Mutation of the YXXL endocytosis motif in the cytoplasmic tail of pseudorabies virus gE

The role of alphaherpesvirus membrane protein internalization during the course of viral infection remains a matter of speculation. To determine the role of internalization of the pseudorabies virus (PRV) gE and gI proteins, we constructed viral mutants encoding specific mutations in the cytoplasmic tail of the gE gene that inhibited internalization of the gE-gI complex. We used these mutants to assess the role of gE-gI endocytosis in incorporation of the proteins into the viral envelope and in gE-mediated spread or gE-promoted virulence. In addition, we report that another viral mutant, PRV 25, which encodes a gE protein defective in endocytosis, contains an additional, previously uncharacterized mutation in the gE gene. We compared PRV 25 to another viral mutant, PRV 107, that does not express the cytoplasmic tail of the gE protein. The gE protein encoded by PRV 107 is also defective in endocytosis. We conclude that efficient endocytosis of gE is not required for gE incorporation into virions, gE-mediated virulence, or spread of virus in the rat central nervous system. However, we do correlate the defect in endocytosis to a small-plaque phenotype in cultured cells.     

A tyrosine-based motif in the cytoplasmic tail of pseudorabies virus glycoprotein B is important for both antibody-induced internalization of viral glycoproteins and efficient cell-to-cell spread

Pseudorabies virus (PRV), a swine alphaherpesvirus, is capable of causing viremia in vaccinated animals. Two mechanisms that may help PRV avoid recognition by the host immune system during this viremia are direct cell-to-cell spread in tissue and antibody-induced internalization of viral cell surface glycoproteins in PRV-infected blood monocytes, the carrier cells of the virus in the blood. PRV glycoprotein B (gB) is crucial during both processes. Here we show that mutating a tyrosine residue located in a YXXPhi motif in the gB cytoplasmic tail results in decreased efficiency of cell-to-cell spread and a strong reduction in antibody-induced internalization of viral cell surface glycoproteins. Mutating the dileucine motif in the gB tail led to an increased cell-to-cell spread of the virus and the formation of large syncytia.     

A novel cytoplasmic tail MXXXL motif mediates the internalization of prostate-specific membrane antigen

Prostate-specific membrane antigen (PSMA) is a transmembrane protein expressed at high levels in prostate cancer and in tumor-associated neovasculature. In this study, we report that PSMA is internalized via a clathrin-dependent endocytic mechanism and that internalization of PSMA is mediated by the five N-terminal amino acids (MWNLL) present in its cytoplasmic tail. Deletion of the cytoplasmic tail abolished PSMA internalization. Mutagenesis of N-terminal amino acid residues at position 2, 3, or 4 to alanine did not affect internalization of PSMA, whereas mutation of amino acid residues 1 or 5 to alanine strongly inhibited internalization. Using a chimeric protein composed of Tac antigen, the alpha-chain of interleukin 2-receptor, fused to the first five amino acids of PSMA (Tac-MWNLL), we found that this sequence is sufficient for PSMA internalization. In addition, inclusion of additional alanines into the MWNLL sequence either in the Tac chimera or the full-length PSMA strongly inhibited internalization. From these results, we suggest that a novel MXXXL motif in the cytoplasmic tail mediates PSMA internalization. We also show that dominant negative micro2 of the adaptor protein (AP)-2 complex strongly inhibits the internalization of PSMA, indicating that AP-2 is involved in the internalization of PSMA mediated by the MXXXL motif.     

Endocytosis and recycling of varicella-zoster virus Fc receptor glycoprotein gE: internalization mediated by a YXXL motif in the cytoplasmic tail.

Varicella-zoster virus (VZV) encodes a cell surface Fc receptor, glycoprotein gE. VZV gE has previously been shown to display several features common to nonviral cell surface receptors. Most recently, VZV gE was reported to be tyrosine phosphorylated on a dimeric form (J. K. Olson, G. A. Bishop, and C. Grose, J. Virol. 71:110-119, 1997). Thereafter, attention focused on the ability of VZV gE to undergo receptor-mediated endocytosis. The current transient transfection studies demonstrated by confocal microscopy and internalization assays that VZV gE was endocytosed when expressed in HeLa cells. Endocytosis of gE was shown to be dependent on clathrin-coated vesicle formation within the cells. Subsequent colocalization studies showed that endocytosis of VZV gE closely mimicked endocytosis of the transferrin receptor. The gE cytoplasmic tail and more specifically tyrosine residue 582 were determined by mutagenesis studies to be important for efficient internalization of the protein; this tyrosine residue is part of a conserved YXXL motif. The amount of gE internalized at any given time reached a steady state of 32%. In addition, like the transferrin receptor, internalized gE recycled to the cell surface. The finding of gE endocytosis provided insight into earlier documentation of gE serine/threonine and tyrosine phosphorylation, since these phosphorylation events may serve as sorting signals for internalized receptors. Taken together with the previous discovery that both human and simian immunodeficiency virus envelope proteins can undergo endocytosis, the gE findings suggest that endocytosis of envelope components may be a posttranslational regulatory mechanism among divergent families of enveloped viruses.     

Role of the cytoplasmic tail of pseudorabies virus glycoprotein E in virion formation

Glycoproteins M (gM), E (gE), and I (gI) of pseudorabies virus (PrV) are required for efficient formation of mature virions. The simultaneous absence of gM and the gE/gI complex results in severe deficiencies in virion morphogenesis and cell-to-cell spread, leading to drastically decreased virus titers and a small-plaque phenotype (A. Brack, J. Dijkstra, H. Granzow, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 73:5364-5372, 1999). Serial passaging in noncomplementing cells of a virus mutant unable to express gM, gE, and gI resulted in a reversion of the small-plaque phenotype and restoration of infectious virus formation to the level of a gM(-) mutant. Genetic analyses showed that reversion of the phenotype was accompanied by a genomic rearrangement which led to the fusion of a portion of the gE gene encoding the cytoplasmic domain to the 3' end of the glycoprotein D gene, resulting in expression of a chimeric gD-gE protein. Since this indicated that the intracytoplasmic domain of gE was responsible for the observed phenotypic alterations, the UL10 (gM) gene was deleted in a PrV mutant, PrV-107, which specifically lacked the cytoplasmic tail of gE. Regarding one-step growth, plaque size, and virion formation as observed under the electron microscope, the mutant lacking gM and the gE cytoplasmic tail proved to be very similar to the gE/I/M triple mutant. Thus, our data indicate that it is the cytoplasmic tail of gE which is responsible for the observed phenotypic effects in conjunction with deletion of gM. We hypothesize that the cytoplasmic domain of gE specifically interacts with components of the capsid and/or tegument, leading to efficient secondary envelopment of intracytoplasmic capsids.     

Intracellular distribution of lysosomal sialidase is controlled by the internalization signal in its cytoplasmic tail

Sialidase (neuraminidase), encoded by the neu-1 gene in the major histocompatibility complex locus catalyzes the intralysosomal degradation of sialylated glycoconjugates. Inherited deficiency of sialidase results in sialidosis or galactosialidosis, both severe metabolic disorders associated with lysosomal storage of oligosaccharides and glycopeptides. Sialidase also plays an important role in cellular signaling and is specifically required for the production of cytokine interleukin-4 by activated T lymphocytes. In these cells, neu-1-encoded sialidase activity is increased on the cell surface, suggesting that a specific mechanism regulates sorting of this enzyme to the plasma membrane. We investigated that mechanism by first showing that sialidase contains the internalization signal found in lysosomal membrane proteins targeted to endosomes via clathrin-coated pits. The signal consists of a C-terminal tetrapeptide (412)YGTL(415), with Tyr(412) and Leu(415) essential for endocytosis of the enzyme. We further demonstrated that redistribution of sialidase from lysosomes to the cell surface of activated lymphocytes is accompanied by increased reactivity of the enzyme with anti-phosphotyrosine antibodies. We speculate that phosphorylation of Tyr(412) results in inhibition of sialidase internalization in activated lymphocytes.     

Internalization of pseudorabies virus glycoprotein B is mediated by an interaction between the YQRL motif in its cytoplasmic domain and the clathrin-associated AP-2 adaptor complex

The cytoplasmic domain of pseudorabies virus (PRV) glycoprotein B (gB) contains three putative internalization motifs. Previously, we demonstrated that the tyrosine-based YQRL motif at positions 902 to 905, but not the YMSI motif at positions 864 to 867 or the LL doublet at positions 887 and 888, is required for correct functioning of gB during antibody-mediated internalization of PRV cell surface-bound glycoproteins. In the present study, we demonstrate that the YQRL motif is also crucial to allow spontaneous internalization of PRV gB, and thus, that spontaneous and antibody-mediated internalizations of PRV gB occur through closely related mechanisms. Furthermore, we found that PRV gB colocalizes with the cellular clathrin-associated AP-2 adaptor complex and that this colocalization depends on the YQRL motif. In addition, by coimmunoprecipitation assays, we found that during both spontaneous and antibody-dependent internalization, PRV gB physically interacts with AP-2, and that efficient interaction between gB and AP-2 required an intact YQRL motif. Collectively, these findings demonstrate for the first time that during internalization of an alphaherpesvirus envelope protein, i.e., PRV gB, a specific amino acid sequence in the cytoplasmic tail of the protein interacts with AP-2 and may constitute a common AP-2-mediated mechanism of internalization of alphaherpesvirus envelope proteins.     

Human transferrin receptor internalization is partially dependent upon an aromatic amino acid on the cytoplasmic domain.

The objective of this work is to identify the elements of the human transferrin receptor that are involved in receptor internalization, intracellular sorting, and recycling. We have found that an aromatic side chain at position 20 on the cytoplasmic portion of the human transferrin receptor is required for efficient internalization. The wild-type human transferrin receptor has a tyrosine at this position. Replacement of the Tyr-20 with an aromatic amino acid does not alter the rate constant of internalization, whereas substitution with the nonaromatic amino acids serine, leucine, or cysteine reduces the internalization rate constant approximately three-fold. These results are consistent with similar studies of other receptor systems that have also documented the requirement for a tyrosine in rapid internalization. The amino terminus of the transferrin receptor is cytoplasmic, with the tyrosine 41 amino acids from the membrane. These two features distinguish the transferrin receptor from the other membrane proteins for which the role of tyrosine in internalization has been examined, because these proteins have the opposite polarity with respect to the membrane and because the tyrosines are located closer to the membrane (within 25 amino acids). The externalization rate for the recycling of the transferrin receptor is not altered by any of these substitutions, demonstrating that the aromatic amino acid internalization signal is not required for the efficient exocytosis of internalized receptor.     

Polybasic KKR motif in the cytoplasmic tail of Nipah virus fusion protein modulates membrane fusion by inside-out signaling

The cytoplasmic tails of the envelope proteins from multiple viruses are known to contain determinants that affect their fusogenic capacities. Here we report that specific residues in the cytoplasmic tail of the Nipah virus fusion protein (NiV-F) modulate its fusogenic activity. Truncation of the cytoplasmic tail of NiV-F greatly inhibited cell-cell fusion. Deletion and alanine scan analysis identified a tribasic KKR motif in the membrane-adjacent region as important for modulating cell-cell fusion. The K1A mutation increased fusion 5.5-fold, while the K2A and R3A mutations decreased fusion 3- to 5-fold. These results were corroborated in a reverse-pseudotyped viral entry assay, where receptor-pseudotyped reporter virus was used to infect cells expressing wild-type or mutant NiV envelope glycoproteins. Differential monoclonal antibody binding data indicated that hyper- or hypofusogenic mutations in the KKR motif affected the ectodomain conformation of NiV-F, which in turn resulted in faster or slower six-helix bundle formation, respectively. However, we also present evidence that the hypofusogenic phenotypes of the K2A and R3A mutants were effected via distinct mechanisms. Interestingly, the K2A mutant was also markedly excluded from lipid rafts, where approximately 20% of wild-type F and the other mutants can be found. Finally, we found a strong negative correlation between the relative fusogenic capacities of these cytoplasmic-tail mutants and the avidities of NiV-F and NiV-G interactions (P = 0.007, r(2) = 0.82). In toto, our data suggest that inside-out signaling by specific residues in the cytoplasmic tail of NiV-F can modulate its fusogenicity by multiple distinct mechanisms.     

The targeting of Lamp1 to lysosomes is dependent on the spacing of its cytoplasmic tail tyrosine sorting motif relative to the membrane

Lamp1 is a type I transmembrane glycoprotein that is localized primarily in lysosomes and late endosomes. Newly synthesized molecules are mostly transported from the trans-Golgi network directly to endosomes and then to lysosomes. A minor pathway involves transport via the plasma membrane. The 11-amino acid cytoplasmic tail of lamp1 contains a tyrosine-based motif that has been previously shown to mediate sorting in the trans-Golgi network and rapid internalization at the plasma membrane. We studied whether this motif also mediates sorting in endosomes. We found that mutant forms of lamp1 in which all the amino acids of the cytoplasmic tail were modified except for the RKR membrane anchor and the YXXI sorting motif still localized to dense lysosomes, indicating that the YXXI motif is sufficient to confer proper intracellular targeting. However, when the spacing of the YXXI motif relative to the membrane was changed by deleting one amino acid or adding five amino acids, lysosomal targeting was almost completely abolished. Kinetic studies showed that these mutants were trapped in a recycling pathway, involving trafficking between the plasma membrane and early endocytic compartments. These findings indicate that the YXXI signal of lamp1 is recognized at several sorting sites, including the trans-Golgi network, the plasma membrane, and the early/sorting endosomes. Small changes in the spacing of this motif relative to the membrane dramatically impair sorting in the early/sorting endosomes but have only a modest effect on internalization at the plasma membrane. The spacing of sorting signals relative to the membrane may prove to be an important determinant in the functioning of these signals.     

The axonal sorting activity of pseudorabies virus Us9 protein depends on the state of neuronal maturation

Alpha-herpesviruses establish a life-long infection in the nervous system of the affected host; while this infection is restricted to peripheral neurons in a healthy host, the reactivated virus can spread within the neuronal circuitry, such as to the brain, in compromised individuals and lead to adverse health outcomes. Pseudorabies virus (PRV), an alpha-herpesvirus, requires the viral protein Us9 to sort virus particles into axons and facilitate neuronal spread. Us9 sorts virus particles by mediating the interaction of virus particles with neuronal transport machinery. Here, we report that Us9-mediated regulation of axonal sorting also depends on the state of neuronal maturation. Specifically, the development of dendrites and axons is accompanied with proteomic changes that influence neuronal processes. Immature superior cervical ganglionic neurons (SCGs) have rudimentary neurites that lack markers of mature axons. Immature SCGs can be infected by PRV, but they show markedly reduced Us9-dependent regulation of sorting, and increased Us9-independent transport of particles into neurites. Mature SCGs have relatively higher abundances of proteins characteristic of vesicle-transport machinery. We also identify Us9-associated neuronal proteins that can contribute to axonal sorting and subsequent anterograde spread of virus particles in axons. We show that SMPD4/nsMase3, a sphingomyelinase abundant in lipid-rafts, associates with Us9 and is a negative regulator of PRV sorting into axons and neuronal spread, a potential antiviral function.     

Fusogenicity of Jaagsiekte sheep retrovirus envelope protein is dependent on low pH and is enhanced by cytoplasmic tail truncations

Jaagsiekte sheep retrovirus (JSRV) envelope (Env) is an active oncogene responsible for neoplastic transformation in animals and cultured cells. In this study, we used syncytium induction and fluorescence-based cell fusion assays to investigate JSRV Env fusion and its modulation by the cytoplasmic tail (CT). We found that JSRV Env induced syncytia in cells overexpressing the receptor for JSRV and that a low pH was required for this process to occur. Fusion kinetics studies revealed that cell-cell fusion by JSRV Env at neutral pH was poor, taking up to a day, in sharp contrast to fusion at low pH, which peaked within 2 min following a low-pH trigger. Deletion of the C-terminal 7 or 16 amino acids of the JSRV Env CT had no or little effect on fusion, yet additional truncation toward the membrane-spanning domain, resulting in mutants retaining as little as 1 amino acid of the CT, led to progressively increased syncytium formation at neutral pH that was further enhanced by low-pH treatment. Notably, the severely truncated mutants showed elevated levels of surface subunits in culture medium, suggesting that the CT truncations resulted in conformational changes in the ectodomain of Env that impaired surface subunit associations. Taken together, this study reveals for the first time that the fusion activity of the JSRV Env protein is dependent on a low pH and is modulated by the CT, whose truncation overcomes, at least partially, the low-pH requirement for fusion and enhances Env fusion activity and kinetics.     

Fusion of enhanced green fluorescent protein to the pseudorabies virus axonal sorting protein Us9 blocks anterograde spread of infection in mammalian neurons

Pseudorabies virus encodes a membrane protein (Us9) that is essential for the axonal sorting of virus particles within neurons and anterograde spread in the mammalian nervous system. Enhanced green fluorescent protein (GFP)-tagged Us9 mimicked the trafficking properties of the wild-type protein in nonneuronal cells. We constructed a pseudorabies virus strain that expressed Us9-GFP and tested its spread capabilities in the rat visual system and in primary neuronal cultures. We report that Us9-EGFP does not promote anterograde spread of infection and may disrupt packing of viral membrane proteins in lipid rafts, an essential step for Us9-mediated axonal sorting.     

Retrograde, transneuronal spread of pseudorabies virus in defined neuronal circuitry of the rat brain is facilitated by gE mutations that reduce virulence

The pseudorabies virus (PRV) gE gene encodes a multifunctional membrane protein found in infected cell membranes and in the virion envelope. Deletion of the gE gene results in marked attenuation of the virus in almost every animal species tested that is permissive for PRV. A common inference is that gE mutants are less virulent because they have reduced ability to spread from cell to cell; e.g., gE mutants infect fewer cells and, accordingly, animals live longer. In this report, we demonstrate that this inference does not hold in a rat experimental model for virus invasion of the brain. We find that animals infected with gE mutants live longer despite extensive retrograde, transneuronal spread of virus in the rat brain. In this model of brain infection, virus is injected into the stomach musculature and virions spread to the brain in long axons of brain stem neurons that give rise to the tenth cranial nerve (the vagus). The infection then spreads from neuron to neuron in well-defined, and physically separated, areas of the brain involved in autonomic regulation of the viscera. We examined the progression of infection of five PRV strains in this circuitry: the wild-type PRV-Becker strain, the attenuated PRV-Bartha vaccine strain, and three gE mutants isogenic with the PRV-Becker strain. By 60 to 67 h after infection, all PRV-Becker-infected animals were dead. Analysis of Becker-infected rats killed prior to virus-induced death demonstrated that the virus had established an infection only in the primary vagal neurons connected directly to the stomach and synaptically linked neurons in the immediate vicinity of the caudal brain stem. There was little spread to other neurons in the vagus circuitry. In contrast, rats infected with PRV-Bartha or PRV-Becker gE mutants survived to at least 96 h and exhibited few overt signs of disease. Despite this long survival and the lack of symptoms, brains of animals sacrificed at this time revealed extensive transsynaptic infection not only of the brain stem but also of areas of the forebrain synaptically linked to neurons in the brain stem. This finding provides evidence that the gE protein plays a role in promoting symptoms of infection and death in animals that is independent of neuron-to-neuron spread during brain infection. When this early virulence function is not active, animals live longer, resulting in more extensive spread of virus in the brain.     

Role of pseudorabies virus Us9, a type II membrane protein, in infection of tissue culture cells and the rat nervous system

The protein product of the pseudorabies virus (PRV) Us9 gene is a phosphorylated, type II membrane protein that is inserted into virion envelopes and accumulates in the trans-Golgi network. It is among a linked group of three envelope protein genes in the unique short region of the PRV genome which are absent from the attenuated Bartha strain. We found that two different Us9 null mutants exhibited no obvious phenotype after infection of PK15 cells in culture. Unlike those of gE and gI null mutants, the plaque size of Us9 null mutants on Madin-Darby bovine kidney cells was indistinguishable from that of wild-type virus. However, both of the Us9 null mutants exhibited a defect in anterograde spread in the visual and cortical circuitry of the rat. The visual system defect was characterized by restricted infection of a functionally distinct subset of visual projections involved in the temporal organization of behavior, whereas decreased anterograde spread of virus to the cortical projection targets was characteristic of animals receiving direct injections of virus into the cortex. Spread of virus through retrograde pathways in the brain was not compromised by a Us9 deletion. The virulence of the Us9 null mutants, as measured by time to death and appearance of symptoms of infection, also was reduced after their injection into the eye, but not after cortical injection. Through sequence analysis, construction of revertants, measurement of gE and gI protein synthesis in the Us9 null mutants, and mixed-infection studies of rats, we conclude that the restricted-spread phenotype after infection of the rat nervous system reflects the loss of Us9 and is not an indirect effect of the Us9 mutations on expression of glycoproteins gE and gI. Therefore, at least three viral envelope proteins, Us9, gE, and gI, function together to promote efficient anterograde transneuronal infection by PRV in the rat central nervous system.     

Contribution of trafficking signals in the cytoplasmic tail of the infectious bronchitis virus spike protein to virus infection

Coronavirus spike (S) proteins are responsible for binding and fusion with target cells and thus play an essential role in virus infection. Recently, we identified a dilysine endoplasmic reticulum (ER) retrieval signal and a tyrosine-based endocytosis signal in the cytoplasmic tail of the S protein of infectious bronchitis virus (IBV). Here, an infectious cDNA clone of IBV was used to address the importance of the S protein trafficking signals to virus infection. We constructed infectious cDNA clones lacking the ER retrieval signal, the endocytosis signal, or both. The virus lacking the ER retrieval signal was viable. However, this virus had a growth defect at late times postinfection and produced larger plaques than IBV. Further analysis confirmed that the mutant S protein trafficked though the secretory pathway faster than wild-type S protein. A more dramatic phenotype was obtained when the endocytosis signal was mutated. Recombinant viruses lacking the endocytosis signal (in combination with a mutated dilysine signal or alone) could not be recovered, even though transient syncytia were formed in transfected cells. Our results suggest that the endocytosis signal of IBV S is essential for productive virus infection.     

Functional characterization of a lysosomal sorting motif in the cytoplasmic tail of HLA-DObeta

HLA-DO is an intracellular non-classical class II major histocompatibility complex molecule expressed in the endocytic pathway of B lymphocytes, which regulates the loading of antigenic peptides onto classical class II molecules such as HLA-DR. The activity of HLA-DO is mediated through its interaction with the peptide editor HLA-DM. Here, our results demonstrate that although HLA-DO is absolutely dependent on its association with DM to egress the endoplasmic reticulum, the cytoplasmic portion of its beta chain encodes a functional lysosomal sorting signal. By confocal microscopy and flow cytometry analysis, we show that reporter transmembrane molecules fused to the cytoplasmic tail of HLA-DObeta accumulated in Lamp-1(+) vesicles of transfected HeLa cells. Mutagenesis of a leucine-leucine motif abrogated lysosomal accumulation and resulted in cell surface redistribution of reporter molecules. Finally, we show that mutation of the di-leucine sequence in DObeta did not alter its lysosomal sorting when associated with DM molecules. Taken together, these results demonstrate that lysosomal expression of the DO-DM complex is mediated primarily by the tyrosine-based motif of HLA-DM and suggest that the DObeta-encoded motif is involved in the fine-tuning of the intracellular sorting.     

Activity of the Mason-Pfizer monkey virus fusion protein is modulated by single amino acids in the cytoplasmic tail

Mason-Pfizer monkey virus (M-PMV) encodes a transmembrane glycoprotein with a 38-amino-acid-long cytoplasmic tail. After the release of the immature virus, a viral protease-mediated cleavage of the cytoplasmic tail (CT) results in the loss of 17 amino acids from the carboxy terminus and renders the envelope protein fusion competent. To investigate the role of individual amino acid residues in the CT in fusion, a series of mutations was introduced, and the effects of these mutations on glycoprotein biosynthesis and fusion were examined. Most of the alanine-scanning mutations in the CT had little effect on fusion activity. However, four amino acid substitutions (threonine 4, lysine 7, glutamine 9, and isoleucine 10) resulted in substantially increased fusogenicity, while six (leucine 2, phenylalanine 5, isoleucine 13, lysine 16, proline 17, and glycine 31) resulted in much-reduced fusion. Interestingly, the bulk of these mutations are located upstream of the CT cleavage site in a region that has the potential to form a coiled-coil in the Env trimer. Substitutions at glutamine 9 and isoleucine 10 with alanine had the most dramatic positive effect and resulted in the formation of large syncytia. Taken together, these data demonstrate that individual residues within the cytoplasmic domain of M-PMV Env can modulate, in both a positive and negative manner, biological functions that are associated with the extracellular domains of the glycoprotein complex.     

A functional YNKI motif in the short cytoplasmic tail of varicella-zoster virus glycoprotein gH mediates clathrin-dependent and antibody-independent endocytosis

The trafficking of varicella-zoster virus (VZV) gH was investigated under both infection and transfection conditions. In initial endocytosis assays performed in infected cells, the three glycoproteins gE, gI, and gB served as positive controls for internalization from the plasma membrane. Subsequently, we discovered that gH in VZV-infected cells was also internalized and followed a similar trafficking pattern. This observation was unexpected because all herpesvirus gH homologues have short endodomains not known to contain trafficking motifs. Further investigation demonstrated that VZV gH, when expressed alone with its chaperone gL, was capable of endocytosis in a clathrin-dependent manner, independent of gE, gI, or gB. Upon inspection of the short gH cytoplasmic tail, we discovered a putative tyrosine-based endocytosis motif (YNKI). When the tyrosine was replaced with an alanine, endocytosis of gH was blocked. Utilizing an endocytosis assay dependent on biotin labeling, we further documented that endocytosis of VZV gH was antibody independent. In control experiments, we showed that gE, gI, and gB also internalized in an antibody-independent manner. Alignment analysis of the VZV gH cytoplasmic tail to other herpesvirus gH homologues revealed two important findings: (i) herpes simplex virus type 1 and 2 homologues lacked an endocytosis motif, while all other alphaherpesvirus gH homologues contained a potential motif, and (ii) the VZV gH and simian varicella virus gH cytoplasmic tails were likely longer in length (18 amino acids) than predicted in the original sequence analyses (12 and 16 amino acids, respectively). The longer tails provided the proper context for a functional endocytosis motif.     

An acidic cluster of the cytoplasmic tail of the RD114 virus glycoprotein controls assembly of retroviral envelopes

Retroviral core proteins, Gag and envelope (Env) glycoproteins are expressed from distinct cellular areas and therefore need to encounter to assemble infectious particles. The intrinsic cell localisation properties of either viral component or their capacity to mutually interact determines the assembly of infectious particles. Here, we address how Env determinants and cellular sorting proteins allow the Env derived from gamma retroviruses, murine leukemia virus (MLV) and RD114, to travel to or from late endosomes (LE), which may represent the Env assembly site of retroviruses in some cells. The individual expression of MLV Env resulted in its accumulation in LE in contrast to RD114 Env that required the presence of gamma retroviral Gag proteins. To discriminate between intrinsic intracellular Env localisation and gamma retroviral Gag/Env interactions in influencing Env viral incorporation, we studied Env assembly on heterologous lentiviral particles on which they are passively recruited. We found that an acidic cluster present at the C-terminus of the RD114 Env cytoplasmic tail determines its sub-cellular localisation and retrograde transport. Mutation of this motif induced late endosomal concentration of the RD114 Env, correlating with increased viral incorporation and infectivity. Reciprocally, the reinforcement of a poorly functional acidic motif in the MLV Env resulted in a marked decrease of its late endosomal localisation, leading to weakly infectious lentiviral particles with low Env densities. Finally, through upregulation versus downregulation of its cellular expression, we show that phosphofurin acidic-cluster-sorting protein 1 (PACS-1) controls the function of the RD114 Env acidic cluster, assigning to this cellular effector a crucial role in modulation of Env assembly of some retroviruses.