the relatively flexible single-stranded RNA into a more rigid filamentous structure elevates the pressure within the core, which triggers the initiation of capsid disassembly.
.
. IMPORTANCE for successful infection, the HIV-1 genome, which is in the form of a single-stranded RNA enclosed inside a capsid shell, must be reverse transcribed into double-stranded DNA and released from the capsid (in a process known as uncoating) before it can be integrated into the target cell genome. The mechanism that triggers uncoating is a pivotal question of long standing. By using atomic force microscopy, we found that during reverse transcription the pressure inside the capsid increases until the internal stress exceeds the strength of the capsid structure and the capsid breaks open. The application of AFM technologies to study purified HIV-1 cores represents a new experimental platform for elucidating additional aspects of capsid disassembly and HIV-1 uncoating.
Abstract.
Ramalho R, Rankovic S, Zhou J, Aiken C, Rousso I (2016). Analysis of the mechanical properties of wild type and hyperstable mutants of the HIV-1 capsid. Retrovirology, 13(1).
Liu C, Perilla JR, Ning J, Lu M, Hou G, Ramalho R, Himes BA, Zhao G, Bedwell GJ, Byeon I-J, et al (2016). Cyclophilin a stabilizes the HIV-1 capsid through a novel non-canonical binding site. Nature Communications, 7(1).
Basto AP, Piedade J, Ramalho R, Alves S, Soares H, Cornelis P, Martins C, Leitão A (2012). A new cloning system based on the OprI lipoprotein for the production of recombinant bacterial cell wall-derived immunogenic formulations.
J Biotechnol,
157(1), 50-63.
Abstract:
A new cloning system based on the OprI lipoprotein for the production of recombinant bacterial cell wall-derived immunogenic formulations.
The conjugation of antigens with ligands of pattern recognition receptors (PRR) is emerging as a promising strategy for the modulation of specific immunity. Here, we describe a new Escherichia coli system for the cloning and expression of heterologous antigens in fusion with the OprI lipoprotein, a TLR ligand from the Pseudomonas aeruginosa outer membrane (OM). Analysis of the OprI expressed by this system reveals a triacylated lipid moiety mainly composed by palmitic acid residues. By offering a tight regulation of expression and allowing for antigen purification by metal affinity chromatography, the new system circumvents the major drawbacks of former versions. In addition, the anchoring of OprI to the OM of the host cell is further explored for the production of novel recombinant bacterial cell wall-derived formulations (OM fragments and OM vesicles) with distinct potential for PRR activation. As an example, the African swine fever virus ORF A104R was cloned and the recombinant antigen was obtained in the three formulations. Overall, our results validate a new system suitable for the production of immunogenic formulations that can be used for the development of experimental vaccines and for studies on the modulation of acquired immunity.
Abstract.
Author URL.
Ayouchi R, Schwarz R, Melo LV, Ramalho R, Alves E, Marques CP, Santos L, Almeida R, Conde O (2009). Morphological and optical properties of silicon thin films by PLD. Applied Surface Science, 255(10), 5299-5302.
Ramalho RR, Soares H, Melo LV (2007). Microtubule behavior under strong electromagnetic fields. Materials Science and Engineering: C, 27(5-8), 1207-1210.
Publications by year
2018
Rankovic S, Ramalho R, Aiken C, Rousso I (2018). PF74 Reinforces the HIV-1 Capsid to Impair Reverse Transcription-Induced Uncoating.
Journal of Virology,
92(20).
Abstract:
PF74 Reinforces the HIV-1 Capsid to Impair Reverse Transcription-Induced Uncoating
ABSTRACTThe RNA genome of human immunodeficiency virus type 1 (HIV-1) is enclosed in a cone-shaped capsid shell that disassembles following cell entry via a process known as uncoating. During HIV-1 infection, the capsid is important for reverse transcription and entry of the virus into the target cell nucleus. The small molecule PF74 inhibits HIV-1 infection at early stages by binding to the capsid and perturbing uncoating. However, the mechanism by which PF74 alters capsid stability and reduces viral infection is presently unknown. Here, we show, using atomic force microscopy (AFM), that binding of PF74 to recombinant capsid-like assemblies and to HIV-1 isolated cores stabilizes the capsid in a concentration-dependent manner. At a PF74 concentration of 10 μM, the mechanical stability of the core is increased to a level similar to that of the intrinsically hyperstable capsid mutant E45A. PF74 also prevented the complete disassembly of HIV-1 cores normally observed during 24 h of reverse transcription. Specifically, cores treated with PF74 only partially disassembled: the main body of the capsid remained intact and stiff, and a cap-like structure dissociated from the narrow end of the core. Moreover, the internal coiled structure that was observed to form during reverse transcriptionin vitropersisted throughout the duration of the measurement (∼24 h). Our results provide direct evidence that PF74 directly stabilizes the HIV-1 capsid lattice, thereby permitting reverse transcription while interfering with a late step in uncoating.IMPORTANCEThe capsid-binding small molecule PF74 inhibits HIV-1 infection at early stages and perturbs uncoating. However, the mechanism by which PF74 alters capsid stability and reduces viral infection is presently unknown. We recently introduced time-lapse atomic force microscopy to study the morphology and physical properties of HIV-1 cores during the course of reverse transcription. Here, we apply this AFM methodology to show that PF74 prevented the complete disassembly of HIV-1 cores normally observed during 24 h of reverse transcription. Specifically, cores with PF74 only partially disassembled: the main body of the capsid remained intact and stiff, but a cap-like structure dissociated from the narrow end of the core HIV-1. Our result provides direct evidence that PF74 directly stabilizes the HIV-1 capsid lattice.
Abstract.
2017
Rankovic S, Varadarajan J, Ramalho R, Aiken C, Rousso I (2017). Reverse Transcription Mechanically Initiates HIV-1 Capsid Disassembly.
Journal of Virology,
91(12).
Abstract:
Reverse Transcription Mechanically Initiates HIV-1 Capsid Disassembly
ABSTRACT
. The HIV-1 core consists of the viral genomic RNA and several viral proteins encased within a conical capsid. After cell entry, the core disassembles in a process termed uncoating. Although HIV-1 uncoating has been linked to reverse transcription of the viral genome in target cells, the mechanism by which uncoating is initiated is unknown. Using time-lapse atomic force microscopy, we analyzed the morphology and physical properties of isolated HIV-1 cores during the course of reverse transcription in vitro. We found that, during an early stage of reverse transcription the pressure inside the capsid increases, reaching a maximum after 7 h. High-resolution mechanical mapping reveals the formation of a stiff coiled filamentous structure underneath the capsid surface. Subsequently, this coiled structure disappears, the stiffness of the capsid drops precipitously to a value below that of a pre-reverse transcription core, and the capsid undergoes partial or complete rupture near the narrow end of the conical structure. We propose that the transcription of the relatively flexible single-stranded RNA into a more rigid filamentous structure elevates the pressure within the core, which triggers the initiation of capsid disassembly.
.
. IMPORTANCE for successful infection, the HIV-1 genome, which is in the form of a single-stranded RNA enclosed inside a capsid shell, must be reverse transcribed into double-stranded DNA and released from the capsid (in a process known as uncoating) before it can be integrated into the target cell genome. The mechanism that triggers uncoating is a pivotal question of long standing. By using atomic force microscopy, we found that during reverse transcription the pressure inside the capsid increases until the internal stress exceeds the strength of the capsid structure and the capsid breaks open. The application of AFM technologies to study purified HIV-1 cores represents a new experimental platform for elucidating additional aspects of capsid disassembly and HIV-1 uncoating.
Abstract.
2016
Ramalho R, Rankovic S, Zhou J, Aiken C, Rousso I (2016). Analysis of the mechanical properties of wild type and hyperstable mutants of the HIV-1 capsid. Retrovirology, 13(1).
Liu C, Perilla JR, Ning J, Lu M, Hou G, Ramalho R, Himes BA, Zhao G, Bedwell GJ, Byeon I-J, et al (2016). Cyclophilin a stabilizes the HIV-1 capsid through a novel non-canonical binding site. Nature Communications, 7(1).
2012
Basto AP, Piedade J, Ramalho R, Alves S, Soares H, Cornelis P, Martins C, Leitão A (2012). A new cloning system based on the OprI lipoprotein for the production of recombinant bacterial cell wall-derived immunogenic formulations.
J Biotechnol,
157(1), 50-63.
Abstract:
A new cloning system based on the OprI lipoprotein for the production of recombinant bacterial cell wall-derived immunogenic formulations.
The conjugation of antigens with ligands of pattern recognition receptors (PRR) is emerging as a promising strategy for the modulation of specific immunity. Here, we describe a new Escherichia coli system for the cloning and expression of heterologous antigens in fusion with the OprI lipoprotein, a TLR ligand from the Pseudomonas aeruginosa outer membrane (OM). Analysis of the OprI expressed by this system reveals a triacylated lipid moiety mainly composed by palmitic acid residues. By offering a tight regulation of expression and allowing for antigen purification by metal affinity chromatography, the new system circumvents the major drawbacks of former versions. In addition, the anchoring of OprI to the OM of the host cell is further explored for the production of novel recombinant bacterial cell wall-derived formulations (OM fragments and OM vesicles) with distinct potential for PRR activation. As an example, the African swine fever virus ORF A104R was cloned and the recombinant antigen was obtained in the three formulations. Overall, our results validate a new system suitable for the production of immunogenic formulations that can be used for the development of experimental vaccines and for studies on the modulation of acquired immunity.
Abstract.
Author URL.
2009
Ayouchi R, Schwarz R, Melo LV, Ramalho R, Alves E, Marques CP, Santos L, Almeida R, Conde O (2009). Morphological and optical properties of silicon thin films by PLD. Applied Surface Science, 255(10), 5299-5302.
2007
Ramalho RR, Soares H, Melo LV (2007). Microtubule behavior under strong electromagnetic fields. Materials Science and Engineering: C, 27(5-8), 1207-1210.