Ruben D R Ramalho | University of Exeter (original) (raw)
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Papers by Ruben D R Ramalho
Journal of Virology
The RNA genome of human immunodeficiency virus type 1 (HIV-1) is enclosed in a cone-shaped capsid... more The 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 tra...
Journal of virology, Jun 5, 2017
The HIV-1 core consists of the viral genomic RNA and several viral proteins encased within a coni... more The HIV-1 core consists of the viral genomic RNA and several viral proteins encased within a conical capsid. Following 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 find that, during an early stage of reverse transcription the pressure inside the capsid increases, reaching a maximum after 7 hours. 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 n...
Journal of Virology
The RNA genome of human immunodeficiency virus type 1 (HIV-1) is enclosed in a cone-shaped capsid... more The 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 tra...
Applied Surface Science, 2009
Silicon thin films have been prepared on sapphire substrates by pulsed laser deposition (PLD) tec... more Silicon thin films have been prepared on sapphire substrates by pulsed laser deposition (PLD) technique. The films were deposited in vacuum from a silicon target at a base pressure of 10−6mbar in the temperature range from 400 to 800°C. A Q-switched Nd:YAG laser (1064nm, 5ns duration, 10Hz) at a constant energy density of 2J×cm−2 has been used. The influence of the substrate temperature on the structural, morphological and optical properties of the Si thin films was investigated.Spectral ellipsometry and atomic force microscopy (AFM) were used to study the thickness and the surface roughness of the deposited films. Surface roughness values measured by AFM and ellipsometry show the same tendency of increasing roughness with increased deposition temperature.
Nature Communications, 2016
The host cell factor cyclophilin A (CypA) interacts directly with the HIV-1 capsid and regulates ... more The host cell factor cyclophilin A (CypA) interacts directly with the HIV-1 capsid and regulates viral infectivity. Although the crystal structure of CypA in complex with the N-terminal domain of the HIV-1 capsid protein (CA) has been known for nearly two decades, how CypA interacts with the viral capsid and modulates HIV-1 infectivity remains unclear. We determined the cryoEM structure of CypA in complex with the assembled HIV-1 capsid at 8-Å resolution. The structure exhibits a distinct CypA-binding pattern in which CypA selectively bridges the two CA hexamers along the direction of highest curvature. EM-guided all-atom molecular dynamics simulations and solid-state NMR further reveal that the CypA-binding pattern is achieved by single-CypA molecules simultaneously interacting with two CA subunits, in different hexamers, through a previously uncharacterized non-canonical interface. These results provide new insights into how CypA stabilizes the HIV-1 capsid and is recruited to facilitate HIV-1 infection.
Journal of Biotechnology, 2012
Background: The human immunodeficiency virus (HIV-1) capsid is a self-assembled protein shell tha... more Background: The human immunodeficiency virus (HIV-1) capsid is a self-assembled protein shell that contains the viral genome. During the stages between viral entry into a host cell and nuclear import of the viral DNA, the capsid dissociates in a process known as uncoating, which leads to the release of the viral genetic material. Mutations that alter the stability of the capsid affect the uncoating rate and impair HIV-1 infectivity. Results: To gain further insight into the role of capsid stability during uncoating, we used atomic force spectroscopy to quantify the stiffness of the capsid. Empty in vitro assemblies of wild type (WT) and mutant recombinant HIV-1 capsid protein (CA) as well as isolated WT and mutant HIV-1 cores (i.e., filled capsids) were analyzed. We find that hyperstable CA mutant assemblies (A204C, A14C/E45C, E45A and E45A/R132T) are significantly stiffer than WT assemblies. However, the hardening effect of disulfide crosslinking (A204C and A14C/E45C) is lower than that of hydropho-bic interactions (E45A and E45A/R132T). Conclusions: Our results demonstrate that mutations that increase the intrinsic stability of the HIV-1 capsid have an increased stiffness of their lattice.
Journal of Virology
The RNA genome of human immunodeficiency virus type 1 (HIV-1) is enclosed in a cone-shaped capsid... more The 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 tra...
Journal of virology, Jun 5, 2017
The HIV-1 core consists of the viral genomic RNA and several viral proteins encased within a coni... more The HIV-1 core consists of the viral genomic RNA and several viral proteins encased within a conical capsid. Following 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 find that, during an early stage of reverse transcription the pressure inside the capsid increases, reaching a maximum after 7 hours. 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 n...
Journal of Virology
The RNA genome of human immunodeficiency virus type 1 (HIV-1) is enclosed in a cone-shaped capsid... more The 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 tra...
Applied Surface Science, 2009
Silicon thin films have been prepared on sapphire substrates by pulsed laser deposition (PLD) tec... more Silicon thin films have been prepared on sapphire substrates by pulsed laser deposition (PLD) technique. The films were deposited in vacuum from a silicon target at a base pressure of 10−6mbar in the temperature range from 400 to 800°C. A Q-switched Nd:YAG laser (1064nm, 5ns duration, 10Hz) at a constant energy density of 2J×cm−2 has been used. The influence of the substrate temperature on the structural, morphological and optical properties of the Si thin films was investigated.Spectral ellipsometry and atomic force microscopy (AFM) were used to study the thickness and the surface roughness of the deposited films. Surface roughness values measured by AFM and ellipsometry show the same tendency of increasing roughness with increased deposition temperature.
Nature Communications, 2016
The host cell factor cyclophilin A (CypA) interacts directly with the HIV-1 capsid and regulates ... more The host cell factor cyclophilin A (CypA) interacts directly with the HIV-1 capsid and regulates viral infectivity. Although the crystal structure of CypA in complex with the N-terminal domain of the HIV-1 capsid protein (CA) has been known for nearly two decades, how CypA interacts with the viral capsid and modulates HIV-1 infectivity remains unclear. We determined the cryoEM structure of CypA in complex with the assembled HIV-1 capsid at 8-Å resolution. The structure exhibits a distinct CypA-binding pattern in which CypA selectively bridges the two CA hexamers along the direction of highest curvature. EM-guided all-atom molecular dynamics simulations and solid-state NMR further reveal that the CypA-binding pattern is achieved by single-CypA molecules simultaneously interacting with two CA subunits, in different hexamers, through a previously uncharacterized non-canonical interface. These results provide new insights into how CypA stabilizes the HIV-1 capsid and is recruited to facilitate HIV-1 infection.
Journal of Biotechnology, 2012
Background: The human immunodeficiency virus (HIV-1) capsid is a self-assembled protein shell tha... more Background: The human immunodeficiency virus (HIV-1) capsid is a self-assembled protein shell that contains the viral genome. During the stages between viral entry into a host cell and nuclear import of the viral DNA, the capsid dissociates in a process known as uncoating, which leads to the release of the viral genetic material. Mutations that alter the stability of the capsid affect the uncoating rate and impair HIV-1 infectivity. Results: To gain further insight into the role of capsid stability during uncoating, we used atomic force spectroscopy to quantify the stiffness of the capsid. Empty in vitro assemblies of wild type (WT) and mutant recombinant HIV-1 capsid protein (CA) as well as isolated WT and mutant HIV-1 cores (i.e., filled capsids) were analyzed. We find that hyperstable CA mutant assemblies (A204C, A14C/E45C, E45A and E45A/R132T) are significantly stiffer than WT assemblies. However, the hardening effect of disulfide crosslinking (A204C and A14C/E45C) is lower than that of hydropho-bic interactions (E45A and E45A/R132T). Conclusions: Our results demonstrate that mutations that increase the intrinsic stability of the HIV-1 capsid have an increased stiffness of their lattice.