Characterizing the Interaction of Human CD4 and the HIV-1 accessory Protein VpU using Liquid State NMR

ABSTRACTThe HIV-1 accessory protein Vpr displays different activities potentially impacting viral replication, including the arrest of the cell cycle in the G2phase and the stimulation of apoptosis and DNA damage response pathways. Vpr also modulates cytokine production by infected cells, but this property remains partly characterized. Here, we investigated the effect of Vpr on the production of the proinflammatory cytokine tumor necrosis factor (TNF). We report that Vpr significantly increases TNF secretion by infected lymphocytes.De novoproduction of Vpr is required for this effect. Vpr mutants known to be defective for G2cell cycle arrest induce lower levels of TNF secretion, suggesting a link between these two functions. Silencing experiments and the use of chemical inhibitors further implicated the cellular proteins DDB1 and TAK1 in this activity of Vpr. TNF secreted by HIV-1-infected cells triggers NF-κB activity in bystander cells and allows viral reactivation in a model of latently infected cells. Thus, the stimulation of the proinflammatory pathway by Vpr may impact HIV-1 replicationin vivo.IMPORTANCEThe role of the HIV-1 accessory protein Vpr remains only partially characterized. This protein is important for viral pathogenesis in infected individuals but is dispensable for viral replication in most cell culture systems. Some of the functions described for Vpr remain controversial. In particular, it remains unclear whether Vpr promotes or instead prevents proinflammatory and antiviral immune responses. In this report, we show that Vpr promotes the release of TNF, a proinflammatory cytokine associated with rapid disease progression. Using Vpr mutants or inhibiting selected cellular genes, we show that the cellular proteins DDB1 and TAK1 are involved in the release of TNF by HIV-infected cells. This report provides novel insights into how Vpr manipulates TNF production and helps clarify the role of Vpr in innate immune responses and inflammation.

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A C-terminal domain of HIV-1 accessory protein Vpr is involved in penetration, mitochondrial dysfunction and apoptosis of human CD4+ lymphocytes

APOPTOSIS ◽

10.1023/a:1026487609215 ◽

1997 ◽

Vol 2 (1) ◽

pp. 69-76 ◽

Cited By ~ 41

Author(s):

C. Arunagiri ◽

I. Macreadie ◽

D. Hewish ◽

A. Azad

Keyword(s):

Mitochondrial Dysfunction ◽

Accessory Protein ◽

Terminal Domain ◽

Hiv 1 ◽

Cd4 Lymphocytes

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CD317/Tetherin Is Enriched in the HIV-1 Envelope and Downregulated from the Plasma Membrane upon Virus Infection

Journal of Virology ◽

10.1128/jvi.02421-09 ◽

2010 ◽

Vol 84 (9) ◽

pp. 4646-4658 ◽

Cited By ~ 74

Author(s):

Anja Habermann ◽

Jacomine Krijnse-Locker ◽

Heike Oberwinkler ◽

Manon Eckhardt ◽

Stefanie Homann ◽

...

Keyword(s):

Plasma Membrane ◽

Accessory Protein ◽

Wild Type ◽

Double Labeling ◽

Recycling Endosomes ◽

Immunodeficiency Virus ◽

Quantitative Immunoelectron Microscopy ◽

Golgi Network ◽

Hiv 1

ABSTRACT CD317/Bst-2/tetherin is a host factor that restricts the release of human immunodeficiency virus type 1 (HIV-1) by trapping virions at the plasma membrane of certain producer cells. It is antagonized by the HIV-1 accessory protein Vpu. Previous light microscopy studies localized CD317 to the plasma membrane and the endosomal compartment and showed Vpu induced downregulation. In the present study, we performed quantitative immunoelectron microscopy of CD317 in cells producing wild-type or Vpu-defective HIV-1 and in control cells. Double-labeling experiments revealed that CD317 localizes to the plasma membrane, to early and recycling endosomes, and to the trans-Golgi network. CD317 largely relocated to endosomes upon HIV-1 infection, and this effect was partly counteracted by Vpu. Unexpectedly, CD317 was enriched in the membrane of viral buds and cell-associated and cell-free viruses compared to the respective plasma membrane, and this enrichment was independent of Vpu. These results suggest that the tethering activity of CD317 critically depends on its density at the cell surface and appears to be less affected by its density in the virion membrane.

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HIV-1 Nef Antagonizes SERINC5 Restriction by Downregulation of SERINC5 via the Endosome/Lysosome System

Journal of Virology ◽

10.1128/jvi.00196-18 ◽

2018 ◽

Vol 92 (11) ◽

Cited By ~ 39

Author(s):

Jing Shi ◽

Ran Xiong ◽

Tao Zhou ◽

Peiyi Su ◽

Xihe Zhang ◽

...

Keyword(s):

Cell Surface ◽

Viral Pathogenesis ◽

Living Cells ◽

Restriction Factor ◽

Accessory Protein ◽

Viral Infectivity ◽

Bafilomycin A1 ◽

Mhc I ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACTThe primate lentiviral accessory protein Nef downregulates CD4 and major histocompatibility complex class I (MHC-I) from the cell surface via independent endosomal trafficking pathways to promote viral pathogenesis. In addition, Nef antagonizes a novel restriction factor, SERINC5 (Ser5), to increase viral infectivity. To explore the molecular mechanism of Ser5 antagonism by Nef, we determined how Nef affects Ser5 expression and intracellular trafficking in comparison to CD4 and MHC-I. We confirm that Nef excludes Ser5 from human immunodeficiency virus type 1 (HIV-1) virions by downregulating its cell surface expression via similar functional motifs required for CD4 downregulation. We find that Nef decreases both Ser5 and CD4 expression at steady-state levels, which are rescued by NH4Cl or bafilomycin A1 treatment. Nef binding to Ser5 was detected in living cells using a bimolecular fluorescence complementation assay, where Nef membrane association is required for interaction. In addition, Nef triggers rapid Ser5 internalization via receptor-mediated endocytosis and relocalizes Ser5 to Rab5+early, Rab7+late, and Rab11+recycling endosomes. Manipulation of AP-2, Rab5, Rab7, and Rab11 expression levels affects the Nef-dependent Ser5 and CD4 downregulation. Moreover, although Nef does not promote Ser5 polyubiquitination, Ser5 downregulation relies on the ubiquitination pathway, and both K48- and K63-specific ubiquitin linkages are required for the downregulation. Finally, Nef promotes Ser5 colocalization with LAMP1, which is enhanced by bafilomycin A1 treatment, suggesting that Ser5 is targeted to lysosomes for destruction. We conclude that Nef uses a similar mechanism to downregulate Ser5 and CD4, which sorts Ser5 into a point-of-no-return degradative pathway to counteract its restriction.IMPORTANCEHuman immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) express an accessory protein called Nef to promote viral pathogenesis. Nef drives immune escapein vivothrough downregulation of CD4 and MHC-I from the host cell surface. Recently, Nef was reported to counteract a novel host restriction factor, Ser5, to increase viral infectivity. Nef downregulates cell surface Ser5, thus preventing its incorporation into virus particles, resulting in disruption of its antiviral activity. Here, we report mechanistic studies of Nef-mediated Ser5 downregulation in comparison to CD4 and MHC-I. We demonstrate that Nef binds directly to Ser5 in living cells and that Nef-Ser5 interaction requires Nef association with the plasma membrane. Subsequently, Nef internalizes Ser5 from the plasma membrane via receptor-mediated endocytosis, and targets ubiquitinated Ser5 to endosomes and lysosomes for destruction. Collectively, these results provide new insights into our ongoing understanding of the Nef-Ser5 arms race in HIV-1 infection.

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Expression of HIV-1 accessory protein Vif is controlled uniquely to be low and optimal by proteasome degradation

Microbes and Infection ◽

10.1016/j.micinf.2004.04.011 ◽

2004 ◽

Vol 6 (9) ◽

pp. 791-798 ◽

Cited By ~ 48

Author(s):

M FUJITA ◽

H AKARI ◽

A SAKURAI ◽

A YOSHIDA ◽

T CHIBA ◽

...

Keyword(s):

Accessory Protein ◽

Proteasome Degradation ◽

Hiv 1

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HIV-1 Accessory Protein Vpu Internalizes Cell-surface BST-2/Tetherin through Transmembrane Interactions Leading to Lysosomes

Journal of Biological Chemistry ◽

10.1074/jbc.m109.058305 ◽

2009 ◽

Vol 284 (50) ◽

pp. 35060-35072 ◽

Cited By ~ 150

Author(s):

Yukie Iwabu ◽

Hideaki Fujita ◽

Masanobu Kinomoto ◽

Keiko Kaneko ◽

Yukihito Ishizaka ◽

...

Keyword(s):

Cell Surface ◽

Accessory Protein ◽

Hiv 1

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Minor contribution of HLA class I-associated selective pressure to the variability of HIV-1 accessory protein Vpu

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2012.04.002 ◽

2012 ◽

Vol 421 (2) ◽

pp. 291-295 ◽

Cited By ~ 6

Author(s):

Zafrul Hasan ◽

Jonathan M. Carlson ◽

Hiroyuki Gatanaga ◽

Anh Q. Le ◽

Chanson J. Brumme ◽

...

Keyword(s):

Selective Pressure ◽

Hla Class I ◽

Class I ◽

Accessory Protein ◽

Minor Contribution ◽

Hiv 1

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S2 from Equine infectious anemia virus is an infectivity factor which counteracts the retroviral inhibitors SERINC5 and SERINC3

10.1101/065078 ◽

2016 ◽

Author(s):

Ajit Chande ◽

Cristiana Cuccurullo ◽

Annachiara Rosa ◽

Serena Ziglio ◽

Susan Carpenter ◽

...

Keyword(s):

Equine Infectious Anemia Virus ◽

Critical Role ◽

Host Factor ◽

Target Cells ◽

Accessory Protein ◽

Post Translational Modification ◽

Virus Particles ◽

Equine Infectious Anemia ◽

Anemia Virus ◽

Hiv 1

ABSTRACTThe lentivirus equine infectious anemia virus (EIAV) encodes S2, a pathogenic determinant important for virus replication and disease progression in horses. No molecular function has yet been linked to this accessory protein. We now report that S2 can replace the activity of Nef on HIV-1 infectivity, being required to antagonize the inhibitory activity of SERINC proteins on Nef-defective HIV-1. Similar to Nef, S2 excludes SERINC5 from virus particles and requires an ExxxLL motif predicted to recruit the clathrin adaptor AP2. Accordingly, a functional endocytic machinery is essential for S2-mediated infectivity enhancement, which is impaired by inhibitors of clathrin-mediated endocytosis. In addition to retargeting SERINC5 to a late endosomal compartment, S2 promotes the host factor degradation. Emphasizing the similarity with Nef, we show that S2 is myristoylated and, compatible with a crucial role of the post-translational modification, its N-terminal glycine is required for the anti-SERINC5 activity.EIAV-derived vectors devoid of S2 are less susceptible than HIV-1 to the inhibitory effect of both human and equine SERINC5. We then identified the envelope glycoprotein of EIAV as a determinant which also modulates retrovirus susceptibility to SERINC5, indicating a bi-modular ability of the equine lentivirus to counteract the host factor.S2 shares no sequence homology with other retroviral factors known to counteract SERINC5. Adding to primate lentivirus Nef and gammaretrovirus glycoGag, the accessory protein from EIAV makes another example of a retroviral virulence determinant which independently evolved SERINC5-antagonizing activity. SERINC5 therefore plays a critical role for the interaction of the host with diverse retrovirus pathogens.Significance StatementSERINC5 and SERINC3 are recently discovered cellular inhibitors of retroviruses, which are incorporated into virus particles and impair their ability to propagate the infection to target cells. Only two groups of viruses (represented by HIV-1 and MLV) have so far been identified to have evolved the ability of counteracting SERINC inhibition. We now discovered that Equine infectious anemia virus, which causes a debilitating disease in horses, also acquired the ability to protect the virus particle from inhibition by SERINC5 and SERINC3, using its small protein S2. The evidence that three different retroviruses have independently evolved the ability to elude inhibition bySERINC5 and SERINC3 indicates that these cellular factors play a fundamental role against various retrovirus pathogens.

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A combined EM and proteomic analysis places HIV-1 Vpu at the crossroads of retromer and ESCRT complexes: PTPN23 is a Vpu-cofactor

PLoS Pathogens ◽

10.1371/journal.ppat.1009409 ◽

2021 ◽

Vol 17 (11) ◽

pp. e1009409

Author(s):

Charlotte A. Stoneham ◽

Simon Langer ◽

Paul D. De Jesus ◽

Jacob M. Wozniak ◽

John Lapek ◽

...

Keyword(s):

Cell Surface ◽

Protein Trafficking ◽

Fusion Proteins ◽

Immune Surveillance ◽

Accessory Protein ◽

Wild Type ◽

Membrane Systems ◽

Membrane Protein Trafficking ◽

Cellular Pathways ◽

Hiv 1

The HIV-1 accessory protein Vpu modulates membrane protein trafficking and degradation to provide evasion of immune surveillance. Targets of Vpu include CD4, HLAs, and BST-2. Several cellular pathways co-opted by Vpu have been identified, but the picture of Vpu’s itinerary and activities within membrane systems remains incomplete. Here, we used fusion proteins of Vpu and the enzyme ascorbate peroxidase (APEX2) to compare the ultrastructural locations and the proximal proteomes of wild type Vpu and Vpu-mutants. The proximity-omes of the proteins correlated with their ultrastructural locations and placed wild type Vpu near both retromer and ESCRT-0 complexes. Hierarchical clustering of protein abundances across the mutants was essential to interpreting the data and identified Vpu degradation-targets including CD4, HLA-C, and SEC12 as well as Vpu-cofactors including HGS, STAM, clathrin, and PTPN23, an ALIX-like protein. The Vpu-directed degradation of BST-2 was supported by STAM and PTPN23 and to a much lesser extent by the retromer subunits Vps35 and SNX3. PTPN23 also supported the Vpu-directed decrease in CD4 at the cell surface. These data suggest that Vpu directs targets from sorting endosomes to degradation at multi-vesicular bodies via ESCRT-0 and PTPN23.

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An amino acid polymorphism within the HIV-1 Nef dileucine motif functionally uncouples cell surface CD4 and SERINC5 downregulation

Journal of Virology ◽

10.1128/jvi.00588-21 ◽

2021 ◽

Author(s):

Mitchell J. Mumby ◽

Aaron L. Johnson ◽

Steven M. Trothen ◽

Cassandra R. Edgar ◽

Richard Gibson ◽

...

Keyword(s):

T Cell ◽

Cell Surface ◽

Disease Progression ◽

Adaptor Protein ◽

Accessory Protein ◽

Functional Impairments ◽

Cell Counts ◽

Dileucine Motif ◽

Cell Decline ◽

Hiv 1

Serine Incorporator 5 (SERINC5) reduces the infectivity of progeny HIV-1 virions by incorporating into the outer host-derived viral membrane during egress. To counter SERINC5, the HIV-1 accessory protein Nef triggers SERINC5 internalization by engaging the Adaptor Protein 2 (AP-2) complex using the [D/E]xxxL[L/I] 167 Nef dileucine motif. Nef also engages AP-2 via its dileucine motif to downregulate the CD4 receptor. Although these two Nef functions are related, the mechanisms governing SERINC5 downregulation are incompletely understood. Here, we demonstrate that two primary Nef isolates, referred to as 2410 and 2391 Nef, acquired from acutely HIV-1 infected women from Zimbabwe, both downregulate CD4 from the cell surface. However, only 2410 Nef retains the ability to downregulate cell surface SERINC5. Using a series of Nef chimeras, we mapped the region of 2391 Nef responsible for the functional uncoupling of these two antagonistic pathways to the dileucine motif. Modifications of the first and second ‘x’ positions of the 2410 Nef dileucine motif to asparagine and aspartic acid residues respectively (ND 164 ), impaired cell surface SERINC5 downregulation, which resulted in reduced infectious virus yield in the presence of SERINC5. The ND 164 mutation additionally partially impaired, but did not completely abrogate, Nef-mediated cell surface CD4 downregulation. Furthermore, the patient infected with HIV-1 encoding 2391 Nef had stable CD4 + T cell counts, whereas infection with HIV-1 encoding 2410 Nef resulted in CD4 + T cell decline and disease progression. Importance A contributing factor to HIV-1 persistence is evasion of the host immune response. HIV-1 uses the Nef accessory protein to evade the anti-viral roles of the adaptive and intrinsic innate immune responses. Nef targets SERINC5, a restriction factor which potently impairs HIV-1 infection by triggering SERINC5 removal from the cell surface. The molecular determinants underlying this Nef function remain incompletely understood. Recent studies have found a correlation between the extent of Nef-mediated SERINC5 downregulation and the rate of disease progression. Furthermore, single residue polymorphisms outside of the known Nef functional motifs can modulate SERINC5 downregulation. The identification of a naturally occurring Nef polymorphism impairing SERINC5 downregulation in this study supports a link between Nef downregulation of SERINC5 and the rate of plasma CD4 + T cell decline. Moreover, the observed functional impairments of this polymorphism could provide clues to further elucidate unknown aspects of the SERINC5 antagonistic pathway via Nef.

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