The human immunodeficiency virus type 1 accessory protein Vpu induces apoptosis by suppressing the nuclear factor kappaB-dependent expression of antiapoptotic factors.

Human immunodeficiency virus (HIV) type 1 Vpu is an integral membrane protein with a unique affinity for betaTrCP (TrCP), a key member of the SkpI-Cullin-F-box E3 ubiquitin ligase complex that is involved in the regulated degradation of cellular proteins, including IkappaB. Remarkably, Vpu is resistant to TrCP-mediated degradation and competitively inhibits TrCP-dependent degradation of IkappaB, resulting in the suppression of nuclear factor (NF)-kappaB activity in Vpu-expressing cells. We now report that Vpu, through its interaction with TrCP, potently contributes to the induction of apoptosis in HIV-infected T cells. Vpu-induced apoptosis is specific and independent of other viral proteins. Mutation of a TrCP-binding motif in Vpu abolishes its apoptogenic property, demonstrating a close correlation between this property of Vpu and its ability to inhibit NF-kappaB activity. The involvement of NF-kappaB in Vpu-induced apoptosis is further supported by the finding that the levels of antiapoptotic factors Bcl-xL, A1/Bfl-1, and TNF receptor-associated factor (TRAF)1, all of which are expressed in an NF-kappaB-dependent manner, are reduced and, at the same time, levels of active caspase-3 are elevated. Thus, Vpu induces apoptosis through activation of the caspase pathway by way of inhibiting the NF-kappaB-dependent expression of antiapoptotic genes.

Adipose tissue lymphocytes: types and roles.

Besides adipocytes, specialized in lipid handling and involved in energy balance regulation, white adipose tissue (WAT) is mainly composed of other cell types among which lymphocytes represent a non-negligible proportion. Different types of lymphocytes (B, alphabetaT, gammadeltaT, NK and NKT) have been detected in WAT of rodents or humans, and vary in their relative proportion according to the fat pad anatomical location. The lymphocytes found in intra-abdominal, visceral fat pads seem representative of innate immunity, while those present in subcutaneous fat depots are part of adaptive immunity, at least in mice. Both the number and the activity of the different lymphocyte classes, except B lymphocytes, are modified in obesity. Several of these modifications in the relative proportions of the lymphocyte classes depend on the degree of obesity, or on leptin concentration, or even fat depot anatomical location. Recent studies suggest that alterations of lymphocyte number and composition precede the macrophage increase and the enhanced inflammatory state of WAT found in obesity. Lymphocytes express receptors to adipokines while several proinflammatory chemokines are produced in WAT, rendering intricate crosstalk between fat and immune cells. However, the evidences and controversies available so far are in favour of an involvement of lymphocytes in the control of the number of other cells in WAT, either adipocytes or immune cells and of their secretory and metabolic activities. Therefore, immunotherapy deserves to be considered as a promising approach to treat the endocrino-metabolic disorders associated to excessive fat mass development.

Influence of acute and chronic administration of benzylamine on glucose tolerance in diabetic and obese mice fed on very high-fat diet.

The combination of vanadate plus benzylamine has been reported to stimulate glucose transport in rodent adipocytes and to mimic other insulin actions in diverse studies. However, benzylamine alone activates glucose uptake in human fat cells and increases glucose tolerance in rabbits. The aim of this work was to unravel the benzylamine antihyperglycemic action and to test whether its chronic oral administration could restore the defective glucose handling of mice rendered slightly obese and diabetic by very high-fat diet (VHFD). When VHFD mice were i.p. injected with benzylamine at 0.7 to 700 micromol/kg before glucose tolerance test, they exhibited reduced hyperglycemic response without alteration of insulin secretion. Whole body glucose turnover, as assessed by the glucose isotopic dilution technique, was unchanged in mice perfused with benzylamine (total dose of 75 micromol/kg). However, their in vivo glycogen synthesis rate was increased. Benzylamine appeared therefore to directly facilitate glucose utilisation in peripheral tissues. When given chronically at 2000 or 4000 micromol/kg/d in drinking water, benzylamine elicited a slight reduction of water consumption but did not change body weight or adiposity and did not modify oxidative stress markers. Benzylamine treatment improved glucose tolerance but failed to normalize the elevated glucose fasting plasma levels of VHFD mice. There was no influence of benzylamine ingestion on lipolytic activity, basal and insulin-stimulated glucose uptake, and on inflammatory adipokine expression in adipocytes. The improvement of glucose tolerance and the lack of adverse effects on adipocyte metabolism, reported here in VHFD mice allow to consider orally given benzylamine as a potential antidiabetic strategy which deserves to be further studied in other diabetic models.

Increased monoamine oxidase and semicarbazide-sensitive amine oxidase activities in white adipose tissue of obese dogs fed a high-fat diet.

Adipocytes express two types of amine oxidases: the cell surface semicarbazide-sensitive amine oxidase (SSAO) and the mitochondrial monoamine oxidase (MAO). In human abdominal subcutaneous adipose tissue, it has been reported that SSAO substrates stimulate glucose transport and inhibit lipolysis while MAO activity is decreased in obese patients when compared to age-matched controls. However, no information has been reported on visceral WAT. To further investigate the obesity-induced regulations of MAO and SSAO in white adipose tissue (WAT) from different anatomical locations, enzyme activities and mRNA abundance have been determined on tissue biopsies from control and high-fat fed dogs, an obesity model already described to be associated with arterial hypertension and hyperinsulinemia. MAO activity was increased in the enlarged omental WAT of diet-induced obese dogs, but not in their mesenteric WAT, another intra-abdominal fat depot. Subcutaneous WAT did not exhibit any change in MAO activity, as did the richest MAO-containing tissue: liver. Similarly, SSAO was increased in omental WAT of diet-induced obese dogs, but was not modified in other WAT and in aorta. The increase in SSAO activity observed in omental WAT likely results from an increased expression of the AOC3 gene since mRNA abundance and maximal benzylamine oxidation velocity were increased. Finally, plasma SSAO was decreased in obese dogs. Although the observed regulations differ from those found in subcutaneous WAT of obese patients, this canine model shows a tissue- and site-specific regulation of peripheral MAO and SSAO in obesity.

Prolonged treatment with the beta3-adrenergic agonist CL 316243 induces adipose tissue remodeling in rat but not in guinea pig: 2) modulation of glucose uptake and monoamine oxidase activity.

Beta3-adrenergic agonists are well-recognited to promote lipid mobilisation and adipose tissue remodeling in rodents, leading to multilocular fat cells enriched in mitochondria. However, effects of beta3-adrenergic agonists on glucose transport are still controversial. In this work, we studied in white adipose tissue (WAT) the influence of sustained beta3-adrenergic stimulation on the glucose transport and on the mitochondrial monoamine oxidase (MAO) activity. As one-week administration of CL 316243 (CL, 1 mg/kg/d) induces beta-adrenergic desensitization in rat but not in guinea pig adipocytes, attention was paid to compare these models. When expressing glucose uptake as nmoles of 2-deoxyglucose/100 mg cell lipids, maximally stimulated uptake was increased in adipocytes of WAT from treated rats but not from treated guinea pigs. However, basal hexose uptake was also increased in CL-treated rats and, as a consequence, the dose-dependent curves for insulin stimulation were similar in control and CL-treated rats when expressed as fold increase over basal. Insulin-induced lipogenesis was unchanged in rat or guinea pig adipocytes after CL-treatment. The glucose carriers GLUT4 and corresponding mRNA were increased in subcutaneous WAT or in brown adipose tissue (BAT) but not in visceral WAT or muscles of CL-treated rats. There was an increase of MAO activity in WAT and BAT, but not in liver, of CL-treated rats while no change was detected in guinea pigs. These findings show that only rat adipocytes, which are beta3-adrenergic-responsive, respond to chronic beta3-AR agonist by an increase of GLUT4 content and MAO activity, despite a desensitization of all beta-adrenoceptor subtypes.

HIV-1 associated down-modulation of CD4 gene expression is differentially restricted in lymphocytic and monocytic cell lines.

We previously demonstrated that chronic infection of a monocytic cell line (U-937) with human immunodeficiency virus type 1 (HIV-1) was not accompanied by down-modulation of CD4 transcription, unlike the situation with CD4+ T lymphocyte lines. To better understand the refractoriness of monocytes to alterations in levels of CD4 mRNA, we treated HIV-IIIB chronically infected U-937 cells with phorbol myristate acetate (PMA), a known stimulus of HIV gene expression. Although PMA caused a significant increase in HIV mRNA levels that was sustained over 7 days, no effect on CD4 transcript levels was noted. Clonal derivatives of HIV-IIIB-infected U-937 cells, which produced a variety of infectious and defective particles, were likewise not affected in ability to produce CD4 mRNA. To rule out the possibility that U-937 cells select out HIV-1 variants unable to modulate CD4 mRNA levels, we passaged infectious virus from a U-937 clonal derivative (UHC1) onto different monocytic and T lymphocytic cell lines. In monocytic cell lines (U-937, PLB-985, THP-1), we observed an avirulent infection that did not affect CD4 mRNA levels, whereas UHC1 infection of each of two T lymphocytic cell lines (CEM-T4, Jurkat) caused both cytopathic replication and reductions in CD4 mRNA levels. In one case (Jurkat), variants expressing low CD4 mRNA may have emerged, because the outgrowth no longer expressed viral products. In the other case (CEM-T4), high expression of viral genes was accompanied by CD4 mRNA down-modulation, suggesting either that low-CD4-expressing variants were selected that maintained viral gene expression or that CD4 gene expression was repressed by viral products.

Influence of high-fat diet on amine oxidase activity in white adipose tissue of mice prone or resistant to diet-induced obesity.

Decreased monoamine oxidase (MAO) activity has been observed in adipose tissue of obese patients. Since substrates of MAO and semicarbazide-sensitive amine oxidase (SSAO) can modify adipocyte metabolism, this work investigates whether changes in amine oxidase activity may occur during white adipose tissue (WAT) development. We evaluated MAO and SSAO activities in WAT of high-fat diet (HFD) and low-fat diet fed mice. To distinguish the effect of HFD on its own from the effect of fat mass enlargement, obesity-prone transgenic line of the FVBn strain lacking beta3-adrenergic receptors (AR) but expressing human beta3-AR and alpha2-AR (mbeta3-/-, hbeta3+/+, halpha2+/-) was compared to its obesity-resistant control (mbeta3-/-, hbeta3+/+). As already reported, the former mice became obese while the latter resisted to HFD. No significant change in SSAO or MAO activity was found in WAT of both strains after HFD when expressing oxidase activity per milligram of protein. However, when considering the overall capacity of the fat depots to oxidize tyramine or benzylamine, there was an increase in MAO and SSAO activity only in the enlarged WAT of HFD-induced obese mice. Therefore, the comparison of these models allowed to demonstrate that the higher amine oxidase capacity hold in enlarged fat stores of obese mice is more likely the consequence of increased fat cell number rather than the result of an increased expression of MAO or SSAO in the adipocyte.

Effects of oral administration of benzylamine on glucose tolerance and lipid metabolism in rats.

Repeated administration of benzylamine plus vanadate have been reported to exhibit anti-hyperglycemic effects in different models of diabetic rats. Likewise oral treatment with Moringa oleifera extracts which contain the alkaloïd moringine, identical to benzylamine, has also been shown to prevent hyperglycemia in alloxan-induced diabetic rats. With these observations we tested whether prolonged oral administration of benzylamine could interact with glucose and/or lipid metabolism. Seven week old male Wistar rats were treated for seven weeks with benzylamine 2.9 g/l in drinking water and were submitted to glucose tolerance tests. A slight decrease in water consumption was observed in benzylamine-treated animals while there was no change in body and adipose tissue weights at the end of treatment. Blood glucose and plasma insulin, triacylglycerol or cholesterol levels were not modified. However, benzylamine treatment resulted in a decrease in plasma free fatty acids in both fed and fasted conditions. Benzylamine treatment improved glucose tolerance as shown by the reduction of hyperglycemic response to intra-peritoneal glucose load. Oral benzylamine treatment did not alter the response of adipocytes to insulin nor to insulin-like actions of benzylamine plus vanadate, via in vitro activation of glucose transport or inhibition of lipolysis. This work demonstrates for the first time that oral administration of benzylamine alone influences glucose and lipid metabolism. However, these results obtained in normoglycemic rats require to be confirmed in diabetic models.

Amine oxidase substrates for impaired glucose tolerance correction.

Amine oxidases are widely distributed from microorganisms to vertebrates and produce hydrogen peroxide plus aldehyde when catabolizing endogenous or xenobiotic amines. Novel roles have been attributed to several members of the amine oxidase families, which cannot be anymore considered as simple amine scavengers. Semicarbazide-sensitive amine oxidase (SSAO) is abundantly expressed in mammalian endothelial, smooth muscle, and fat cells, and plays a role in lymphocyte adhesion to vascular wall, arterial fiber elastic maturation, and glucose transport, respectively. This latter role was studied in detail and the perspectives of insulin-like actions of amine oxidase substrates are discussed in the present review. Independent studies have demonstrated that SSAO substrates and monoamine oxidase substrates mimic diverse insulin effects in adipocytes: glucose transport activation, lipogenesis stimulation and lipolysis inhibition. These substrates also stimulate in vitro adipogenesis. Acute in vivo administration of amine oxidase substrates improves glucose tolerance in rats, mice and rabbits, while chronic treatments with benzylamine plus vanadate exert an antihyperglycaemic effect in diabetic rats. Dietary supplementations with methylamine, benzylamine or tyramine have been proven to influence metabolic control in rodents by increasing glucose tolerance or decreasing lipid mobilisation, without noticeable changes in the plasma markers of lipid peroxidation or protein glycation, despite adverse effects on vasculature. Thus, the ingested amines are not totally metabolized at the intestinal level and can act on adipose and vascular tissues. In regard with this influence on metabolic control, more attention must be paid to the composition or supplementation in amines in foods and nutraceutics.

Diminution of CD4 surface protein but not CD4 messenger RNA levels in monocytic cells infected by HIV-1.

As expected, the productive infection of several monocytic cell lines by HIV-1 led to a diminution of cell-surface CD4 antigen. However, unlike findings reported for HIV-1-infected T cells, this decrease was not accompanied by a similar reduction in levels of CD4 transcripts. OKT4 monoclonal antibodies (MAbs) to CD4 were used in immunoprecipitation experiments to show that intracellular CD4 levels were diminished in U-937 monocytic cells that had been infected by HIV-1. These MAbs also coprecipitated viral gp120, indicating that CD4-gp120 complexes are present in infected monocytes. Our results therefore demonstrate that cell-surface down-modulation of CD4 is exclusively a post-transcriptional event in HIV-1 infected monocytic cells. These data suggest that HIV-1-mediated depletion of cell-surface CD4 in monocytes does not involve transcript down-modulation as has been reported in T lymphocytes.

Clinical correlates and molecular basis of HIV drug resistance.

It has been widely reported that zidovudine (ZDV)-resistant variants of human immunodeficiency virus type 1 (HIV-1) can be isolated from patients undergoing prolonged therapy with this drug. At the same time, treatment of HIV-infected individuals with ZDV and other forms of nucleotide therapy, including didanosine (ddI), have enabled patients to live longer than would otherwise be the case and to enjoy improved quality of life. HIV resistance to ZDV, ddI, and other nucleosides is attributable to a series of point mutations within the pol gene of HIV-1 that encodes the viral enzyme, reverse transcriptase (RT). This is not surprising as the virus is known to replicate at high rates in infected individuals; moreover the RT that mediates transcription of proviral DNA from viral genomic RNA is known to be highly error prone. Thus, mutants of HIV-1, which possess a drug-resistance phenotype and genotype, may be expected to emerge under the selective pressure of long-term antiviral chemotherapy. This article describes a novel mutation at site 184 within the pol gene that accounts for resistance against both ddI and zalcitibine (ddC). HIV drug resistance occurs most commonly in individuals with low CD4 cell counts who have progressed to more serious forms of disease. Moreover, viruses obtained from patients with AIDS generally display higher levels of resistance, relative to pretreatment isolates, than do viruses from patients with more-limited illness. Although observations of drug resistance can be correlated with disease progression and a weakened immune system, it is still unclear whether a cause-and-effect relationship exists. Because of the error-prone nature of viral RT and the fact that the HIV-1 genome can mutate efficiently, it can be anticipated that viral drug resistance may emerge for all forms of nucleotide therapy to be offered in the future. In addition, resistance may also become apparent with regard to drugs that block HIV replication by acting at sites within the viral replication cycle other than RT.

The role and fate of the CD4 molecule in lymphocytes and monocytes infected by HIV-1.

Infection by HIV-1 of monocyte cell lines, in contrast to T lymphocytes, did not lead to decreased steady-state levels of CD4 mRNA. Similar results were also obtained using clonal derivatives of infected U-937 cells that produced either competent, highly replicative progeny viruses or defective non-infectious particles. In each case, the infected U-937 cells or clonal derivatives were found to be significantly deficient with regard to surface representation of CD4 protein, in spite of the presence of high levels of CD4 mRNA. However, both HIV-1-infected U-937 cells, as well as clonal derivatives which produced high levels of viral env mRNA and non-infectious viral structures that lacked envelope glycoproteins, contained diminished levels of OKT4-immunoprecipitable CD4 protein, in comparison with uninfected U-937 cells. Thus, expression of viral env mRNA but neither the efficient synthesis or packaging of viral glycoproteins or viral assembly is required for disappearance of cell surface CD4 to occur. Furthermore, viral gp160 co-precipitated with CD4 in both the parental and cloned cell lines. We have also shown that the generation of intracellular complexes of gp160 and CD4 is directly responsible for the disappearance of cell surface CD4 in HIV-1-infected U-937 cells. In this system, expression of gp160 was both necessary and sufficient to result in CD4 receptor down-modulation. Finally, in vitro co-translation studies revealed that the presence or synthesis of viral gp160 led to a failure to efficiently generate CD4 protein.

Moderate weight-lowering effect of octopamine treatment in obese Zucker rats.

Octopamine is proposed as a substitution product of synephrine by diverse drug industries that advertise new weight-lowering products or medicinal plants enriched in this biogenic amine. We have already reported that octopamine is able to activate in vitro lipolysis in rat adipocytes via beta3-adrenergic receptor activation, while it activates glucose uptake in human fat cells via its oxidation by amine oxidases. In this work, we tested whether a chronic challenge with octopamine could exert anti-obesity effects. A treatment consisting in daily i.p. administration of octopamine (81 micromol/kg) was compared on a four-week period with calorie restriction in the genetically obese Zucker rat. Octopamine treatment resulted in a 19% decrease in body weight gain, when compared to the 177 g gained by controls during the same period. The decrease in body weight gain was detectable only after three weeks of treatment and was apparently not due to a pronounced and sustainable anorectic effect of octopamine since: 1) cumulated food consumption was only reduced by 10%; 2) the experimental 18% reduction of food intake provoked a rapid decrease in body weight gain, significant in less than two weeks. The lipolytic responses to isoprenaline or octopamine and the stimulation of glucose transport by insulin or by the amine oxidase substrate tyramine were unmodified by the treatments. Noteworthy, the elevated plasma insulin of obese rats was lowered by octopamine. This study shows that octopamine can reduce body weight gain in obese rats, without apparent adverse effects, but with less efficacy than beta3-AR agonists.

Effect of prolonged treatment with tyramine on glucose tolerance in streptozotocin-induced diabetic rats.

The biogenic amine tyramine has been reported to stimulate in vitro glucose transport in adipocytes, cardiomyocytes and skeletal muscle, and to improve in vivo glucose utilization in rats. These effects were dependent on amine oxidation, since they were blocked by inhibitors of monoamine oxidase (MAO) and semicarbazide-sensitive amine oxidase (SSAO). We thus tested in this work whether a prolonged treatment with tyramine could improve glucose tolerance in streptozotocin-induced diabetic rats. First, tyramine content of standard rodent chow was determined by HPLC and daily tyramine intake of control rats was estimated to be around 26 micromol/kg body weight. Then, tyramine was administred during 3 weeks in streptozotocin-induced diabetic rats at 29 micromol/kg by daily i.p. injection alone or together with vanadate 0.02 micromol/kg. In another group of diabetic rats, tyramine was subcutaneously delivered at 116 micromol/kg/day by osmotic minipumps. All tyramine treatments resulted in a decrease of the hyperglycemic responses to an i.p. glucose load. Adipocytes isolated from either untreated or treated diabetic rats were sensitive to the stimulation of glucose uptake by tyramine. However, diabetic animals receiving tyramine for three weeks did not recover from their hyperglycemia, hypoinsulinemia and glucosuria. These results show that the improvement of glucose tolerance induced by prolonged tyramine administration occurs in an insulin-depleted model and probably results from peripheral insulin-like actions of the oxidation of MAO/SSAO substrates, such as the stimulation of glucose uptake into adipocytes.

Semicarbazide-sensitive amine oxidase substrates fail to induce insulin-like effects in fat cells from AOC3 knockout mice.

Substrates of semicarbazide-sensitive amine oxidases (SSAO) stimulate glucose transport in adipocytes. To definitively demonstrate the involvement of SSAO in this insulin-like effect, glucose transport has been studied in fat cells from mice with a targeted deletion of AOC3, a gene encoding a SSAO called vascular adhesion protein-1. SSAO activity was present in white adipose tissues of wild type (WT) but was absent in AOC3KO mice. The SSAO-substrates benzylamine and methylamine were unable to stimulate hexose transport in adipocytes isolated from AOC3KO mice while they were active in WT adipocytes, especially in combination with vanadate. Impairment of amine-dependent glucose uptake was also observed with tyramine while there was no change in insulin responsiveness. These observations prove that the effects of exogenous or biogenic amines on glucose transport are not receptor-mediated but are oxidation-dependent. They also confirm that the major SSAO form expressed in mouse adipocytes is encoded by the AOC3 gene.

Reduction of fat deposition by combined inhibition of monoamine oxidases and semicarbazide-sensitive amine oxidases in obese Zucker rats.

Semicarbazide-sensitive amine oxidase (SSAO) and monoamine oxidases (MAO) are highly expressed in adipocytes and generate hydrogen peroxide when activated. Consequently, high concentrations of MAO- or SSAO-substrates acutely stimulate glucose transport and inhibit lipolysis in isolated adipocytes in a hydrogen peroxide-dependent manner. Chronic treatments with MAO and SSAO substrates also increase in vitro adipogenesis and in vivo glucose utilization and fat deposition in diabetic rodents. To further investigate the interplay between amine oxidases, energy balance and fat deposition, prolonged MAO and/or SSAO blockade was performed in obese rats. Pargyline (P, MAO inhibitor), semicarbazide (S, SSAO inhibitor), alone or in combination (P+S), were daily i.p. administered for 3-5 weeks to obese Zucker rats at doses ranging from 20 to 300 micromol/kg. P+S treatments abolished MAO and SSAO activities in any tested tissue. P and S led to a 12-17% reduction of food intake when given in combination but were inactive when given separately. Despite a similar body weight gain reduction in P+S-treated and pair-fed rats, the mitigation of fat deposition was greater in rats receiving both inhibitors. Adipocytes from P+S-treated rats responded as control to insulin but exhibited impaired responses to tyramine, benzylamine or methylamine plus vanadate when considering glucose transport activation or lipolysis inhibition. Although our results did not directly demonstrate that amines are able to spontaneously produce in vivo the insulin-like effects described in vitro, we propose that P+S-induced reduction of fat deposition results from decreased food intake and from impaired MAO- and SSAO-dependent lipogenic and antilipolytic actions of endogenous or alimentary amines.

The human immunodeficiency virus (HIV) type 2 envelope protein is a functional complement to HIV type 1 Vpu that enhances particle release of heterologous retroviruses.

We have recently shown that the envelope glycoprotein of the ROD10 isolate of human immunodeficiency virus type 2 (HIV-2) has the ability to positively regulate HIV-2 viral particle release. The activity provided by the ROD10 Env was remarkably similar to that of the HIV-1 Vpu protein, thus raising the possibility that the two proteins act in a related fashion. We now show that the ROD10 Env can functionally replace Vpu to enhance the rate of HIV-1 particle release. When provided in trans, both Vpu and the ROD10 Env restored wild-type levels of particle release in a Vpu-deficient mutant of the NL4-3 molecular clone with indistinguishable efficiencies. This effect was independent of the presence of the HIV-1 envelope protein. The ROD10 Env also enhanced HIV-1 particle release in the context of HIV-2 chimeric viruses containing the HIV-1 gag-pol, indicating a lack of need for additional HIV-1 products in this process. In addition, we show for the first time that HIV-1 Vpu, as well as ROD10 Env, has the ability to enhance simian immunodeficiency virus (SIV) particle release. The effects of Vpu and ROD10 Env on SIV particle release were indistinguishable and were observed in the context of full-length SIVmac239 and simian-human immunodeficiency virus chimeras. These results further demonstrate that ROD10 Env can functionally complement Vpu with respect to virus release. In contrast, we found no evidence of a destabilizing activity of ROD10 Env on the CD4 molecule. HIV-1 and HIV-2 thus appear to have evolved genetically distinct but functionally similar strategies to resolve the common problem of efficient release of progeny virus from infected cells.

The human immunodeficiency virus type 1 Vpu protein specifically binds to the cytoplasmic domain of CD4: implications for the mechanism of degradation.

We have recently demonstrated that coexpression of Vpu and CD4 in HeLa cells results in the degradation of CD4 in the endoplasmic reticulum. The sensitivity of CD4 to Vpu-mediated degradation is conferred by the presence of specific sequences located between amino acids 402 and 420 in the CD4 cytoplasmic domain. Using an in vitro translation system, we also showed that degradation of CD4 by Vpu requires the two proteins to be present in the same membrane compartment. Although these results suggest that spatial proximity between CD4 and Vpu may be critical in triggering degradation, it remains unknown whether the two molecules have the ability to interact with each other. In order to better define the mechanisms involved in CD4 degradation, we investigated the existence and functional relevance of direct interactions between CD4 and Vpu. Coimmunoprecipitation experiments showed that Vpu specifically binds to the cytoplasmic tail of CD4. This phenomenon is relevant to the mechanism of CD4 degradation since the ability of CD8/CD4 chimeric molecules and various CD4 mutants to form complexes with Vpu correlates with their sensitivity to degradation. Accordingly, we found that amino acid residues in the CD4 cytoplasmic tail previously shown to be important for degradation are necessary for Vpu binding. We further demonstrate that a deletion mutant of Vpu as well as a phosphorylation mutant, both biologically inactive with regard to CD4 degradation, retained the capacity to interact with the CD4 cytoplasmic domain. Taken together, these results indicate that Vpu binding is necessary to trigger CD4 degradation. However, the binding to target molecules is not sufficient per se to cause degradation. Interaction between CD4 and Vpu is thus likely to be an early event critical in triggering a multistep process leading to CD4 degradation.