A conserved dileucine motif mediates clathrin and AP-2-dependent endocytosis of the HIV-1 envelope protein

During the assembly of enveloped viruses viral and cellular components essential for infectious particles must colocalize at specific membrane locations. For the human and simian immunodeficiency viruses (HIV and SIV), sorting of the viral envelope proteins (Env) to assembly sites is directed by trafficking signals located in the cytoplasmic domain of the transmembrane protein gp41 (TM). A membrane proximal conserved GYxx? motif mediates endocytosis through interaction with the clathrin adaptor AP-2. However, experiments with SIV(mac239) Env indicate the presence of additional signals. Here we show that a conserved C-terminal dileucine in HIV(HxB2) also mediates endocytosis. Biochemical and morphological assays demonstrate that the C-terminal dileucine motif mediates internalization as efficiently as the GYxx? motif and that both must be removed to prevent Env internalization. RNAi experiments show that depletion of the clathrin adaptor AP-2 leads to increased plasma membrane expression of HIV Env and that this adaptor is required for efficient internalization mediated by both signals. The redundancy of conserved endocytosis signals and the role of the SIV(mac239) Env GYxx? motif in SIV pathogenesis, suggest that these motifs have functions in addition to endocytosis, possibly related to Env delivery to the site of viral assembly and/or incorporation into budding virions.     

Regulation of human immunodeficiency virus type 1 envelope glycoprotein fusion by a membrane-interactive domain in the gp41 cytoplasmic tail

Truncation of the human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) gp41 cytoplasmic tail (CT) can modulate the fusogenicity of the envelope glycoprotein (Env) on infected cells and virions. However, the CT domains involved and the underlying mechanism responsible for this "inside-out" regulation of Env function are unknown. HIV and SIV CTs are remarkably long and contain amphipathic alpha-helical domains (LLP1, LLP2, and LLP3) that likely interact with cellular membranes. Using a cell-cell fusion assay and a panel of HIV Envs with stop codons at various positions in the CT, we show that truncations of gp41 proximal to the most N-terminal alpha helix, LLP2, increase fusion efficiency and expose CD4-induced epitopes in the Env ectodomain. These effects were not seen with a truncation distal to this domain and before LLP1. Using a dye transfer assay to quantitate fusion kinetics, we found that these truncations produced a two- to fourfold increase in the rate of fusion. These results were observed for X4-, R5-, and dual-tropic Envs on CXCR4- and CCR5-expressing target cells and could not be explained by differences in Env surface expression. These findings suggest that distal to the membrane-spanning domain, an interaction of the gp41 LLP2 domain with the cell membrane restricts Env fusogenicity during Env processing. As with murine leukemia viruses, where cleavage of a membrane-interactive R peptide at the C terminus is required for Env to become fusogenic, this restriction of Env function may serve to protect virus-producing cells from the membrane-disruptive effects of the Env ectodomain.     

Interactions of the Cytoplasmic Domains of Human and Simian Retroviral Transmembrane Proteins with Components of the Clathrin Adaptor Complexes Modulate Intracellular and Cell Surface Expression of Envelope Glycoproteins

The cytoplasmic domains of the transmembrane (TM) envelope proteins (TM-CDs) of most retroviruses have a Tyr-based motif, YXXØ, in their membrane-proximal regions. This signal is involved in the trafficking and endocytosis of membrane receptors via clathrin-associated AP-1 and AP-2 adaptor complexes. We have used CD8-TM-CD chimeras to investigate the role of the Tyr-based motif of human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and human T-leukemia virus type 1 (HTLV-1) TM-CDs in the cell surface expression of the envelope glycoprotein. Flow cytometry and confocal microscopy studies showed that this motif is a major determinant of the cell surface expression of the CD8-HTLV chimera. The YXXØ motif also plays a key role in subcellular distribution of the envelope of lentiviruses HIV-1 and SIV. However, these viruses, which encode TM proteins with a long cytoplasmic domain, have additional determinants distal to the YXXØ motif that participate in regulating cell surface expression. We have also used the yeast two-hybrid system and in vitro binding assays to demonstrate that all three retroviral YXXØ motifs interact with the μ1 and μ2 subunits of AP complexes and that the C-terminal regions of HIV-1 and SIV TM proteins interact with the β2 adaptin subunit. The TM-CDs of HTLV-1, HIV-1, and SIV also interact with the whole AP complexes. These results clearly demonstrate that the cell surface expression of retroviral envelope glycoproteins is governed by interactions with adaptor complexes. The YXXØ-based signal is the major determinant of this interaction for the HTLV-1 TM, which contains a short cytoplasmic domain, whereas the lentiviruses HIV-1 and SIV have additional determinants distal to this signal that are also involved.     

HSV‐1 Glycoproteins Are Delivered to Virus Assembly Sites Through Dynamin‐Dependent Endocytosis

Herpes simplex virus‐1 (HSV‐1) is a large enveloped DNA virus that belongs to the family of Herpesviridae. It has been recently shown that the cytoplasmic membranes that wrap the newly assembled capsids are endocytic compartments derived from the plasma membrane. Here, we show that dynamin‐dependent endocytosis plays a major role in this process. Dominant‐negative dynamin and clathrin adaptor AP180 significantly decrease virus production. Moreover, inhibitors targeting dynamin and clathrin lead to a decreased transport of glycoproteins to cytoplasmic capsids, confirming that glycoproteins are delivered to assembly sites via endocytosis. We also show that certain combinations of glycoproteins colocalize with each other and with the components of clathrin‐dependent and ‐independent endocytosis pathways. Importantly, we demonstrate that the uptake of neutralizing antibodies that bind to glycoproteins when they become exposed on the cell surface during virus particle assembly leads to the production of non‐infectious HSV‐1. Our results demonstrate that transport of viral glycoproteins to the plasma membrane prior to endocytosis is the major route by which these proteins are localized to the cytoplasmic virus assembly compartments. This highlights the importance of endocytosis as a major protein‐sorting event during HSV‐1 envelopment.      

Mutation of the dominant endocytosis motif in human immunodeficiency virus type 1 gp41 can complement matrix mutations without increasing Env incorporation

The human immunodeficiency virus type 1 transmembrane glycoprotein (TM) is efficiently endocytosed in a clathrin-dependent manner. Internalization is mediated by a tyrosine-containing motif within the cytoplasmic domain, and replacement of the cytoplasmic tyrosine by cysteine or phenylalanine increased expression of mutant glycoprotein on the surface of transfected cells by as much as 2.5-fold. Because interactions between the cytoplasmic domain of Env and the matrix protein (MA) have been suggested to mediate incorporation of Env in virus particles, we examined whether perturbation of endocytosis would alter incorporation. Proviruses were constructed to contain the wild-type or mutant Env in conjunction with point mutations in MA that had previously been shown to block Env incorporation. These constructs were used to evaluate the effect of glycoprotein endocytosis on incorporation into virus particles and to test the necessity for a specific interaction between Env and MA to mediate incorporation. Viruses produced from transfected 293T cells were used to infect various cell lines, including MAGI, H9, and CEMx174. Viruses encoding both a disrupted endocytosis motif signal and mutations within MA were significantly more infectious in MAGI cells than their counterparts encoding a mutant MA and wild-type Env. This complementation of infectivity for the MA incorporation mutant viruses was not due to increased glycoprotein incorporation into particles but instead reflected an enhanced fusogenicity of the mutated Env proteins. Our findings further support the concept that a specific interaction between the long cytoplasmic domain of TM and MA is required for efficient incorporation of Env into assembling virions. Alteration of the endocytosis signal of Env, and the resulting increase in cell surface glycoprotein, has no effect on incorporation despite demonstrable effects on fusion, virus entry, and infectivity.     

Surface stability and immunogenicity of the human immunodeficiency virus envelope glycoprotein: role of the cytoplasmic domain

The effects of two functional domains, the membrane-proximal YXXPhi motif and the membrane-distal inhibitory sequence in the long cytoplasmic tail of the human immunodeficiency virus type 1 (HIV-1) envelope protein (Env), on immunogenicity of the envelope protein were investigated. Genes with codons optimized for mammalian expression were synthesized for the HIV 89.6 Env and a truncated Env with 50 amino acids in the cytoplasmic domain to delete the membrane distal inhibitory sequence for surface expression. Additional genes were generated in which the tyrosine residue in the YXXPhi motif was changed into a serine. Pulse-chase radioactive labeling and immunoprecipitation studies indicated that both domains can mediate endocytosis of the HIV Env, and removal of both domains is required to enhance HIV Env protein surface stability. Analysis of immune responses induced by DNA immunization of mice showed that the DNA construct for the mutant Env exhibiting enhanced surface stability induced significantly higher levels of antibody responses against the HIV Env protein. Our results suggest that the HIV Env cytoplasmic domain may play important roles in virus infection and pathogenesis by modulating its immunogenicity.     

Regulation of clathrin adaptor function in endocytosis: novel role for the SAM domain

During clathrin‐mediated endocytosis, adaptor proteins play central roles in coordinating the assembly of clathrin coats and cargo selection. Here we characterize the binding of the yeast endocytic adaptor Sla1p to clathrin through a variant clathrin‐binding motif that is negatively regulated by the Sla1p SHD2 domain. The crystal structure of SHD2 identifies the domain as a sterile α‐motif (SAM) domain and shows a propensity to oligomerize. By co‐immunoprecipitation, Sla1p binds to clathrin and self‐associates in vivo. Mutations in the clathrin‐binding motif that abolish clathrin binding and structure‐based mutations in SHD2 that impede self‐association result in endocytosis defects and altered dynamics of Sla1p assembly at the sites of endocytosis. These results define a novel mechanism for negative regulation of clathrin binding by an adaptor and suggest a role for SAM domains in clathrin‐mediated endocytosis.     

An internalization signal in the simian immunodeficiency virus transmembrane protein cytoplasmic domain modulates expression of envelope glycoproteins on the cell surface

A Tyr to Cys mutation at amino acid position 723 in the cytoplasmic domain of the simian immunodeficiency virus (SIV) transmembrane (TM) molecule has been shown to increase expression of envelope glycoproteins on the surface of infected cells. Here we show that Tyr- 723 contributes to a sorting signal that directs the rapid endocytosis of viral glycoproteins from the plasma membrane via coated pits. On cells infected by SIVs with a Tyr at position 723, envelope glycoproteins were transiently expressed on the cell surface and then rapidly endocytosed. Similar findings were noted for envelope molecules expressed in the absence of other viral proteins. Immunoelectron microscopy demonstrated that these molecules were localized in patches on the cell surface and were frequently associated with coated pits. In contrast, envelope glycoproteins containing a Y723C mutation were diffusely distributed over the entire plasma membrane. To determine if an internalization signal was present in the SIV TM, chimeric molecules were constructed that contained the CD4 external and membrane spanning domains and a SIV TM cytoplasmic tail with a Tyr or other amino acids at SIV position 723. In Hela cells stably expressing these molecules, chimeras with a Tyr-723 were rapidly endocytosed, while chimeras containing other amino acids at position 723, including a Phe, were internalized at rates only slightly faster than a CD4 molecule that lacked a cytoplasmic domain. In addition, the biological effects of the internalization signal were evaluated in infectious viruses. A mutation that disrupted the signal and as a result, increased the level of viral envelope glycoprotein on infected cells, was associated with accelerated infection kinetics and increased cell fusion during viral replication. These results demonstrate that a Tyr-dependent motif in the SIV TM cytoplasmic domain can function as an internalization signal that can modulate expression of the viral envelope molecules on the cell surface and affect the biological properties of infectious viruses. The conservation of an analogous Tyr in all human and simian immunodeficiency viruses suggests that this signal may be present in other primate lentiviruses and could be important in the pathogenesis of these viruses in vivo.     

Endocytosis of synaptotagmin 1 is mediated by a novel,tryptophan-containing motif

The rate at which a membrane protein is internalized from the plasma membrane can be regulated by revealing a latent internalization signal in response to an appropriate stimulus. Internalization of the synaptic vesicle membrane protein, synaptotagmin 1, is controlled by two distinct regions of its intracytoplasmic C2B domain, an internalization signal present in the 29 carboxyterminal (CT) amino acids and a separate regulatory region. We have now characterized the internalization motif by mutagenesis and found that it involves an essential tryptophan in the last beta strand of the C2B domain, a region that is distinct from the AP2-binding site previously described. Internalization through the tryptophan-based motif is sensitive to eps15 and dynamin mutants and is therefore likely to be clathrin mediated. A tryptophan-to-phenylalanine mutation had no effect on internalization of the CT domain alone, but completely inhibited endocytosis of the folded C2B domain. This result suggests that recognition of sorting motifs can be influenced by their structural context. We conclude that endocytosis of synaptotagmin 1 requires a novel type of internalization signal that is subject to regulation by the rest of the C2B domain.     

Characterization of BIV Env core: implication for mechanism of BIV-mediated cell fusion

Entry of lentiviruses, such as human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV), requires folding of two heptad repeat regions (HR1 and HR2) of gp41 into a trimer-of-hairpins, which subsequently brings virus and cell membrane into fusion. This motif is a generalized feature of viral fusion proteins and has been exploited in generating antiviral fusion agents. In the present paper, we report structural characters of Env protein from another lentivirus, bovine immunodeficiency virus (BIV), which contributes to a good animal model of HIV. BIV HR1 and HR2 regions are predicted by two different programs and expressed separately or conjointly in Escherichia coli. Biochemical and biophysical analyses show that the predicted HRs of BIV Env can form a stable trimer-of-hairpins or six-helix bundle just like that formed by feline immunodeficiency virus Env. Cell fusion assay demonstrates that the HR2 peptide of BIV can efficiently inhibit the virus-mediated cell fusion.     

Calcyon, a mammalian specific NEEP21 family member, interacts with adaptor protein complex 3 (AP-3) and regulates targeting of AP-3 cargoes

Calcyon is a neural enriched, single transmembrane protein that interacts with clathrin light chain and stimulates clathrin assembly and clathrin‐mediated endocytosis. A similar property is shared by the heterotetrameric adaptor protein (AP) complexes AP‐1, AP‐2, and AP‐3 which recruit cargoes for insertion into clathrin coated transport vesicles. Here we report that AP medium (μ) subunits interact with a YXXØ‐type tyrosine motif located at residues 133–136 in the cytoplasmic domain of calcyon. Site specific mutagenesis of the critical tyrosine and bulky hydrophobic residues tyrosine 133 and methionine 136 preferentially abrogated binding of the ubiquitous and neuronal isoforms of μ3, and also impacted μ1 and μ2 binding to a lesser degree. The relevance of these interactions was explored in vivo using mice harboring null alleles of calcyon. As seen in the mutagenesis studies, calcyon deletion in mice preferentially altered the subcellular distribution of AP‐3 suggesting that calcyon could regulate membrane‐bound pools of AP‐3 and AP‐3 function. To test this hypothesis, we focused on the hilar region of hippocampus, where levels of calcyon, AP‐3, and AP‐3 cargoes are abundant. We analyzed brain cryosections from control and calcyon null mice for zinc transporter 3 (ZnT3), and phosphatidylinositol‐4‐kinase type II alpha (PI4KIIα), two well‐defined AP‐3 cargoes. Confocal microscopy indicated that ZnT3 and PI4KIIα are significantly reduced in the hippocampal mossy fibers of calcyon knock‐out brain, a phenotype previously described in AP‐3 deficiencies. Altogether, our data suggest that calcyon directly interacts with μ3A and μ3B, and regulates the subcellular distribution of AP‐3 and the targeting of AP‐3 cargoes.     

Cooperative interactions of simian immunodeficiency virus Nef,AP-2, and CD3-zeta mediate the selective induction of T-cell receptor-CD3 endocytosis

The Nef proteins of human immunodeficiency virus and simian immunodeficiency virus (SIV) bind the AP-1 and AP-2 clathrin adaptors to downmodulate the expression of CD4 and CD28 by recruiting them to sites of AP-2 clathrin-dependent endocytosis. Additionally, SIV Nef directly binds the CD3-zeta subunit of the CD3 complex and downmodulates the T-cell receptor (TCR)-CD3 complex. We report here that SIV mac239 Nef induces the endocytosis of TCR-CD3 in Jurkat T cells. SIV Nef also induces the endocytosis of a chimeric CD8-CD3-zeta protein containing only the CD3-zeta cytoplasmic domain (8-zeta), in the absence of other CD3 subunits. Thus, the interaction of SIV Nef with CD3-zeta likely mediates the induction of TCR-CD3 endocytosis. In cells expressing SIV Nef and 8-zeta, both proteins colocalize with AP-2, indicating that Nef induces 8-zeta internalization via this pathway. Surprisingly, deletion of constitutively strong AP-2 binding determinants (CAIDs) in SIV Nef had little effect on its ability to induce TCR-CD3, or 8-zeta endocytosis, even though these determinants are required for the induction of CD4 and CD28 endocytosis via this pathway. Fluorescent microscopic analyses revealed that while neither the mutant SIV Nef protein nor 8-zeta colocalized with AP-2 when expressed independently, both proteins colocalized with AP-2 when coexpressed. In vitro binding studies using recombinant SIV Nef proteins lacking CAIDs and recombinant CD3-zeta cytoplasmic domain demonstrated that SIV Nef and CD3-zeta cooperate to bind AP-2 via a novel interaction. The fact that Nef uses distinct AP-2 interaction surfaces to recruit specific membrane receptors demonstrates how Nef independently selects distinct types of target receptors and recruits them to AP-2 for endocytosis.     

The Surface Density of the Glutamate Transporter EAAC1 is Controlled by Interactions with PDZK1 and AP2 Adaptor Complexes

The glutamate transporter excitatory amino acid carrier (EAAC1/EAAT3) mediates the absorption of dicarboxylic amino acids in epithelial cells as well as the uptake of glutamate from the synaptic cleft. Its cell‐surface density is regulated by interaction with accessory proteins which remain to be identified. We detected a consensus sequence for interaction with post‐synaptic density‐95/Discs large/Zonula occludens (PDZ) proteins (‐SQF) and a tyrosine‐based internalization signal (‐YVNG‐) in the C‐terminus of EAAC1, and investigated their role in the transporter localization. We demonstrated that PDZ interactions are required for the efficient delivery to and the retention in the plasma membrane of EAAC1 and we identified PDZK1/NHERF3 (Na+/H+‐exchanger regulatory factor 3) as a novel EAAC1 interacting protein. Expression of PDZK1 in Madin‐Darby canine kidney (MDCK) cells tethered EAAC1 to filopodia and increased its surface activity. Removal of the PDZ‐target motif promoted the EAAC1 binding to α‐adaptin and clathrin and the transporter internalization in endocytic/degradative compartments. This defect was largely prevented by hypertonic treatment or overexpression of the dominant‐negative µ2‐W421A‐subunit of AP‐2 clathrin‐adaptor. The rate of transporter endocytosis was attenuated following tyrosine mutagenesis in the internalization signal, thus indicating that this motif can regulate the transporter endocytosis. We suggest that EAAC1 density is controlled by balanced interactions with PDZK1 and adaptor protein 2 (AP2): the former promotes the transporter expression at the cell surface, and the latter mediates its constitutive endocytosis.     

In vivo attenuation of simian immunodeficiency virus by disruption of a tyrosine-dependent sorting signal in the envelope glycoprotein cytoplasmic tail

Attenuated simian immunodeficiency viruses (SIVs) have been described that produce low levels of plasma virion RNA and exhibit a reduced capacity to cause disease. These viruses are particularly useful in identifying viral determinants of pathogenesis. In the present study, we show that mutation of a highly conserved tyrosine (Tyr)-containing motif (Yxxphi) in the envelope glycoprotein (Env) cytoplasmic tail (amino acids YRPV at positions 721 to 724) can profoundly reduce the in vivo pathogenicity of SIVmac239. This domain constitutes both a potent endocytosis signal that reduces Env expression on infected cells and a sorting signal that directs Env expression to the basolateral surface of polarized cells. Rhesus macaques were inoculated with SIVmac239 control or SIVmac239 containing either a Tyr-721-to-Ile mutation (SIVmac239Y/I) or a deletion of Tyr-721 and the preceding glycine (DeltaGY). To assess the in vivo replication competence, all viruses contained a stop codon in nef that has been shown to revert during in vivo but not in vitro replication. All three control animals developed high viral loads and disease. One of two animals that received SIVmac239Y/I and two of three animals that received SIVmac239DeltaGY remained healthy for up to 140 weeks with low to undetectable plasma viral RNA levels and normal CD4(+) T-cell percentages. These animals exhibited ongoing viral replication as determined by detection of viral sequences and culturing of mutant viruses from peripheral blood mononuclear cells and persistent anti-SIV antibody titers. In one animal that received SIVmac239Y/I, the Ile reverted to a Tyr and was associated with a high plasma RNA level and disease, while one animal that received SIVmac239DeltaGY also developed a high viral load that was associated with novel and possibly compensatory mutations in the TM cytoplasmic domain. In all control and experimental animals, the nef stop codon reverted to an open reading frame within the first 2 months of inoculation, indicating that the mutant viruses had replicated well enough to repair this mutation. These findings indicate that the Yxxphi signal plays an important role in SIV pathogenesis. Moreover, because mutations in this motif may attenuate SIV through mechanisms that are distinct from those caused by mutations in nef, this Tyr-based sorting signal represents a novel target for future models of SIV and human immunodeficiency virus attenuation that could be useful in new vaccine strategies.     

Human immunodeficiency virus type 1 envelope protein endocytosis mediated by a highly conserved intrinsic internalization signal in the cytoplasmic domain of gp41 is suppressed in the presence of the Pr55gag precursor protein.

The mechanisms involved in the incorporation of viral glycoproteins into virions are incompletely understood. For retroviruses, incorporation may involve interactions between the Gag proteins of these viruses and the cytoplasmic domains of the relevant envelope (Env) glycoproteins. Recent studies have identified within the cytoplasmic tail of the human immunodeficiency virus type 1 (HIV-1) Env protein a tyrosine-containing internalization motif similar to those found in the cytoplasmic domains of certain cell surface proteins that undergo rapid constitutive endocytosis in clathrin-coated pits. Given that surface expression of the HIV-1 Env protein is essential for the production of infectious virus, the presence of this internalization motif is surprising. We show here that in contrast to the rapid rate of Env protein internalization observed in cells expressing the Env protein in the absence of other HIV-1 proteins, the rate of internalization of Env protein from the surfaces of HIV-1-infected cells is extremely slow. The presence of the Pr55gag precursor protein is necessary and sufficient for inhibition of Env protein internalization, while a mutant Pr55-gag that is incapable of mediating Env incorporation into virions is also unable to inhibit endocytosis of the Env protein. The failure of the Env protein to undergo endocytosis from the surface of an HIV-1-infected cell may reflect the fact that the proposed interaction of the matrix domain of the Gag protein with Env during assembly prevents the interaction of Env with host adaptin molecules that recruit plasma membrane molecules such as the transferrin receptor into clathrin-coated pits. When the normal ratio of Gag and Env proteins in the infected cells is altered by overexpression of Env protein, this mechanism allows removal of excess Env protein from the cell surface. Taken together, these results suggest that a highly conserved system to reduce surface levels of the Env protein functions to remove Env protein that is not associated with Gag and that is therefore not destined for incorporation into virions. This mechanism for the regulation of surface levels of Env protein may protect infected cells from Env-dependent cytopathic effects or Env-specific immune responses.     

Tetherin/BST-2 Antagonism by Nef Depends on a Direct Physical Interaction between Nef and Tetherin,and on Clathrin-mediated Endocytosis

Nef is the viral gene product employed by the majority of primate lentiviruses to overcome restriction by tetherin (BST-2 or CD317), an interferon-inducible transmembrane protein that inhibits the detachment of enveloped viruses from infected cells. Although the mechanisms of tetherin antagonism by HIV-1 Vpu and HIV-2 Env have been investigated in detail, comparatively little is known about tetherin antagonism by SIV Nef. Here we demonstrate a direct physical interaction between SIV Nef and rhesus macaque tetherin, define the residues in Nef required for tetherin antagonism, and show that the anti-tetherin activity of Nef is dependent on clathrin-mediated endocytosis. SIV Nef co-immunoprecipitated with rhesus macaque tetherin and the Nef core domain bound directly to a peptide corresponding to the cytoplasmic domain of rhesus tetherin by surface plasmon resonance. An analysis of alanine-scanning substitutions identified residues throughout the N-terminal, globular core and flexible loop regions of Nef that were required for tetherin antagonism. Although there was significant overlap with sequences required for CD4 downregulation, tetherin antagonism was genetically separable from this activity, as well as from other Nef functions, including MHC class I-downregulation and infectivity enhancement. Consistent with a role for clathrin and dynamin 2 in the endocytosis of tetherin, dominant-negative mutants of AP180 and dynamin 2 impaired the ability of Nef to downmodulate tetherin and to counteract restriction. Taken together, these results reveal that the mechanism of tetherin antagonism by Nef depends on a physical interaction between Nef and tetherin, requires sequences throughout Nef, but is genetically separable from other Nef functions, and leads to the removal of tetherin from sites of virus release at the plasma membrane by clathrin-mediated endocytosis.     

Endocytic activity of HIV‐1 Vpu: Phosphoserine‐dependent interactions with clathrin adaptors

HIV‐1 Vpu modulates cellular transmembrane proteins to optimize viral replication and provide immune‐evasion, triggering ubiquitin‐mediated degradation of some targets but also modulating endosomal trafficking to deplete them from the plasma membrane. Interactions between Vpu and the heterotetrameric clathrin adaptor protein (AP) complexes AP‐1 and AP‐2 have been described, yet the molecular basis and functional roles of such interactions are incompletely defined. To investigate the trafficking signals encoded by Vpu, we fused the cytoplasmic domain (CD) of Vpu to the extracellular and transmembrane domains of the CD8 α‐chain. CD8‐VpuCD was rapidly endocytosed in a clathrin‐ and AP‐2‐dependent manner. Multiple determinants within the Vpu CD contributed to endocytic activity, including phosphoserines of the β‐TrCP binding site and a leucine‐based ExxxLV motif. Using recombinant proteins, we confirmed ExxxLV‐dependent binding of the Vpu CD to the α/σ2 subunit hemicomplex of AP‐2 and showed that this is enhanced by serine‐phosphorylation. Remarkably, the Vpu CD also bound directly to the medium (μ) subunits of AP‐2 and AP‐1; this interaction was dependent on serine‐phosphorylation of Vpu and on basic residues in the μ subunits. We propose that the flexibility with which Vpu binds AP complexes broadens the range of cellular targets that it can misdirect to the virus' advantage.      

The highly conserved C-terminal dileucine motif in the cytosolic domain of the human immunodeficiency virus type 1 envelope glycoprotein is critical for its association with the AP-1 clathrin adaptor [correction of adapter

Short amino acid sequences in the cytosolic domains of transmembrane proteins are recognized by specialized adaptor [corrected] proteins which are part of coated vesicles utilized to transport membrane proteins between the trans-Golgi network (TGN) and the plasma membrane (forward and backward). Previously, we and others reported that the membrane-proximal tyrosine residues Y712 (human immunodeficiency virus [HIV]) and Y721 (simian immunodeficiency virus [SIV]) in the envelope glycoprotein (Env) of the primate lentiviruses are crucial for the association of Env with clathrin-associated adaptor [corrected] complex AP-2. The same tyrosine-based endocytosis motifs in the cytosolic domains (EnvCD) of transmembrane gp41 of HIV type 1 (HIV-1) and SIV, respectively, were also shown to modulate the interaction with TGN- and endosome-based clathrin-associated complex AP-1. Our findings suggested that EnvCD binding to AP-1, unlike the association of EnvCD with AP-2, is dependent largely on residues other than Y712 and Y721. Here, we tested if motifs downstream of Y712 affect HIV-1 EnvCD-AP-1 binding and Env trafficking. Mutational analysis revealed that the C-terminal leucine-based motif in Env was crucial for the recruitment of AP-1 in vitro and in Env-expressing cells. In addition to affecting Env-AP-1 association, mutations at the C terminus of Env also altered the subcellular localization of Env, suggesting that proper post-Golgi routing of Env depends on its recruitment of AP-1. Finally, the C-terminal dileucine was shown to assist the membrane-proximal Y712 motif in restricting the cell surface expression of Env.     

Intracellular Interaction of Simian Immunodeficiency Virus Gag and Env Proteins

In polarized epithelial cells, the assembly and release of human immunodeficiency virus type 1 (HIV-1) occur at the basolateral side of the plasma membrane, and the site of assembly is determined by the site of expression of the Env protein. In order to investigate whether the expression of the Env proteins exclusively in the endoplasmic reticulum (ER) can alter the site of virus assembly, we coexpressed the simian immunodeficiency virus (SIV) Gag protein and mutant SIV Env proteins having an ER retrieval signal (KKXX motif). In cells expressing the wild-type (wt) Env protein or coexpressing Env and Gag proteins, the Env protein was processed into the surface (SU) and transmembrane (TM) proteins. In contrast, in cells expressing the mutant Env proteins alone or in combination with Gag, the Env proteins were retrieved to the ER and were not proteolytically processed. Coexpression of the Gag and ER-retained mutant Env proteins resulted in a transient decrease in the release of the Gag protein into the medium, suggesting an interaction between the Gag and ER-retrieved Env proteins. Using saponin-permeabilized cells coexpressing Gag and Env proteins, we obtained further evidence for Env-Gag interaction. A monoclonal antibody specific to the SIV Gag protein was found to coimmunoprecipitate both the Gag and Env proteins. The interaction was specific, as coexpressed SIV Env proteins without the cytoplasmic tail or a chimeric HIV-1 Env proteins with the CD4 cytoplasmic tail were not coimmunoprecipitated by the Gag-specific antibody. Electron microscopic analyses indicated that assembly of virus particles occurred only at the surfaces of cells in which the Gag protein was coexpressed with either the wt or ER-retrieved mutant Env protein. These data indicate that although the Env and Gag proteins interact intracellularly, the site of assembly of SIV is not redirected to an intracellular organelle by the retrieval of the Env protein to the ER.     

The Rous sarcoma virus Env glycoprotein contains a highly conserved motif homologous to tyrosine-based endocytosis signals and displays an unusual internalization phenotype

The cytoplasmic domains of retroviral transmembrane (TM) glycoproteins contain conserved sequence motifs that resemble tyrosine-based (YXXO-type) endocytosis signals. We have previously described a mutant Rous sarcoma virus (RSV) Env protein, Env-mu26, with an L165R mutation in the membrane-spanning domain (MSD) of TM, that exhibited dramatically decreased steady-state surface expression (G. L. Davis and E. Hunter, J. Cell Biol. 105:1191-1203, 1987; P. B. Johnston, J. Y. Dong, and E. Hunter, Virology 206:353-361, 1995). We now demonstrate that the tyrosine of the Y(190)RKM motif in the RSV TM cytoplasmic domain is crucial for the mu26 phenotype and is part of an efficient internalization signal in the context of a mutant MSD. In contrast, despite the presence of the Y(190)RKM motif, wild-type RSV Env is constitutively internalized at a slow rate (1.1%/min) more characteristic of bulk uptake during membrane turnover than of active clustering into endocytic vesicles. The mu26 mutation and two MSD mutations that abrogate palmitoylation of TM resulted in enhanced Env endocytosis indicative of active concentration into coated pits. Surprisingly, an Env-Y190A mutant was apparently excluded from coated pits since its uptake rate of 0.3%/min was significantly below that expected for the bulk rate. We suggest that in RSV Env an inherently functional endocytosis motif is silenced by a counteracting determinant in the MSD that acts to prevent clustering of Env into endocytic vesicles. Mutations in either the cytoplasmic tail or the MSD that inactivate one of the two counteracting signals would thus render the remaining determinant dominant.