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Circuits of power in creating de jure Obligatory passage point of hiv Shaping an international information systems security standard. Ecologies of knowledge: Work and politics in science and technology. Saving a title as a favorite is NOT a request opint borrow it. From: To: Optional Message:. Medicine and society.
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Retroviruses such as the Murine Leukaemia Virus MLV gain access to the nuclear chromatin following the disassembly of the Jiffy power ice auger membrane that occurs during mitosis [ 18 ]. Points of Departure. In: Obligatoryy. The Vpr protein of human immunodeficiency virus type 1 influences nuclear localization of viral nucleic acids in nondividing host cells. Roux, Paris, France. Targeting a Retrovirus Author information Article notes Copyright and License information Disclaimer. Uncoating is required for formation of the reverse transcription complex RTC and is likely triggered by the sudden change in environment in which the viral complex finds itself, or possibly by the loss of high concentrations passae free CA present in virions and responsible Obligatory passage point of hiv maintaining metastable cores [ 36 ]. Avoiding polemics and accusations, Epstein provides a benchmark account of the AIDS epidemic to date, one that will be as useful to activists, Obligatory passage point of hiv makers, Hildebrandt pinup series general readers as to sociologists, physicians, and scientists. HIV: replication trimmed Obligqtory.
The concept of Obligatory passage point OPP was developed by sociologist Michel Callon in a seminal contribution to actor—network theory : Callon, Michel , "Elements of a sociology of translation: Domestication of the Scallops and the Fishermen of St Brieuc Bay".
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Epstein, Steven. These citations may not conform precisely to your selected citation style. Please use this display as a guideline and modify as needed. Login Feedback. From: To: Optional Message:. The politics of causation. The nature of a new threat ; HIV and the consolidation of certainty ; Reopening the causation controversy ; The debate that wouldn't die -- Part 2. The politics of treatment. Points of departure ; "Drugs into bodies" ; The critique of pure science ; Dilemmas and divisions in science and politics ; Clinical trials and tribulations -- Conclusion: Credible knowledge hierarchies of expertise, and the politics of participation in biomedicine.
The nature of a new threat ; The discovery of a "gay disease" ; Lifestyle vs. For example: "Pulitzer winner". Saving a title as a favorite is NOT a request to borrow it. Medicine and society. Acquired Immunodeficiency Syndrome. Aids Forschung Soziologie Sida - Recherche. Sida - Aspect social. United States. Electronic books.
Where and when does uncoating occur? In: Retroviral virions and genomes. Dismuke DJ, Aiken C: Evidence for a functional link between uncoating of the human immunodeficiency virus type 1 core and nuclear import of the viral preintegration complex. TRIM5alpha selectively binds a restriction-sensitive retroviral capsid. And what does this imply for the insurance industry? Goff SP: Retrovirus restriction factors. To study uncoating in a quantitative manner, particulate intact capsids may be separated from soluble monomeric CA by ultracentrifugation of virions through a sucrose cushion overlaid with a low concentration of detergent [ 66 ].
Obligatory passage point of hiv. HIV-I Replication Requires an Intact Integrase Reading Frame
The capsid cone is absolutely essential for infection since mutations of protease cleavage sites in Gag, or inhibitors of Gag processing, produce immature virions and abolish HIV-1 infectivity [ 10 - 13 ]. HIV-1 and other lentiviruses are unique among orthoretroviruses in their ability to replicate efficiently in metabolically active non-dividing cells [ 15 , 16 ] as a result of the active nuclear import of their genome across the nuclear membrane of interphasic nuclei [ 17 ].
Retroviruses such as the Murine Leukaemia Virus MLV gain access to the nuclear chromatin following the disassembly of the nuclear membrane that occurs during mitosis [ 18 ]. For such retroviruses, evidence suggests that the viral capsid accompanies the viral genome into the nuclear compartment and participates in interaction with the chromatin [ 19 ] indicating that uncoating is not required prior to nuclear import.
HIV and other lentiviruses enter the nuclei via the nuclear pore; and, although commonly assumed, it is by no means certain that they can use an alternative route of entry during mitosis. In addition, HIV-1 mutants with a nuclear import defect in cell cycle-arrested cells often maintain this defect in cycling cells [ 22 - 25 ]. Finally, the assumption that HIV-1 might passively gain access to the chromatin upon mitosis, if based on the belief that cytoplasmic and nuclear contents mix homogeneously throughout mitosis, is not valid.
Indeed, evidence suggests that mitotic cells maintain spatial information through gradients, such as the RanGTP gradient that surrounds chromatin [ 26 , 27 ]. Taken together, it is probable that HIV-1 enters nuclei only through the nuclear pore whether cells divide or not. Consistent with this notion, previous work has reported a substantial difference in mass between cytoplasmic and nuclear HIV-1 complexes [ 28 , 29 ] and the absence of CA within pre-integration complexes [ 30 , 31 ].
Uncoating is required for formation of the reverse transcription complex RTC and is likely triggered by the sudden change in environment in which the viral complex finds itself, or possibly by the loss of high concentrations of free CA present in virions and responsible for maintaining metastable cores [ 36 ].
In this model, the absence of significant amounts of CA within intracellular HIV-1 complexes soon after inoculation [ 28 , 29 , 37 - 39 ] and the inability to detect capsids in the cytoplasm of infected cells using transmission electron microscopy TEM [ 40 ] led to the conclusion that the primary function of HIV-1 capsid is to deliver the viral genome into the cytoplasm, after which it can and must be discarded for productive infection to proceed, although it is not excluded that initial disassembly is partial [ 32 - 35 ].
A second model proposes that capsid remains intact for some time post-entry, at least for the initiation of reverse transcription, and that uncoating occurs gradually during transport towards the nucleus and reverse transcription [ 41 ].
In support of this hypothesis are studies that report a broad range of different sizes and shapes for cytoplasmic HIV-1, both greater and smaller than mature extracellular cores This suggests a complex series of transformations accompanying reverse transcription and transport to the nucleus [ 39 , 41 - 43 ] although it cannot be excluded that the observed variations are due to the preparation or isolation protocol [ 44 ] , immunofluorescent microscopy showing association of CA with RTCs [ 42 ], and the demonstration that capsids with increased or decreased core stability has impaired reverse transcription [ 14 ].
A third model, which we favour, proposes that capsids remain intact until HIV-1 incoming complexes reach the nuclear membrane and that uncoating occurs at the nuclear pore upon completion of reverse transcription. In this model, the HIV-1 capsid is all-important for maintaining a high stoichiometry of HIV-1 reverse transcriptase enzyme relative to the viral genome during reverse transcription to counteract its tendency to dissociate from its template [ 45 ], since dilution of reverse transcriptase in the cytoplasm would lead to highly ineffective reverse transcription.
While it organises the HIV-1 viral genome and proteins, it offers no impermeable environment from small macromolecules of the cytoplasm: the capsid lattice is an open structure, with inter-ring spacings of up to 10 nm [ 6 ], which allow small macromolecules, such as nucleotide triphosphates and indeed reverse transcriptase inhibitors, to access the reverse transcription complex in the cytoplasm of infected cells.
While the first model proposes that uncoating at the plasma membrane is required to trigger viral reverse transcription, this third model suggests that it is the successful completion of reverse transcription at the nuclear pore that triggers uncoating. Recent evidence suggests that the integrity and timely disassembly of the HIV-1 capsid are essential for routing to the nuclear compartment, reverse transcription and successful nuclear import [ 14 , 29 , 46 - 48 ].
For this reason, there is an increasing appeal to determine up to which point the viral capsid is required for infection and at which point in space and time it is disassembled. They are formed in the cytoplasm upon synthesis of full-length viral DNA and then translocate into the nucleus where they form the integrated provirus. Schematic representation of reverse transcription in lentiviruses and other orthoretroviruses such as MLV.
The conversion of the single-strand RNA genome represented as a black line into double stranded DNA genome at the bottom of the diagram is the hallmark of retroviruses.
The minus-strand strong-stop DNA thus synthesised is then transferred to the 3' end of the genome through complementarity with the R Repeated region of the LTR region Long-Terminal Region thus allowing synthesis of the minus-strand DNA to be completed. In the case of lentiviruses, initiation also takes place at the cPPT. In the case of lentiviruses, plus-strand initiation in two distinct sites leads to a displacement of the downstream strand over ca nucleotides, terminating at the CTS and thus generating a discrete strand displacement called the central DNA Flap.
It is assumed that reverse transcription is triggered by the exposure of the viral complex to non-limiting deoxyribonucleotides in the cytoplasm [ 44 ]. Reverse transcription involves firstly the formation of the minus strand strong-stop DNA, a strand transfer event, and the synthesis of the minus strand DNA with concomitant degradation of the RNA template. Reverse transcription proceeds with synthesis of plus-strand DNA, involves a second strand transfer event, and terminates at a central termination sequence CTS in the centre of the genome.
The duration of reverse transcription varies according to the metabolic state of the cell and in the case of asynchronous infection. Full-length linear DNA may be detected as early as 4 h post-infection but reaches its peak at h post-infection [ 22 , 55 , 56 ]. Upon DNA Flap formation and completion of reverse transcription, the viral complex becomes a PIC, competent for import into the nucleus and integration within the host cell chromatin.
Although the complete identification of PIC components remains elusive due to the difficulty to isolate PICs from infected cells, many viral and cellular factors have been identified as PIC components [ 34 ]. Although interaction of these cellular factors with HIV-1 PICs may occur in the cytoplasm, their role in HIV-1 infection becomes apparent in the nucleus where they may assist tethering of the PIC to the chromatin, determine integration site selection and assist integration [ 31 , 58 , 59 ].
Clearly, the transition between RTC and PIC is associated with uncoating, however the fragile nature of the HIV-1 capsid and the complexity of the early phases of HIV-1 infection have made it particularly difficult to pinpoint when this occurs.
Using these approaches, CA was not found to be substantially associated with the viral genome within the cytoplasm of infected cells, thus leading to the conclusion that the viral capsid is discarded from RTCs rapidly after cell entry [ 28 , 29 , 37 - 39 ]. However, the HIV-1 capsid is inherently unstable and disassembles readily in the presence of non-ionic detergents and upon ultracentrifugation [ 2 , 60 ].
Therefore, it cannot be excluded that the complexes analysed by biochemical isolation approaches have in fact lost their capsid during the isolation procedure. This is the case of viral particles that have entered by endocytosis [ 62 ] and of functional RTCs that are lost in the routing process towards the nucleus [ 42 ]. As a result, the majority of cytoplasmic RTCs isolated early after infection likely represent complexes that were damaged or engaged in a pathway of degradation at the time of isolation or observation.
In order to circumvent the need to isolate RTCs from infected cells, some groups have attempted to visualise HIV-1 capsids by in situ ultrastructural electron microscopy in infected cells.
Generally speaking however, it is difficult to follow the fate of viral complexes inside the cytoplasm using morphological criteria in sections of electron microscopy EM [ 63 ]. Any other cut will lead to heterogeneous circular and ovoid structures. Using this ultrastructural approach with TEM, few [ 47 ] or no [ 40 ] intact virus cores were observed in the cytoplasm of infected cells. The intrinsic difficulty of studying HIV-1 uncoating has driven the development of alternative approaches.
Using in situ immunohistochemical approaches, HIV-1 CA is readily detected throughout the cytoplasm of infected cells and co-localises with the viral genome [ 42 , 47 ]. Nevertheless, in order to demonstrate that this CA signal corresponds to capsid cores rather than soluble CA, immunolabelling of CA or detection of the viral genome must be combined with ultrastructural observations.
This is especially difficult to achieve using TEM since preparations generally favour either ultrastructural observations or immunolabelling. One approach to overcome this involves detection of the viral DNA using in situ hybridisation with electron microscopy [ 64 ] without the usual protease treatment in order to preserve proteinaceous structures as much as possible. Using this approach, capsid shells could be detected around the viral genome but with weak intensity [ 47 ]. An alternative approach involves observing intracellular complexes, in situ via a scanning EM SEM , in cells stripped of their plasma membrane [ 47 , 65 ].
To study uncoating in a quantitative manner, particulate intact capsids may be separated from soluble monomeric CA by ultracentrifugation of virions through a sucrose cushion overlaid with a low concentration of detergent [ 66 ]. This cell-free assay enabled to analyse the effects of mutations on capsid stability [ 14 , 67 , 68 ] and of reverse transcription on capsid integrity [ 47 ].
A variation of this assay enables the study of capsid uncoating in infected cells by carefully designed ultracentrifugation of cell lysates through a sucrose cushion, which separates cytosolic cores from soluble CA [ 69 ]. This fate-of-capsid assay has been used for example to establish a correlation between retroviral restriction and accelerated uncoating [ 69 , 70 ], to study capsid stability in infected cells [ 71 ], and quantify retroviral restriction potency and kinetics [ 72 , 73 ].
Given the complexity and fragile nature of the HIV-1 capsids, there is still a need for sensitive, specific and reliable assays for uncoating. No assay may be relied upon solely when interpreting uncoating events.
Retroviral restriction mechanisms are thought to have evolved in many species including primates as a result of evolutionary pressure exerted by continual exposure to retroviruses [ 77 ]. The startling evolutionary conservation of recognition of capsid cores as restriction mechanism suggests that their structure, composition and stability are key to retroviral infections. These findings indicated for the first time that premature uncoating of HIV-1, far from being beneficial for initiation of reverse transcription and infection, is in fact detrimental to both and is the molecular cornerstone for potent species-specific retroviral restrictions.
This suggests that the stability and integrity of HIV-1 capsids during the early steps of infection is key to effective replication. Schematic representation of the fates of viral capsids in the cytoplasm of newly infected cells. After entry into the cytoplasm, HIV-1 capsids that are on a path of productive infection remain intact and are transported towards the nucleus along the cytoskeleton.
They uncoat at the nuclear membrane upon completion of reverse transcription. Similarly, compromised uncoating, in the case of incomplete reverse transcription or of hyperstable capsid mutants, also leads to a dead-end infection event. One hypothesis proposes that uncoating might occur in response to changes in viral nucleic acid nature and structure, rather than in changes in cellular environment. If this were the case, then inhibiting reverse transcription would be expected to arrest uncoating at a very defined and reproducible step.
Strikingly, blocking reverse transcription by a reverse transcriptase inhibitor Nevirapine resulted in the accumulation of conical capsid cores in proximity to and at the nuclear membrane and nuclear pores at late time points post-infection [ 47 ].
These were formally identified as HIV-1 capsids based on specific anti-CA labelling and morphological criteria, and were shown to contain the viral genome using EM in situ hybridisation. Therefore, although incoming HIV-1 capsids may undergo stepwise destabilisation during cell entry and cytoplasmic transport, these data suggest that HIV-1 uncoating is not progressive but occurs upon completion of reverse transcription. They also suggest that progression through reverse transcription and uncoating is independent of transport towards the nucleus.
The search for the determinants of nuclear import that allow HIV-1 and other lentiviruses to infect non-dividing cells is an active and controversial field of investigation [ 85 ]. Based on the search of nuclear localization sequences, a number of HIV-1 proteins have been proposed to contribute in a redundant manner to the karyophilic properties of the HIV-1 PIC but the actual participation of these proteins in HIV-1 genome nuclear import has been a matter of strong debate [ 85 ].
The integrase protein, which is tightly associated with PICs until the integration of the viral DNA into the host chromosomes, is karyophilic and may participate in HIV-1 nuclear import. The cis-acting sequences cPPT and CTS, which form the central DNA Flap during reverse transcription, have also been identified as determinants of HIV-1 genome nuclear import [ 22 ] and are as a result systematically inserted within lentiviral vectors to enhance gene transfer efficiencies.
Intriguingly, recent work based on capsid mutants or chimeras has introduced the existence of a functional link between the HIV-1 CA and nuclear import [ 46 , 48 , 86 ], underlying the importance of timely uncoating for nuclear import.
Uncoating is necessary for passage through the nuclear pore, and HIV-1 complexes that fail to uncoat will accumulate at the cytoplasmic face of the nuclear membrane [ 47 ]. However, these data further suggest that HIV-1 CA may also be essential to mediate interaction with the nuclear pore, with transport proteins such as transportin-SR2, or with nucleoporins prior to uncoating. Previous work led us to suppose that the HIV-1 capsid core, although all-essential for initial delivery into the cytoplasm, is then discarded immediately post-fusion to stimulate reverse transcription.
Others propose that uncoating probably occurs gradually, possibly in response to multiple cellular cues such as interaction with cellular proteins or subcellular localisation, or viral cues such as the progress of reverse transcription. Although incoming capsids may undergo progressive destabilisation during their transport towards the nucleus, since hyperstable capsid mutants have impaired reverse transcription [ 14 ], recent independent experiments suggest that the position and timing of uncoating may in fact be tightly regulated and have a trigger.
Although the intricacies of HIV-1 uncoating - its timing, location and mechanism - are by no means resolved, recent work enables us to etch a possible model for the early steps of HIV-1 infection. Entry of HIV-1 into target cells delivers the intact capsid core into the cytoplasm and exposure of the viral nucleoprotein complex to non-limiting deoxyribonucleotides triggers reverse transcription. This likely occurs within the intact capsid core, which is essential for maintaining a high concentration of enzyme around the nucleic acid while being entirely permeable to the necessary deoxyribonucleotides.
During reverse transcription, HIV-1 RTCs move rapidly toward the nuclear compartment, using microtubules then actin filaments to reach the nuclear pore [ 42 , 47 ]. This implies that subcellular fractionation experiments that do not distinguish between nuclear membrane and nucleoplasm are in fact incapable of distinguishing nuclear from cytoplasmic HIV-1 complexes. Indeed, HIV-1 complexes docked at the nuclear membrane will appear in the nuclear fraction even though they are in fact in the cytoplasm.
Using a yeast-two-hybrid screen and interaction assays with capsid cores, we identified several components of the microtubule and actin network as interaction partners for HIV-1 capsid and essential co-factors of HIV-1 infection A.
Becker, S. Munier, N. Arhel, unpublished data. Therefore, as well as being essential for reverse transcription, the capsid shell may also be key to bringing viral complexes to their site of replication.
One hypothesis brought forward is that the completion of reverse transcription and the formation of the central DNA Flap trigger or facilitate uncoating [ 47 ]. If this is the case, then the trigger for uncoating is not a cellular cue, as is the case for adenoviruses [ 91 ] or herpes simplex virus type 1 [ 92 ], but a viral signal. Concordant with this, uncoating can occur in vitro upon synthesis of full-length viral DNA by endogenous reverse transcription, suggesting that any cellular factors required for uncoating are present within HIV-1 virions [ 47 ].
Apart from the DNA Flap, other viral and cellular factors have been proposed to participate in uncoating, including IN [ 93 ], prolyl isomerases Pin1 [ 94 ] and Cyclophilin A [ 72 ], and cellular factors present in non-resting cells [ 95 ]. A further hypothesis is that uncoating occurs at the nuclear pore [ 47 ] and allows PICs to be imported into the nucleus.
Similarly, complexes that fail to uncoat, such as hyperstable capsid mutants or in the case of inhibited reverse transcription, cannot be imported into the nucleus. Much of previous work was interpreted in light of the assumption that HIV-1 uncoating occurred immediately post-fusion and the association of CA with intracellular HIV-1 was understood to be detrimental for HIV-1 infection.
If we accept the premise that HIV-1 capsids uncoat at the nuclear pore upon completion of reverse transcription, our interpretation of data must be reversed: the loss of capsid cores after entry then corresponds to early degradation products of abortive complexes and the maintaining of intact capsids to complexes on the path of productive infection.
The molecular mechanisms underlying the destabilisation and uncoating of HIV-1 in the cytoplasm of infected cells remain to be elucidated. Both cytoplasmic environment and major rearrangements of the RTC at the end of reverse transcription could contribute to the disassembly of capsids prior to nuclear import.
Apologies are extended to those colleagues whose studies could not be mentioned due to space limitation. National Center for Biotechnology Information , U.
Journal List Retrovirology v. Published online Nov Nathalie Arhel 1. Author information Article notes Copyright and License information Disclaimer. Corresponding author. Nathalie Arhel: rf. Received Sep 7; Accepted Nov This article has been cited by other articles in PMC. Abstract HIV uncoating is defined as the loss of viral capsid that occurs within the cytoplasm of infected cells before entry of the viral genome into the nucleus.
Open in a separate window. Figure 1. The necessity of uncoating for HIV-1 and other lentiviruses HIV-1 and other lentiviruses are unique among orthoretroviruses in their ability to replicate efficiently in metabolically active non-dividing cells [ 15 , 16 ] as a result of the active nuclear import of their genome across the nuclear membrane of interphasic nuclei [ 17 ].
Where and when does uncoating occur? Figure 2. Figure 3. Lessons from reverse transcription: unsuccessful reverse transcription precludes uncoating One hypothesis proposes that uncoating might occur in response to changes in viral nucleic acid nature and structure, rather than in changes in cellular environment.
Lessons from nuclear import: timely uncoating underlies the ability of HIV-1 to infect non-dividing cells The search for the determinants of nuclear import that allow HIV-1 and other lentiviruses to infect non-dividing cells is an active and controversial field of investigation [ 85 ].
Conclusion: rethinking HIV-1 uncoating Previous work led us to suppose that the HIV-1 capsid core, although all-essential for initial delivery into the cytoplasm, is then discarded immediately post-fusion to stimulate reverse transcription. Perspectives The molecular mechanisms underlying the destabilisation and uncoating of HIV-1 in the cytoplasm of infected cells remain to be elucidated. Competing interests The author declares that they have no competing interests.
Spatial visualization of the maturing HIV-1 core and its linkage to the envelope. Biochemical and structural analysis of isolated mature cores of human immunodeficiency virus type 1.
J Virol. Structural organization of authentic, mature HIV-1 virions and cores. Avoiding polemics and accusations, Epstein provides a benchmark account of the AIDS epidemic to date, one that will be as useful to activists, policy makers, and general readers as to sociologists, physicians, and scientists.
The work on which this book is based won the American Sociological Association's award for best dissertation of the year. Virus The Triumph of Retrovirology 2. The Dynamics of Closure: Whither the Controversy? Inside and Outside the System 9. Books Digital Products Journals. Disciplines Health Health Care. About the Book In the short, turbulent history of AIDS research and treatment, the boundaries between scientist insiders and lay outsiders have been crisscrossed to a degree never before seen in medical history.
The concept of Obligatory passage point OPP was developed by sociologist Michel Callon in a seminal contribution to actor—network theory : Callon, Michel , "Elements of a sociology of translation: Domestication of the Scallops and the Fishermen of St Brieuc Bay". In John Law Ed. London, Routledge: Obligatory passage points are a feature of actor-networks, usually associated with the initial problematization phase of a translation process.
An OPP can be thought of as the narrow end of a funnel, that forces the actors to converge on a certain topic, purpose or question. The OPP thereby becomes a necessary element for the formation of a network and an action program. The OPP thereby mediates all interactions between actors in a network and defines the action program. Obligatory passage points allow for local networks to set up negotiation spaces that allow them a degree of autonomy from the global network of involved actors.
If a project is unable to impose itself as a strong OPP between the global and local networks, it has no control over global resources such as financial and political support, which can be misused or withdrawn. Additionally, a weak OPP is unable to take credit for the successes achieved within the local network, as outside actors are able to bypass its control and influence the local network directly. An action program can comprise a number of different OPP's.
An OPP can also be redefined as the problematization phase is revisited. In Callon and Law's '"Engineering and Sociology in a Military Aircraft Project"  the project management of a project to design a new strategic jet fighter for the British Military became an obligatory passage point between representatives of government and aerospace engineers.
In recent years, the notion of the Obligatory Passage Point has taken hold in the Information Systems Security and Information Privacy disciplines and journals. Backhouse et al. From Wikipedia, the free encyclopedia. Please help improve this article by adding citations to reliable sources. Unsourced material may be challenged and removed. In Wiebe E. Bijker and John Law ed. Ecologies of knowledge: Work and politics in science and technology.
State University of New York Press. Circuits of power in creating de jure standards: Shaping an international information systems security standard. Management Information Systems Quarterly, 30 3 , — Categories : Actor-network theory Sociology stubs. Hidden categories: Articles needing additional references from December All articles needing additional references All stub articles.