Jacek Skowronski

Vpx relieves inhibition of HIV-1 infection of macrophages mediated by the SAMHD1 protein

Macrophages and dendritic cells have key roles in viral infections, providing virus reservoirs that frequently resist antiviral therapies and linking innate virus detection to antiviral adaptive immune responses. Human immunodeficiency virus 1 (HIV-1) fails to transduce dendritic cells and has a reduced ability to transduce macrophages, due to an as yet uncharacterized mechanism that inhibits infection by interfering with efficient synthesis of viral complementary DNA. In contrast, HIV-2 and related simian immunodeficiency viruses (SIVsm/mac) transduce myeloid cells efficiently owing to their virion-associated Vpx accessory proteins, which counteract the restrictive mechanism. Here we show that the inhibition of HIV-1 infection in macrophages involves the cellular SAM domain HD domain-containing protein 1 (SAMHD1). Vpx relieves the inhibition of lentivirus infection in macrophages by loading SAMHD1 onto the CRL4DCAF1 E3 ubiquitin ligase, leading to highly efficient proteasome-dependent degradation of the protein. Mutations in SAMHD1 cause Aicardi–Goutières syndrome, a disease that produces a phenotype that mimics the effects of a congenital viral infection. Failure to dispose of endogenous nucleic acid debris in Aicardi–Goutières syndrome results in inappropriate triggering of innate immune responses via cytosolic nucleic acids sensors. Thus, our findings show that macrophages are defended from HIV-1 infection by a mechanism that prevents an unwanted interferon response triggered by self nucleic acids, and uncover an intricate relationship between innate immune mechanisms that control response to self and to retroviral pathogens.

Altered T cell activation and development in transgenic mice expressing the HIV-1 nef gene.

The nef gene, which encodes related cytoplasmic proteins in both human (HIV) and simian (SIV) immunodeficiency viruses is dispensable for viral replication in vitro. In contrast, in vivo experiments have revealed that SIV nef is required for efficient viral replication and development of AIDS in SIV infected rhesus monkeys, thus indicating that nef plays an essential role in the natural infection. We show that expression of the Nef protein from the HIV‐1 NL43 isolate in transgenic mice perturbs development of CD4+ T cells in the thymus and elicits depletion of peripheral CD4+ T cells. Thymic T cells expressing NL43 Nef show altered activation responses. In contrast, Nef protein of the HIV‐1 HxB3 isolate does not have an overt effect on T cells when expressed in transgenic animals. The differential effects of the two HIV‐1 nef alleles in transgenic mice correlate with down‐regulation of CD4 antigen expression on thymic T cells. The differential interactions of the NL43 and HxB3 nef alleles with CD4 were reproduced in a transient assay in human CD4+ CEM T cells. Down‐regulation of CD4 by nef in both human and transgenic murine T cells indicates that the relevant interactions are conserved in these two systems and suggests that the consequences of Nef expression on the host cell function can be analyzed in vivo in the murine system. Our observations from transgenic mice suggest that nef‐elicited perturbations in T cell signalling play an important role in the viral life cycle in vivo, perhaps resulting in elimination of infected CD4+ T cells.

A dileucine motif in HIV-1 Nef is essential for sorting into clathrin-coated pits and for downregulation of CD4

Nef, a approximately 200 residue multifunctional regulatory protein of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), interacts with components of host cell signal transduction and clathrin-dependent protein sorting pathways. The downregulation of surface CD4 molecules and major histocompatibility complex (MHC) class I antigens by Nef is believed to be important in AIDS pathogenesis [1-7]. Nef contains a globular core domain and two disordered segments--a myristylated arm at the amino terminus and a carboxy-terminal loop projecting from the globular core [8,9]. Here, we aimed to determine the sorting signals in HIV-1 Nef that were responsible for its involvement in the clathrin-mediated pathway. We found that a sequence in the carboxy-terminal disordered loop of Nef is essential for downregulation of CD4. This sequence resembles the dileucine motif, one of two well-characterized sorting signals that target membrane proteins to clathrin-coated vesicles. The dileucine-motif-containing segment of Nef bound directly and specifically to the beta-adaptin subunit of the clathrin adaptor complexes AP-1 and AP-2, which are responsible for recruiting sorted proteins into coated pits. Unlike wild-type Nef, a mutant form of Nef that lacked the dileucine motif did not localize to clathrin-coated pits and did not downregulate CD4 expression, although it could downregulate MHC class I surface expression. Thus, the dileucine motif in HIV-1 is required for CD4 downregulation and for interaction with clathrin adaptor complexes.

Co-localization of HIV-1 Nef with the AP-2 adaptor protein complex correlates with Nef-induced CD4 down-regulation

The nef gene of human and simian immunodeficiency viruses is critical for AIDS pathogenesis. Its function in vivo is unknown, but in vitro natural isolates of Nef down‐regulate expression of the cell surface CD4 molecule, a component of the T cell antigen receptor and the viral receptor, by accelerating its endocytosis. We have used chimeric proteins comprised of the natural HIV‐1 NA7 Nef fused to a strongly fluorescing mutant of green fluorescent protein (GFP) to correlate Nef function with intracellular localization in human CD4‐positive Jurkat T cells. The NA7–GFP fusion protein co‐localizes with components of the clathrin coat, including clathrin and the β‐subunit of the AP‐2 adaptor protein complex, at discrete locations that are consistent with the normal cellular distribution of clathrin coats at the plasma membrane. The NA7–GFP protein is also found in the perinuclear region of the cell, which is likely to reflect the Golgi apparatus. Evidence from a CD4‐negative fibroblast cell line indicates that co‐localization of NA7–GFP with components of the clathrin coat does not require expression of the CD4 molecule. Analysis of a large panel of chimeric molecules containing mutant Nef moieties demonstrated that the N‐terminal membrane targeting signal cooperates with additional element(s) in the disordered loops in the Nef molecule to co‐localize the Nef protein with AP‐2 adaptor complexes at the cell margin. This localization of NA7–GFP correlates with, but is not sufficient for, down‐regulation of surface CD4 and at least one additional function of Nef is required. In T cells co‐expressing CD4 and NA7–GFP, CD4 at the cell surface is redistributed into a discrete pattern that co‐localizes with that of NA7–GFP. Our observations place NA7–GFP in physical proximity to AP‐2‐containing clathrin coat at the plasma membrane and imply that Nef interacts, either directly or indirectly, with a component of the AP‐2‐containing coat at this location. This evidence supports a model whereby Nef recruits CD4 to the endocytic machinery via AP‐2‐containing clathrin coats at the plasma membrane.

Separable functions of Nef disrupt two aspects of T cell receptor machinery: CD4 expression and CD3 signaling

The Nef protein alters T cell receptor (TCR) signaling in T cells and is critical for the pathogenesis of AIDS. We used a transient expression assay in a human CD4+ T cell line to analyze the interaction of Nef with the TCR machinery. We show that, in addition to down‐regulating CD4 expression on the cell surface, Nef blocks a receptor‐proximal event in CD3 signaling. Analysis of a large number of mutant Nef proteins demonstrated that the effects of Nef on CD4 expression and on CD3 signaling are separable. The ability of Nef to block CD3 signaling was selectively abolished by mutations in the central part of the Nef protein and in particular by those known to disrupt the SH3 binding surface in the structured core of Nef. In contrast, the ability of Nef to down‐regulate CD4 expression was selectively abolished by two clusters of mutations, one in the N‐terminal and one in the C‐terminal region of Nef. These two regions correspond to the two flexible loops in Nef as predicted by solution NMR analysis. We show that this general functional organization is conserved between the Nef proteins of the human and simian immunodeficiency viruses (HIV‐1 and SIV). Our data demonstrate that Nef has at least two independent mechanisms to alter TCR function and thus may interfere with a range of T cell responses.

Mechanism for down-regulation of CD28 by Nef

SIV and HIV Nef proteins disrupt T‐cell receptor machinery by down‐modulating cell surface expression of CD4 and expression or signaling of CD3‐TCR. Nef also down‐modulates class I major histocompatibility complex (MHC) surface expression. We show that SIV and HIV‐1 Nefs down‐modulate CD28, a major co‐stimulatory receptor that mediates effective T‐cell activation, by accelerating CD28 endocytosis. The effects of Nef on CD28, CD4, CD3 and class I MHC expression are all genetically separable, indicating that all are selected independently. In cells expressing a Nef‐green fluorescent protein (GFP) fusion, CD28 co‐localizes with the AP‐2 clathrin adaptor and Nef‐GFP. Mutations that disrupt Nef interaction with AP‐2 disrupt CD28 down‐regulation. Furthermore, HIV and SIV Nefs use overlapping but distinct target sites in the membrane‐proximal region of the CD28 cytoplasmic domain. Thus, Nef probably induces CD28 endocytosis via the AP‐2 pathway, and this involves a ternary complex containing Nef, AP‐2 and CD28. The likely consequence of the concerted down‐regulation of CD28, CD4 and/or CD3 by Nef is disruption of antigen‐specific signaling machineries in infected T cells following a productive antigen recognition event.

Lentiviral Vpx Accessory Factor Targets VprBP/DCAF1 Substrate Adaptor for Cullin 4 E3 Ubiquitin Ligase to Enable Macrophage Infection

Vpx is a small virion-associated adaptor protein encoded by viruses of the HIV-2/SIVsm lineage of primate lentiviruses that enables these viruses to transduce monocyte-derived cells. This probably reflects the ability of Vpx to overcome an as yet uncharacterized block to an early event in the virus life cycle in these cells, but the underlying mechanism has remained elusive. Using biochemical and proteomic approaches, we have found that Vpx protein of the pathogenic SIVmac 239 strain associates with a ternary protein complex comprising DDB1 and VprBP subunits of Cullin 4–based E3 ubiquitin ligase, and DDA1, which has been implicated in the regulation of E3 catalytic activity, and that Vpx participates in the Cullin 4 E3 complex comprising VprBP. We further demonstrate that the ability of SIVmac as well as HIV-2 Vpx to interact with VprBP and its associated Cullin 4 complex is required for efficient reverse transcription of SIVmac RNA genome in primary macrophages. Strikingly, macrophages in which VprBP levels are depleted by RNA interference resist SIVmac infection. Thus, our observations reveal that Vpx interacts with both catalytic and regulatory components of the ubiquitin proteasome system and demonstrate that these interactions are critical for Vpx ability to enable efficient SIVmac replication in primary macrophages. Furthermore, they identify VprBP/DCAF1 substrate receptor for Cullin 4 E3 ubiquitin ligase and its associated protein complex as immediate downstream effector of Vpx for this function. Together, our findings suggest a model in which Vpx usurps VprBP-associated Cullin 4 ubiquitin ligase to enable efficient reverse transcription and thereby overcome a block to lentivirus replication in monocyte-derived cells, and thus provide novel insights into the underlying molecular mechanism.

Lentiviral Vpr usurps Cul4–DDB1[VprBP] E3 ubiquitin ligase to modulate cell cycle

The replication of viruses depends on the cell cycle status of the infected cells. Viruses have evolved functions that alleviate restrictions imposed on their replication by the host. Vpr, an accessory factor of primate lentiviruses, arrests cells at the DNA damage checkpoint in G2 phase of the cell cycle, but the mechanism underlying this effect has remained elusive. Here we report that Vpr proteins of both the human (HIV-1) and the distantly related simian (SIVmac) immunodeficiency viruses specifically associate with a protein complex comprising subunits of E3 ubiquitin ligase assembled on Cullin-4 scaffold (Cul4–DDB1[VprBP]). We show that Vpr binding to Cul4–DDB1[VprBP] leads to increased neddylation and elevated intrinsic ubiquitin ligase activity of this E3. This effect is mediated through the VprBP subunit of the complex, which recently has been suggested to function as a substrate receptor for Cul4. We also demonstrate that VprBP regulates G1 phase and is essential for the completion of DNA replication in S phase. Furthermore, the ability of Vpr to arrest cells in G2 phase correlates with its ability to interact with Cul4–DDB1[VprBP] E3 complex. Our studies identify the Cul4–DDB1[VprBP] E3 ubiquitin ligase complex as the downstream effector of lentiviral Vpr for the induction of cell cycle arrest in G2 phase and suggest that Vpr may use this complex to perturb other aspects of the cell cycle and DNA metabolism in infected cells.

HIV/simian immunodeficiency virus (SIV) accessory virulence factor Vpx loads the host cell restriction factor SAMHD1 onto the E3 ubiquitin ligase complex CRL4DCAF1.

Background: Human SAMHD1 protein restricts HIV/SIV infection of myeloid cells and is targeted for proteasomal degradation by HIV-2 Vpx protein. Results: Vpx binds the divergent C terminus of human SAMHD1 and loads it onto DCAF1 substrate receptor of CRL4 E3 ubiquitin ligase. Conclusion: Vpx programs SAMHD1 degradation by loading it onto CRL4DCAF1. Significance: Learning how viruses overcome innate anti-viral mechanisms is critical for the conception of new antiviral therapeutics. The sterile alpha motif and HD domain-containing protein-1 (SAMHD1) inhibits infection of myeloid cells by human and related primate immunodeficiency viruses (HIV and SIV). This potent inhibition is counteracted by the Vpx accessory virulence factor of HIV-2/SIVsm viruses, which targets SAMHD1 for proteasome-dependent degradation, by reprogramming cellular CRL4DCAF1 E3 ubiquitin ligase. However, the precise mechanism of Vpx-dependent recruitment of human SAMHD1 onto the ligase, and the molecular interfaces on the respective molecules have not been defined. Here, we show that human SAMHD1 is recruited to the CRL4DCAF1-Vpx E3 ubiquitin ligase complex by interacting with the DCAF1 substrate receptor subunit in a Vpx-dependent manner. No stable association is detectable with DCAF1 alone. The SAMHD1 determinant for the interaction is a short peptide located distal to the SAMHD1 catalytic domain and requires the presence of Vpx for stable engagement. This peptide is sufficient to confer Vpx-dependent recruitment to CRL4DCAF1 and ubiquitination when fused to heterologous proteins. The precise amino acid sequence of the peptide diverges among SAMHD1 proteins from different vertebrate species, explaining selective down-regulation of human SAMHD1 levels by Vpx. Critical amino acid residues of SAMHD1 and Vpx involved in the DCAF1-Vpx-SAMDH1 interaction were identified by mutagenesis. Our findings show that the N terminus of Vpx, bound to DCAF1, recruits SAMHD1 via its C terminus to CRL4, in a species-specific manner for proteasomal degradation.

HIV-1 Nef Binds the DOCK2–ELMO1 Complex to Activate Rac and Inhibit Lymphocyte Chemotaxis

The infectious cycle of primate lentiviruses is intimately linked to interactions between cells of the immune system. Nef, a potent virulence factor, alters cellular environments to increase lentiviral replication in the host, yet the mechanisms underlying these effects have remained elusive. Since Nef likely functions as an adaptor protein, we exploited a proteomic approach to directly identify molecules that Nef targets to subvert the signaling machinery in T cells. We purified to near homogeneity a major Nef-associated protein complex from T cells and identified by mass spectroscopy its subunits as DOCK2–ELMO1, a key activator of Rac in antigen- and chemokine-initiated signaling pathways, and Rac. We show that Nef activates Rac in T cell lines and in primary T cells following infection with HIV-1 in the absence of antigenic stimuli. Nef activates Rac by binding the DOCK2–ELMO1 complex, and this interaction is linked to the abilities of Nef to inhibit chemotaxis and promote T cell activation. Our data indicate that Nef targets a critical switch that regulates Rac GTPases downstream of chemokine- and antigen-initiated signaling pathways. This interaction enables Nef to influence multiple aspects of T cell function and thus provides an important mechanism by which Nef impacts pathogenesis by primate lentiviruses.