Macrophage Internal HIV-1 Is Protected from Neutralizing Antibodies

Koppensteiner, Herwig; Banning, Carina; Schneider, Carola; Hohenberg, Heinz; Schindler, Michael

doi:10.1128/jvi.05915-11

Cited by 70 publications

(101 citation statements)

References 47 publications

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“…peYFP-and peCFP-C1/N1 plasmids, the peCFP-eYFP Förster resonance energy transfer (FRET) positive control, the membrane-associated cyan fluorescent protein (MEM-CFP) control, and vectors encoding CD81 and HIV-1 NL4-3 Nef and Vpu fusion proteins have been described previously (38,39). A plasmid expressing CD82 N-terminally labeled with yellow fluorescent protein (YFP) was generated by PCR amplifying CD82 from a HeLa cell-derived cDNA library, introducing 5= XhoI and 3= EcoRI restriction sites.…”

Section: Methodsmentioning

confidence: 99%

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Vpu Is the Main Determinant for Tetraspanin Downregulation in HIV-1-Infected Cells

Lambelé

Koppensteiner

Symeonides

et al. 2015

J Virol

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Tetraspanins constitute a family of cellular proteins that organize various membrane-based processes. Several members of this family, including CD81, are actively recruited by HIV-1 Gag to viral assembly and release sites. Despite their enrichment at viral exit sites, the overall levels of tetraspanins are decreased in HIV-1-infected cells. Here, we identify Vpu as the main viral determinant for tetraspanin downregulation. We also show that reduction of CD81 levels by Vpu is not a by-product of CD4 or BST-2/ tetherin elimination from the surfaces of infected cells and likely occurs through an interaction between Vpu and CD81. Finally, we document that Vpu-mediated downregulation of CD81 from the surfaces of infected T cells can contribute to preserving the infectiousness of viral particles, thus revealing a novel Vpu function that promotes virus propagation by modulating the host cell environment. IMPORTANCEThe HIV-1 accessory protein Vpu has previously been shown to downregulate various host cell factors, thus helping the virus to overcome restriction barriers, evade immune attack, and maintain the infectivity of viral particles. Our study identifies tetraspanins as an additional group of host factors whose expression at the surfaces of infected cells is lowered by Vpu. While the downregulation of these integral membrane proteins, including CD81 and CD82, likely affects more than one function of HIV-1-infected cells, we document that Vpu-mediated lowering of CD81 levels in viral particles can be critical to maintaining their infectiousness. Tetraspanins are integral membrane proteins that span the lipid bilayer four times. The 33 members (in humans) of this protein family, by homo-and hetero-oligomerizing and by laterally interacting with other proteins and with lipids, form a web that serves as the basis for their involvement in the organization of membranes. When triggered by intra-or extracellular cues, socalled tetraspanin-enriched microdomains (TEMs) can form, and these platforms then support or modulate various membranebased processes, including cell adhesion, membrane fusion, signaling, and protein sorting. Consequently, tetraspanins play roles in a wide range of biological activities, such as fertilization, muscle formation and repair, generation of synaptic contacts at neuromuscular junctions, maintenance of skin integrity, and induction of immune responses (1-4). They are also implicated in pathologies, including cancer (e.g., metastasis [5]) and inherited disorders (6), as well as in the propagation and pathogenesis of numerous infectious agents (parasites, bacteria, and viruses) (7-11).While one member of the tetraspanin family (CD63) was shown more than 2 decades ago to be specifically acquired by HIV-1 particles released from infected cells (12)(13)(14), only during the past decade has work by several groups documented that tetraspanins play roles during different stages of the viral replication cycle (for recent reviews, see references 9 and 15). The tetraspanins CD9, CD53, CD63, CD81, CD...

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Section: Methodsmentioning

confidence: 99%

“…FACS-FRET was essentially done as described previously (38,39). In brief, 150,000 293T cells were seeded per 12-well plate and transfected with 2.5 g total DNA (1.25 g of each construct) using the calcium phosphate technique.…”

Section: Methodsmentioning

confidence: 99%

Vpu Is the Main Determinant for Tetraspanin Downregulation in HIV-1-Infected Cells

Lambelé

Koppensteiner

Symeonides

et al. 2015

J Virol

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“…Long-lived HIV-1-infected macrophages resist viral cytopathic effects and shelter replicationcompetent virions in surface-accessible (3)(4)(5)(6), but antibody-occluding (7,8), virus-containing compartments (VCCs) (6) for several weeks (9), which strongly implicates macrophages in HIV-1 persistence (10,11). Moreover, by efficiently spreading HIV-1 to uninfected CD4 ϩ T cells through direct cell-to-cell contact (4,12), macrophages may actively contribute to viral pathogenesis (10).…”

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confidence: 99%

High-Multiplicity HIV-1 Infection and Neutralizing Antibody Evasion Mediated by the Macrophage-T Cell Virological Synapse

Duncan

Williams

Schiffner

et al. 2014

J Virol

100

113

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“…We established and used this technique in the past to demonstrate and map, for instance, the interaction of HIV-1 Vpu and Ebola GP2 with the antiviral factor Tetherin/Bst-2 (6,18,19) and to show the interplay between HIV-1 Gag and tetraspanins (34). Also, other groups used our FACS-FRET approach to show direct protein interactions, confirming independently the robustness of the assay (35)(36)(37)(38).…”

Section: Hcv Protein Interactions Determined Via Facs-fret-tomentioning

confidence: 55%

The Intraviral Protein Interaction Network of Hepatitis C Virus

Hagen

Bayer

Rösch

et al. 2014

Molecular & Cellular Proteomics

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Hepatitis C virus (HCV) is a global health problem and one of the main reasons for chronic liver diseases such as cirrhosis and hepatocellular carcinoma. The HCV genome is translated into a polyprotein which is proteolytically processed into 10 viral proteins. The interactome of the HCV proteins with the host cell has been worked out; however, it remains unclear how viral proteins interact with each other. We aimed to generate the interaction network of these 10 HCV proteins using a flow-cytometrybased FRET assay established in our laboratory (Banning, C., Votteler, J., Hoffmann, D., Koppensteiner, H., Warmer, M., Reimer, R., Kirchhoff, F., Schubert, U., Hauber, J., and Hepatitis C virus (HCV)1 belongs to the family of Flaviviridae and is the only member of the genus Hepacivirus. The ϳ9.5-kB positive-strand RNA genome is directly translated via an internal ribosomal entry site into a polyprotein. This is proteolytically processed by cellular and viral proteases into structural (Core, E1, E2) and nonstructural (p7, NS2, NS3, NS4A/B, and NS5A/B) proteins (1). In recent decades, light was shed on the importance and biological relevance of most HCV proteins, which ultimately led to the development of the first specific antiviral therapy involving inhibition of the NS3 serine protease (2). However, because HCV is highly variable and because of the rapid emergence of drug resistance, additional therapeutic approaches are urgently needed (2). An impressive body of data was derived from protein interaction or siRNA screens investigating the interplay of HCV proteins with cellular factors (3-5). Although these screens are essential in order for researchers to understand how HCV manipulates the host cell, their potential benefit for novel therapeutic approaches could be limited. HCV is a chronic viral infection, and targeting host factors might result in drugs with severe adverse effects. Thus, a promising strategy would be to specifically inhibit interactions among viral proteins. Surprisingly, until now, a comprehensive analysis of the putative interactions and the interplay of HCV proteins with each other in living human cells has been lacking.In the present work, we did an extensive and thorough analysis of intra-HCV protein interactions. We used our novel flow-cytometry-based FRET assay that allows rapid assessment of the interplay between proteins in thousands of living cells (6). Therefore, this experimental approach enables quantification and statistical evaluation of all results. From the total of 20 interactions established by FACS-FRET, we chose to further investigate three that were not yet described in the literature. The putative HCV viroporin p7 binds to the structural proteins, and this was verified via biochemical methods in cells expressing fully infectious HCV.The established network of intra-HCV protein interactions in living mammalian cells provides new insights into the biology of this important human pathogen. Furthermore, we identified several HCV protein interactions that could be targeted for an...

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Macrophage Internal HIV-1 Is Protected from Neutralizing Antibodies

Cited by 70 publications

References 47 publications

Vpu Is the Main Determinant for Tetraspanin Downregulation in HIV-1-Infected Cells

Vpu Is the Main Determinant for Tetraspanin Downregulation in HIV-1-Infected Cells

High-Multiplicity HIV-1 Infection and Neutralizing Antibody Evasion Mediated by the Macrophage-T Cell Virological Synapse

The Intraviral Protein Interaction Network of Hepatitis C Virus

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