Requirements for the selective degradation of CD4 receptor molecules by the human immunodeficiency virus type 1 Vpu protein in the endoplasmic reticulum

BACKGROUND: HIV-1 Vpu targets newly synthesized CD4 receptor for rapid degradation by a process reminiscent of endoplasmic reticulum (ER)-associated protein degradation (ERAD). Vpu is thought to act as an adaptor protein, connecting CD4 to the ubiquitin (Ub)-proteasome degradative system through an interaction with beta-TrCP, a component of the SCFbeta-TrCP E3 Ub ligase complex. RESULTS: Here, we provide direct evidence indicating that Vpu promotes trans-ubiquitination of CD4 through recruitment of SCFbeta-TrCP in human cells. To examine whether Ub conjugation occurs on the cytosolic tail of CD4, we substituted all four Ub acceptor lysine residues for arginines. Replacement of cytosolic lysine residues reduced but did not prevent Vpu-mediated CD4 degradation and ubiquitination, suggesting that Vpu-mediated CD4 degradation is not entirely dependent on the ubiquitination of cytosolic lysines and as such might also involve ubiquitination of other sites. Cell fractionation studies revealed that Vpu enhanced the levels of ubiquitinated forms of CD4 detected in association with not only the ER membrane but also the cytosol. Interestingly, significant amounts of membrane-associated ubiquitinated CD4 appeared to be fully dislocated since they could be recovered following sodium carbonate salt treatment. Finally, expression of a transdominant negative mutant of the AAA ATPase Cdc48/p97 involved in the extraction of ERAD substrates from the ER membrane inhibited Vpu-mediated CD4 degradation. CONCLUSION: Taken together, these results are consistent with a model whereby HIV-1 Vpu targets CD4 for degradation by an ERAD-like process involving most likely poly-ubiquitination of the CD4 cytosolic tail by SCFbeta-TrCP prior to dislocation of receptor molecules across the ER membrane by a process that depends on the AAA ATPase Cdc48/p97.This work was performed by JB in partial fulfillment of her doctoral thesis and was supported by grants from the Canadian Institutes of Health Research (CIHR) (MOP-14228) and from the Fonds de la Recherche en Santé du Québec (FRSQ) to EAC. JB is the recipient of a studentship from the FRSQ and EAC holds the Canada Research Chair in Human Retrovirology

Association between disruption of CD4 receptor dimerization and increased human immunodeficiency virus type 1 entry

BACKGROUND: Human immunodeficiency virus (HIV) enters target cells by a membrane fusion process that involves a series of sequential interactions between its envelope glycoproteins, the CD4 receptor and CXCR4/CCR5 coreceptors. CD4 molecules are expressed at the cell surface of lymphocytes and monocytes mainly as monomers, but basal levels of CD4 dimers are also present at the cell surface of these cells. Previous evidence indicates that the membrane distal and proximal extracellular domains of CD4, respectively D1 and D4, are involved in receptor dimerization. RESULTS: Here, we have used A201 cell lines expressing two CD4 mutants, CD4-E91K, E92K (D1 mutant) and CD4-Q344E (D4 mutant), harboring dimerization defects to analyze the role of CD4 dimerization in HIV-1 entry. Using entry assays based on β-lactamase-Vpr or luciferase reporter activities, as well as virus encoding envelope glycoproteins derived from primary or laboratory-adapted strains, we obtained evidence suggesting an association between disruption of CD4 dimerization and increased viral entry efficiency. CONCLUSION: Taken together, our results suggest that monomeric forms of CD4 are preferentially used by HIV-1 to gain entry into target cells, thus implying that the dimer/monomer ratio at the cell surface of HIV-1 target cells may modulate the efficiency of HIV-1 entry

HIV-1 Vpr-Mediated G2 Arrest Involves the DDB1-CUL4AVPRBP E3 Ubiquitin Ligase

Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) has been shown to cause G2 cell cycle arrest in human cells by inducing ATR-mediated inactivation of p34cdc2, but factors directly engaged in this process remain unknown. We used tandem affinity purification to isolate native Vpr complexes. We found that damaged DNA binding protein 1 (DDB1), viral protein R binding protein (VPRBP), and cullin 4A (CUL4A)—components of a CUL4A E3 ubiquitin ligase complex, DDB1-CUL4AVPRBP—were able to associate with Vpr. Depletion of VPRBP by small interfering RNA impaired Vpr-mediated induction of G2 arrest. Importantly, VPRBP knockdown alone did not affect normal cell cycle progression or activation of ATR checkpoints, suggesting that the involvement of VPRBP in G2 arrest was specific to Vpr. Moreover, leucine/isoleucine-rich domain Vpr mutants impaired in their ability to interact with VPRBP and DDB1 also produced strongly attenuated G2 arrest. In contrast, G2 arrest–defective C-terminal Vpr mutants were found to maintain their ability to associate with these proteins, suggesting that the interaction of Vpr with the DDB1-VPRBP complex is necessary but not sufficient to block cell cycle progression. Overall, these results point toward a model in which Vpr could act as a connector between the DDB1-CUL4AVPRBP E3 ubiquitin ligase complex and an unknown cellular factor whose proteolysis or modulation of activity through ubiquitination would activate ATR-mediated checkpoint signaling and induce G2 arrest

Visuoperceptual deficits and participation in older adults after stroke

Introduction: Visuoperceptual deficits frequently occur after a stroke but little is known about how they evolve over time. These deficits may have an impact on participation in daily activities and social roles. Objectives: The aims were to 1) track changes over six months in the visual perception of older adults with persistent visuoperceptual deficits after a stroke; 2) examine if these changes differed between participants who had and had not received rehabilitation services; and 3) verify if participation differed between participants with and without visuoperceptual deficits. Methods: Visual perception as well as participation of 189 older adults who had had a stroke were evaluated in the first month (T1) after being discharged home from an acute care hospital (NO REHAB group) or rehabilitation unit (REHAB group). For visual perception, only participants presenting deficits at T1 were re-evaluated at 3 months (T2; n=93), and those with deficits at T2 were re-evaluated at 6 months (T3; n=61). Results: A total of 57 people (30.2%) had visuoperceptual deficits six months after discharge home. Despite persistent deficits, approximately 45% of the participants in the two groups improved while 50% of the NO REHAB group and 24.3% of the REHAB group deteriorated. Changes in the mean scores on the MVPT-V were similar in the two groups. Participation, and especially participation in social roles, was more restricted in participants with visuoperceptual deficits (p<0.001), whatever the severity of the stroke. Conclusion: Visuoperceptual deficits are common post-stroke. However, they evolve differently in different people and are associated with a reduction in participation

Bringing patient advisors to the bedside: a promising avenue for improving partnership between patients and their care team

This paper presents an innovative model of care, which brings patients who have already been through a similar experience of illness (patient advisors) directly to the bedside of patients, where they are viewed as full-fledged members of the clinical team. As part of a pilot project, three patient advisors were recruited and met with patients who had sustained a traumatic amputation and were admitted to the only center of expertise in replantation of the upper limb in Canada. Several individual interviews and focus groups with patients and patient advisors have revealed very promising results. Indeed, patients have expressed tremendous appreciation for their meetings and interactions with patient advisors. They have stated feeling less isolated, having a better morale and increased hopefulness regarding the outcome of the care pathway. Patient advisors also felt a positive impact of their involvement. A larger study needs to be conducted to determine the impact of this model of care on patient adherence to treatment and on members of the health care team

Antagonism of Tetherin Restriction of HIV-1 Release by Vpu Involves Binding and Sequestration of the Restriction Factor in a Perinuclear Compartment

The Vpu accessory protein promotes HIV-1 release by counteracting Tetherin/BST-2, an interferon-regulated restriction factor, which retains virions at the cell-surface. Recent reports proposed β-TrCP-dependent proteasomal and/or endo-lysosomal degradation of Tetherin as potential mechanisms by which Vpu could down-regulate Tetherin cell-surface expression and antagonize this restriction. In all of these studies, Tetherin degradation did not, however, entirely account for Vpu anti-Tetherin activity. Here, we show that Vpu can promote HIV-1 release without detectably affecting Tetherin steady-state levels or turnover, suggesting that Tetherin degradation may not be necessary and/or sufficient for Vpu anti-Tetherin activity. Even though Vpu did not enhance Tetherin internalization from the plasma membrane (PM), it did significantly slow-down the overall transport of the protein towards the cell-surface. Accordingly, Vpu expression caused a specific removal of cell-surface Tetherin and a re-localization of the residual pool of Tetherin in a perinuclear compartment that co-stained with the TGN marker TGN46 and Vpu itself. This re-localization of Tetherin was also observed with a Vpu mutant unable to recruit β-TrCP, suggesting that this activity is taking place independently from β-TrCP-mediated trafficking and/or degradation processes. We also show that Vpu co-immunoprecipitates with Tetherin and that this interaction involves the transmembrane domains of the two proteins. Importantly, this association was found to be critical for reducing cell-surface Tetherin expression, re-localizing the restriction factor in the TGN and promoting HIV-1 release. Overall, our results suggest that association of Vpu to Tetherin affects the outward trafficking and/or recycling of the restriction factor from the TGN and as a result promotes its sequestration away from the PM where productive HIV-1 assembly takes place. This mechanism of antagonism that results in TGN trapping is likely to be augmented by β-TrCP-dependent degradation, underlining the need for complementary and perhaps synergistic strategies to effectively counteract the powerful restrictive effects of human Tetherin

Effect of Calcium-Modulating Cyclophilin Ligand on Human Immunodeficiency Virus Type 1 Particle Release and Cell Surface Expression of Tetherin▿

The human immunodeficiency virus type 1 (HIV-1) accessory protein Vpu enhances virus particle release by counteracting a host factor that retains virions at the surfaces of infected cells. It was recently demonstrated that cellular protein BST-2/CD317/Tetherin restricts HIV-1 release in a Vpu-dependent manner. Calcium-modulating cyclophilin ligand (CAML) was also proposed to be involved in this process. We investigated whether CAML is involved in cell surface expression of Tetherin. Here, we show that CAML overexpression in permissive Cos-7 cells or CAML depletion in restrictive HeLa cells has no effect on HIV-1 release or on Tetherin surface expression, indicating that CAML is not required for Tetherin-mediated restriction of HIV-1 release