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Live-Cell Imaging of Early Steps of Single HIV-1 Infection

Published on May 19, 2018in Viruses3.811
· DOI :10.3390/v10050275
Ashwanth C. Francis5
Estimated H-index: 5
,
Gregory B. Melikyan23
Estimated H-index: 23
Abstract
Live-cell imaging of single HIV-1 entry offers a unique opportunity to delineate the spatio-temporal regulation of infection. Novel virus labeling and imaging approaches enable the visualization of key steps of HIV-1 entry leading to nuclear import, integration into the host genome, and viral protein expression. Here, we discuss single virus imaging strategies, focusing on live-cell imaging of single virus fusion and productive uncoating that culminates in HIV-1 infection.
  • References (81)
  • Citations (6)
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References81
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#1Ashwanth C. Francis (Emory University)H-Index: 5
#2Gregory B. Melikyan (Emory University)H-Index: 23
Summary The HIV-1 core consists of capsid proteins (CA) surrounding viral genomic RNA. After virus-cell fusion, the core enters the cytoplasm and the capsid shell is lost through uncoating. CA loss precedes nuclear import and HIV integration into the host genome, but the timing and location of uncoating remain unclear. By visualizing single HIV-1 infection, we find that CA is required for core docking at the nuclear envelope (NE), whereas early uncoating in the cytoplasm promotes proteasomal deg...
20 CitationsSource
#1Alan Engelman (Harvard University)H-Index: 65
#2Parmit Kumar Singh (Harvard University)H-Index: 5
Integration is central to HIV-1 replication and helps mold the reservoir of cells that persists in AIDS patients. HIV-1 interacts with specific cellular factors to target integration to interior regions of transcriptionally active genes within gene-dense regions of chromatin. The viral capsid interacts with several proteins that are additionally implicated in virus nuclear import, including cleavage and polyadenylation specificity factor 6, to suppress integration into heterochromatin. The viral...
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#1Chetan Sood (Emory University)H-Index: 4
#2Ashwanth C. Francis (Emory University)H-Index: 5
Last. Gregory B. Melikyan (Emory University)H-Index: 23
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Abstract Enveloped viruses transfer their genomes into host cells by fusing their membrane to that of the cell. To visualize single-virus fusion in living cells, researchers take advantage of HIV-1's proteolytic maturation, which can generate free fluorescent proteins within the viral particle. Co-labeling viruses with a content marker and a fluorescently tagged Vpr (a viral core protein) enables detection of single-virus fusions, but a major limitation of this approach is that not all viral par...
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#1Yingxin MaH-Index: 3
#2Mingxiu WangH-Index: 2
Last. Xian-En ZhangH-Index: 35
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HIV latency is one of the major problems in HIV/AIDS cure. Imaging single-copy integrated proviral HIV DNA in host cell has both virology and clinical significance but remains technical challenge. Here, we developed a dual-color labeled CRISPR system to image the HIV-1 integrated proviral DNA in latently infected cells. The pair of CRISPRs was fluorescently labeled with two different color QDs using two alternative bioorthogonal ligation reactions. Integrated HIV-sequences are successfully mappe...
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#1Masahiro Yamashita (Aaron Diamond AIDS Research Center)H-Index: 22
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After invasion of a susceptible target cell, HIV-1 completes the early phase of its life cycle upon integration of reverse-transcribed viral DNA into host chromatin. The viral capsid, a conical shell encasing the viral ribonucleoprotein complex, along with its constitutive capsid protein, plays essential roles at virtually every step in the early phase of the viral life cycle. Recent work has begun to reveal how the viral capsid interacts with specific cellular proteins to promote these processe...
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#1Joao Filipe Inacio Mamede (NU: Northwestern University)H-Index: 5
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After fusion, HIV delivers its conical capsid into the cytoplasm. To release the contained reverse-transcribing viral genome, the capsid must disassemble in a process termed uncoating. Defining the kinetics, dynamics, and cellular location of uncoating of virions leading to infection has been confounded by defective, noninfectious particles and the stochastic minefield blocking access to host DNA. We used live-cell fluorescent imaging of intravirion fluid phase markers to monitor HIV-1 uncoating...
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#1Ryan C. BurdickH-Index: 13
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#1Daniel M. Jones (University of Oxford)H-Index: 10
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Summary One of the key research areas surrounding HIV-1 concerns the regulation of the fusion event that occurs between the virus particle and the host cell during entry. Even if it is universally accepted that the large GTPase dynamin-2 is important during HIV-1 entry, its exact role during the first steps of HIV-1 infection is not well characterized. Here, we have utilized a multidisciplinary approach to study the DNM2 role during fusion of HIV-1 in primary resting CD4 T and TZM-bl cells. We h...
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#2Lieve DirixH-Index: 4
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We are grateful to Dr. J. Demeulemeester and Dr. J. De Rijck for critical reading and thank P. Van de Velde, B. Vanremoortel and NJ. Van der Veeken for their technical assistance. Viral vector production was performed at the Leuven Viral Vector Core. LEDGINs were synthesized by Cistim/CD3 (courtesy of Dr. A. Marchand). Personal fellowship from 'Agentschap voor Innovatie door Wetenschap en Technologie' [IWT 111595 to D.B.]; Personal fellowship from 'Fonds Wetenschappelijk Onderzoek' [FWO 11M0713N...
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Enveloped viruses infect target cells by fusing their membrane with cellular membrane through a process that is mediated by specialized viral glycoproteins. The inefficient and highly asynchronous nature of viral fusion complicates studies of virus entry on a population level. Single virus imaging in living cells has become an important tool for delineating the entry pathways and for mechanistic studies of viral fusion. We have previously demonstrated that incorporation of fluorescent labels int...
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