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Mark Marsh

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Kevin E Yarasheski, PhD

Washington University in St. Louis

Armando Marques-Guedes

UNL - New University of Lisbon

Prof.Amjad Al-Nasser

Yarmouk University

Michael J. Gonzalez

Universidad de Puerto Rico Rio

Angel Berenguer

University of Alicante / Universidad de Alicante

University of the Basque Country, Euskal Herriko Unibertsitatea

Isidro Martínez Mira

University of Alicante / Universidad de Alicante

Nicolas A Karakatsanis

Weill Cornell Medicine

Santosh K. Bharti

Johns Hopkins University School of Medicine

Prof. Dr. Hesham N Mustafa 🩺

King AbdulAziz University (KAU) Jeddah, Saudi Arabia

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Papers by Mark Marsh

Cluster of Differentiation Antigen 4 (CD4) Endocytosis and Adaptor Complex Binding Require Activation of the CD4 Endocytosis Signal by Serine Phosphorylation

Molecular Biology of the Cell, Mar 1, 1999

Cluster of differentiation antigen 4 (CD4), the T lymphocyte antigen receptor component and human... more Cluster of differentiation antigen 4 (CD4), the T lymphocyte antigen receptor component and human immunodeficiency virus coreceptor, is down-modulated when cells are activated by antigen or phorbol esters. During down-modulation CD4 dissociates from p56 lck , undergoes endocytosis through clathrin-coated pits, and is then sorted in early endosomes to late endocytic organelles where it is degraded. Previous studies have suggested that phosphorylation and a dileucine sequence are required for down-modulation. Using transfected HeLa cells, in which CD4 endocytosis can be studied in the absence of p56 lck , we show that the dileucine sequence in the cytoplasmic domain is essential for clathrin-mediated CD4 endocytosis. However, this sequence is only functional as an endocytosis signal when neighboring serine residues are phosphorylated. Phosphoserine is required for rapid endocytosis because CD4 molecules in which the cytoplasmic domain serine residues are substituted with glutamic acid residues are not internalized efficiently. Using surface plasmon resonance, we show that CD4 peptides containing the dileucine sequence bind weakly to clathrin adaptor protein complexes 2 and 1. The affinity of this interaction is increased 350-to 700-fold when the peptides also contain phosphoserine residues.

Endosomes, exosomes and Trojan viruses

Trends in Microbiology, Jul 1, 2004

Retroviruses are enveloped viruses that are generally assumed to bud at the plasma membrane of in... more Retroviruses are enveloped viruses that are generally assumed to bud at the plasma membrane of infected cells. Recently it has become apparent that some of these viruses use the endocytic pathway to coordinate their assembly and release. In addition, these and some other enveloped viruses exploit the machinery that generates the internal membranes of multivesicular bodies (MVB). These observations and others have led to the suggestion that retroviruses be regarded as 'viral exosomes'. Here we discuss this concept and the emerging evidence that compartments of the endocytic pathway play important roles in the biogenesis of both the internal vesicles of MVB and viruses.

Intracellular trafficking of proteins

Trends in Cell Biology, 1992

Endosomes—Key Components in Viral Entry and Replication

Springer eBooks, May 6, 2008

ABSTRACT Endosomes play key roles in the cellular infection cycles of many viruses. Initially imp... more ABSTRACT Endosomes play key roles in the cellular infection cycles of many viruses. Initially implicated in virus entry, recent research has demonstrated that endosomes can also be required at other stages in viral replication. Endosomes can provide platforms for viral nucleic acid replication and virus assembly, or play roles in modulating anti-viral immune responses. To these ends viruses exploit various attributes of endosomes such as the low luminal pH, unique trafficking properties, cellular location and composition. In turn, viruses have become remarkable tools for analysing endosome function.

Fix Your Membrane Receptor Imaging: Actin Cytoskeleton and CD4 Membrane Organization Disruption by Chemical Fixation

Frontiers in Immunology, Apr 5, 2019

Single-molecule localization microscopy (SMLM) techniques allow near molecular scale resolution (... more Single-molecule localization microscopy (SMLM) techniques allow near molecular scale resolution (∼ 20 nm) as well as precise and robust analysis of protein organization at different scales. SMLM hardware, analytics and probes have been the focus of a variety of studies and are now commonly used in laboratories across the world. Protocol reliability and artifact identification are increasingly seen as important aspects of super-resolution microscopy. The reliability of these approaches thus requires in-depth evaluation so that biological findings are based on solid foundations. Here we explore how different fixation approaches that disrupt or preserve the actin cytoskeleton affect membrane protein organization. Using CD4 as a model, we show that fixation-mediated disruption of the actin cytoskeleton correlates with changes in CD4 membrane organization. We highlight how these artifacts are easy to overlook and how careful sample preparation is essential for extracting meaningful results from super-resolution microscopy.

Localization of HCMV UL33 and US27 in Endocytic Compartments and Viral Membranes

Traffic, Mar 1, 2002

The human cytomegalovirus genome encodes four putative seven transmembrane domain chemokine recep... more The human cytomegalovirus genome encodes four putative seven transmembrane domain chemokine receptor-like proteins. Although important in viral pathogenesis, little is known about the properties or functions of these proteins. We previously reported that US28 is located in endocytic vesicles and undergoes constitutive endocytosis and recycling. Here we studied the cellular distributions and trafficking of two other human cytomegalovirus chemokine receptor-like proteins, UL33 and US27, in transfected and human cytomegalovirus-infected cells. Immunofluorescence staining indicated that UL33 and US27 are located at the cell surface, although the majority of both proteins was seen in intracellular organelles located in the perinuclear region of the cell. The intracellular pools of UL33 and US27 showed overlap with markers for endocytic organelles. Antibody-feeding experiments indicated that cell surface US27 undergoes endocytosis. By immunogold labeling of cryosections and electron microscopy, UL33 was seen to localize to multivesicular bodies (MVBs or multivesicular endosomes). Electron microscopy analysis of human cytomegalovirus-infected cells showed that most virus particles wrapped individually into short membrane cisternae, although virus particles were also occasionally seen within and budding into MVBs. Electron microscopy immunolocalization of viral UL33 and US27 on ultrathin cryosections of human cytomegalovirus-infected cells showed gold particles over the membranes into which virions were wrapping, in small membrane tubules and vesicles and in MVBs. Labeling of the human cytomegalovirus glycoproteins gB and gH indicated that these proteins were also present in the same membrane structures. This first electron microscopy analysis of human cytomegalovirus assembly using immunolabeling suggests that the localization of UL33, US27 and US28 to endosomes may allow these proteins to be incorporated into the viral membrane during the final stages of human cytomegalovirus assembly.

The cell biology of receptor-mediated virus entry

Journal of Cell Biology, Nov 28, 2011

Roles for endocytosis in lentiviral replication

Trends in Cell Biology, 1997

Endocytosis of the Viral Chemokine Receptor US28 Does Not Require Beta-Arrestins But Is Dependent on the Clathrin-Mediated Pathway

Traffic, Apr 1, 2003

Arrestins bind phosphorylated G‐protein coupled‐receptors (GPCR) and inhibit agonist‐induced sign... more Arrestins bind phosphorylated G‐protein coupled‐receptors (GPCR) and inhibit agonist‐induced signal transduction by uncoupling the receptors from their cognate G‐proteins. β‐arrestins also act as adaptors that target GPCR to endocytic clathrin‐coated vesicles. Unlike cellular GPCRs, the human cytomegalovirus GPCRs and chemokine receptor, US28, shows constitutive signal transduction activity and undergoes constitutive endocytosis. To determine the role of β‐arrestins in US28 trafficking, we used embryonic fibroblasts derived from β‐arrestin knockout mice. In these cells, the internalization of transfected β2‐adrenergic receptor and of the cellular chemokine receptor CCR5 was impaired. By contrast, US28 distribution was unaffected, and US28‐mediated RANTES internalization was similar in normal and knockout cell lines. To investigate whether a clathrin‐mediated pathway is involved in US28 endocytosis, we developed small interfering RNA against the μ2‐adaptin subunit of the AP‐2 adaptor complex. In cells transfected with μ2 small interfering RNA transferrin endocytosis was severely inhibited. Antibody‐feeding experiments and biochemical analysis showed that US28 internalization was also inhibited. Together, these data indicate that US28 endocytosis occurs via a clathrin‐mediated mechanism but is independent of β‐arrestins.

A Conserved Dileucine Motif Mediates Clathrin and AP-2–dependent Endocytosis of the HIV-1 Envelope Protein

Molecular Biology of the Cell, Feb 1, 2007

During the assembly of enveloped viruses viral and cellular components essential for infectious p... more 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 GYxxØ 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 GYxxØ 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 GYxxØ 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.

Distinct intracellular localization of Lck and Fyn protein tyrosine kinases in human T lymphocytes

Journal of Cell Biology, May 1, 1994

Two src family kinases, lck and fyn, participate in the activation of T lymphocytes. Both of thes... more Two src family kinases, lck and fyn, participate in the activation of T lymphocytes. Both of these protein tyrosine kinases are thought to function via their interaction with cell surface receptors. Thus, lck is associated with CD4, CDS, and Thy-1, whereas fyn is associated with the T cell antigen receptor and Thy-1. In this study, the intracellular localization of these two protein tyrosine kinases in T cells was analyzed by immunofluorescence and confocal microscopy. Lck was present at the plasma membrane, consistent with its proposed role in transmembrane signalling, and was also associated with pericentrosomal vesicles which co-localized with the cationindependent mannose 6-phosphate receptor. Surprisingly, fyn was not detected at the plasma membrane in either Jurkat T cells or T lymphoblasts but was closely associated with the centrosome and to microtubule bundles radiating from the centrosome. In mitotic cells, fyn co-localized with the mitotic spindle and poles. The essentially non-overlapping intracellular distributions of lck and fyn suggest that these kinases may be accessible to distinct regulatory proteins and substrates and, therefore, may regulate different aspects of T cell activation. Anti-phosphotyrosine antibody staining at the plasma membrane increases dra-maticaUy after CD3 cross-lb~king of Jurkat T cells. The localization of lck to the plasma membrane suggests that it may participate in mediating this increase in tyrosine phosphorylation, rather than fyn. Furthermore, the distribution of fyn in mitotic cells raises the possibility that it functions at the M phase of the cell cycle. HE T cell antigen receptor (TcR) ~ comprises the products of six genes (ot/53,~e~'2; . The ¢x/3 heterodimer recognizes an antigenic peptide bound to a major histocompatibiLity complex (MHC) molecule on the surface of an antigen-presenting ceil. The CD3 complex ('r&) and [" chains are involved in the signal transduction function of the TcR. Analysis of chimeric polypeptides containing the cytoplasmic tails of the ~" chain or of the CD3 e chain has characterized a sequence motif which is responsible for the signal transduction capability of these chains . This motif is triplicated in the ~" chain and present as a single copy in each of the CD3 chains and couples the TcR to downstream signalling pathways. Stimulation of the TcR induces the rapid tyrosine phosphorylation of multiple proteins on tyrosine (Hsi et al.,

SFV infection in CHO cells: cell-type specific restrictions to productive virus entry at the cell surface

Journal of Cell Science, 1997

Alphaviruses, such as Semliki Forest virus, normally enter cells by penetration from acidic organ... more Alphaviruses, such as Semliki Forest virus, normally enter cells by penetration from acidic organelles of the endocytic pathway. The virions are internalised intact from the cell surface before undergoing acid-induced fusion in endosomes. To investigate the possibility that endocytosis might play a role in delivering virions to specific sites for replication, we compared SFV infection of baby hamster kidney (BHK) cells and Chinese hamster ovary (CHO) cells following either normal virus fusion in endosomes or experimentally-induced fusion at the cell surface. Whereas baby hamster kidney cells were infected efficiently following fusion in endosomes or at the plasma membrane, Chinese hamster ovary cells were only infected following fusion from endocytic organelles. Virions fused at the plasma membrane of CHO cells failed to initiate viral RNA and protein synthesis. Similar results were observed when CHO cells were challenged with a rhabdovirus, vesicular stomatitis virus. These data suggest that in certain cell types a barrier, other than the plasma membrane, can prevent infection by alpha-and rhabdoviruses fused at the cell surface. Moreover, they suggest the endocytic pathway provides a mechanism for bringing viral particles to a site, or sites, in the cell where replication can proceed.

Taking the Rabs off endocytosis The Rab family of small CTP-binding proteins have been implicated in the regulation of membrane traffic. Recent results suggest that Rab? and Rab5 govern rate-limiting steps in the endocytic pathway

Transport between compartments of the exocytic and en- docytic pathways that is mediated by vesic... more Transport between compartments of the exocytic and en- docytic pathways that is mediated by vesicular carriers, as first proposed by Palade [l], requires the regulation of both vesicle formation and the specificity of subsequent vesicle targetting. This dual regulation maintains the struc- tural and biochemical integrity of each compartment and imparts directionality on the transport process. To date, the molecular mechanisms that ensure transport vesicles dock and fuse with their correct targets have remained ob- scure. The Ras-related Rab family of low molecular weight (21-26kD) GTP-binding proteins appear to have at least some of the credentials for such a role. The Rab proteins - so-called because the first members were identified as Ras-like proteins in a rat brain cDNA library - are the mammalian counterparts of two small GTP-binding proteins, Yptl and Sec4, of Saccharomyces cerevkiae. Work on these yeast proteins provided the first insight into the function of Rabs, when mutations of the WTl and SEC4 genes were found to cause defects in the exocytic pathway. SEC4 mutations are associated with the accumulation of stage-specific vesicular intermediates

Signal Peptide-Binding Drug as a Selective Inhibitor of Co-Translational Protein Translocation

PLOS Biology, Dec 2, 2014

In eukaryotic cells, surface expression of most type I transmembrane proteins requires translatio... more In eukaryotic cells, surface expression of most type I transmembrane proteins requires translation and simultaneous insertion of the precursor protein into the endoplasmic reticulum (ER) membrane for subsequent routing to the cell surface. This co-translational translocation pathway is initiated when a hydrophobic N-terminal signal peptide (SP) on the nascent protein emerges from the ribosome, binds the cytosolic signal recognition particle (SRP), and targets the ribosome-nascent chain complex to the Sec61 translocon, a universally conserved protein-conducting channel in the ER-membrane. Despite their common function in Sec61 targeting and ER translocation, SPs have diverse but unique primary sequences. Thus, drugs that recognise SPs could be exploited to inhibit translocation of specific proteins into the ER. Here, through flow cytometric analysis the small-molecule macrocycle cyclotriazadisulfonamide (CADA) is identified as a highly selective human CD4 (hCD4) down-modulator. We show that CADA inhibits CD4 biogenesis and that this is due to its ability to inhibit cotranslational translocation of CD4 into the lumen of the ER, both in cells as in a cell-free in vitro translation/translocation system. The activity of CADA maps to the cleavable N-terminal SP of hCD4. Moreover, through surface plasmon resonance analysis we were able to show direct binding of CADA to the SP of hCD4 and identify this SP as the target of our drug. Furthermore, CADA locks the SP in the translocon during a post-targeting step, possibly in a folded state, and prevents the translocation of the associated protein into the ER lumen. Instead, the precursor protein is routed to the cytosol for degradation. These findings demonstrate that a synthetic, cell-permeable small-molecule can be developed as a SP-binding drug to selectively inhibit protein translocation and to reversibly regulate the expression of specific target proteins.

Tetherin Can Restrict Cell-Free and Cell-Cell Transmission of HIV from Primary Macrophages to T Cells

PLOS Pathogens, Jul 3, 2014

Bst-2/Tetherin inhibits the release of HIV by tethering newly formed virus particles to the plasm... more Bst-2/Tetherin inhibits the release of HIV by tethering newly formed virus particles to the plasma membrane of infected cells. Although the mechanisms of Tetherin-mediated restriction are increasingly well understood, the biological relevance of this restriction in the natural target cells of HIV is unclear. Moreover, whether Tetherin exerts any restriction on the direct cell-cell spread of HIV across intercellular contacts remains controversial. Here we analyse the restriction endogenous Tetherin imposes on HIV transmission from primary human macrophages, one of the main targets of HIV in vivo. We find that the mRNA and protein levels of Tetherin in macrophages are comparable to those in T cells from the same donors, and are highly upregulated by type I interferons. Improved immunocytochemistry protocols enable us to demonstrate that Tetherin localises to the cell surface, the trans-Golgi network, and the macrophage HIV assembly compartments. Tetherin retains budded virions in the assembly compartments, thereby impeding the release and cell-free spread of HIV, but it is not required for the maintenance of these compartments per se. Notably, using a novel assay to quantify cell-cell spread, we show that Tetherin promotes the transfer of virus clusters from macrophages to T cells and thereby restricts the direct transmission of a dual-tropic HIV-1. Kinetic analyses provide support for the notion that this direct macrophage-T cell spread is mediated, at least in part, by so-called virological synapses. Finally, we demonstrate that the viral Vpu protein efficiently downregulates the cell surface and overall levels of Tetherin, and thereby abrogates this HIV restriction in macrophages. Together, our study shows that Tetherin, one of the most potent HIV restriction factors identified to date, can inhibit virus spread from primary macrophages, regardless of the mode of transmission.

No ESCRTs for Exosomes

Science, Feb 29, 2008

Dendritic cells: New roles for Cdc42 and Rac in antigen uptake?

Current Biology, Oct 1, 2000

In dendritic cells, macropinocytosis is downregulated during maturation and antigen presentation.... more

Clathrin-coated Vesicles and Receptor-mediated Endocytosis

Receptor-mediated endocytosis (RME) is the highly efficient uptake of extracellular molecules int... more Receptor-mediated endocytosis (RME) is the highly efficient uptake of extracellular molecules into cells in membrane-bound vesicles formed by invagination of the plasma membrane. RME frequently occurs through clathrin-coated vesicles. Keywords: clathrin; coated pits; endocytosis; endocytic pathway; adaptors

Trafficking of Viral Membrane Proteins

Springer eBooks, Oct 24, 2005

Many viruses express membrane proteins. For enveloped viruses in particular, membrane proteins ar... more Many viruses express membrane proteins. For enveloped viruses in particular, membrane proteins are frequently structural components of the virus that mediate the essential tasks of receptor recognition and membrane fusion. The functional activities of these proteins require that they are sorted correctly in infected cells. These sorting events often depend on the ability of the virus to mimic cellular protein trafficking signals and to interact with the cellular trafficking machinery. Importantly, loss or modification of these signals can influence virus infectivity and pathogenesis.

The Nef Protein of the Macrophage Tropic HIV-1 Strain AD8 Counteracts Human BST-2/Tetherin

Viruses, Apr 18, 2020

Bone Marrow Stromal Cell Antigen 2 (BST-2)/tetherin inhibits the release of numerous enveloped vi... more Bone Marrow Stromal Cell Antigen 2 (BST-2)/tetherin inhibits the release of numerous enveloped viruses by physically tethering nascent particles to infected cells during the process of viral budding from the cell surface. Tetherin also restricts human immunodeficiency virus (HIV), and pandemic main (M) group HIV type 1s (HIV-1s) are thought to rely exclusively on their Vpu proteins to overcome tetherin-mediated restriction of virus release. However, at least one M group HIV-1 strain, the macrophage-tropic primary AD8 isolate, is unable to express Vpu due to a mutation in its translation initiation codon. Here, using primary monocyte-derived macrophages (MDMs), we show that AD8 Nef protein can compensate for the absence of Vpu and restore virus release to wild type levels. We demonstrate that HIV-1 AD8 Nef reduces endogenous cell surface tetherin levels, physically separating it from the site of viral budding, thus preventing HIV retention. Mechanistically, AD8 Nef enhances internalisation of the long isoform of human tetherin, leading to perinuclear accumulation of the restriction factor. Finally, we show that Nef proteins from other HIV strains also display varying degrees of tetherin antagonism. Overall, we show that M group HIV-1s can use an accessory protein other than Vpu to antagonise human tetherin.

Cluster of Differentiation Antigen 4 (CD4) Endocytosis and Adaptor Complex Binding Require Activation of the CD4 Endocytosis Signal by Serine Phosphorylation

Molecular Biology of the Cell, Mar 1, 1999

Cluster of differentiation antigen 4 (CD4), the T lymphocyte antigen receptor component and human... more Cluster of differentiation antigen 4 (CD4), the T lymphocyte antigen receptor component and human immunodeficiency virus coreceptor, is down-modulated when cells are activated by antigen or phorbol esters. During down-modulation CD4 dissociates from p56 lck , undergoes endocytosis through clathrin-coated pits, and is then sorted in early endosomes to late endocytic organelles where it is degraded. Previous studies have suggested that phosphorylation and a dileucine sequence are required for down-modulation. Using transfected HeLa cells, in which CD4 endocytosis can be studied in the absence of p56 lck , we show that the dileucine sequence in the cytoplasmic domain is essential for clathrin-mediated CD4 endocytosis. However, this sequence is only functional as an endocytosis signal when neighboring serine residues are phosphorylated. Phosphoserine is required for rapid endocytosis because CD4 molecules in which the cytoplasmic domain serine residues are substituted with glutamic acid residues are not internalized efficiently. Using surface plasmon resonance, we show that CD4 peptides containing the dileucine sequence bind weakly to clathrin adaptor protein complexes 2 and 1. The affinity of this interaction is increased 350-to 700-fold when the peptides also contain phosphoserine residues.

Endosomes, exosomes and Trojan viruses

Trends in Microbiology, Jul 1, 2004

Retroviruses are enveloped viruses that are generally assumed to bud at the plasma membrane of in... more Retroviruses are enveloped viruses that are generally assumed to bud at the plasma membrane of infected cells. Recently it has become apparent that some of these viruses use the endocytic pathway to coordinate their assembly and release. In addition, these and some other enveloped viruses exploit the machinery that generates the internal membranes of multivesicular bodies (MVB). These observations and others have led to the suggestion that retroviruses be regarded as 'viral exosomes'. Here we discuss this concept and the emerging evidence that compartments of the endocytic pathway play important roles in the biogenesis of both the internal vesicles of MVB and viruses.

Intracellular trafficking of proteins

Trends in Cell Biology, 1992

Endosomes—Key Components in Viral Entry and Replication

Springer eBooks, May 6, 2008

ABSTRACT Endosomes play key roles in the cellular infection cycles of many viruses. Initially imp... more ABSTRACT Endosomes play key roles in the cellular infection cycles of many viruses. Initially implicated in virus entry, recent research has demonstrated that endosomes can also be required at other stages in viral replication. Endosomes can provide platforms for viral nucleic acid replication and virus assembly, or play roles in modulating anti-viral immune responses. To these ends viruses exploit various attributes of endosomes such as the low luminal pH, unique trafficking properties, cellular location and composition. In turn, viruses have become remarkable tools for analysing endosome function.

Fix Your Membrane Receptor Imaging: Actin Cytoskeleton and CD4 Membrane Organization Disruption by Chemical Fixation

Frontiers in Immunology, Apr 5, 2019

Single-molecule localization microscopy (SMLM) techniques allow near molecular scale resolution (... more Single-molecule localization microscopy (SMLM) techniques allow near molecular scale resolution (∼ 20 nm) as well as precise and robust analysis of protein organization at different scales. SMLM hardware, analytics and probes have been the focus of a variety of studies and are now commonly used in laboratories across the world. Protocol reliability and artifact identification are increasingly seen as important aspects of super-resolution microscopy. The reliability of these approaches thus requires in-depth evaluation so that biological findings are based on solid foundations. Here we explore how different fixation approaches that disrupt or preserve the actin cytoskeleton affect membrane protein organization. Using CD4 as a model, we show that fixation-mediated disruption of the actin cytoskeleton correlates with changes in CD4 membrane organization. We highlight how these artifacts are easy to overlook and how careful sample preparation is essential for extracting meaningful results from super-resolution microscopy.

Localization of HCMV UL33 and US27 in Endocytic Compartments and Viral Membranes

Traffic, Mar 1, 2002

The human cytomegalovirus genome encodes four putative seven transmembrane domain chemokine recep... more The human cytomegalovirus genome encodes four putative seven transmembrane domain chemokine receptor-like proteins. Although important in viral pathogenesis, little is known about the properties or functions of these proteins. We previously reported that US28 is located in endocytic vesicles and undergoes constitutive endocytosis and recycling. Here we studied the cellular distributions and trafficking of two other human cytomegalovirus chemokine receptor-like proteins, UL33 and US27, in transfected and human cytomegalovirus-infected cells. Immunofluorescence staining indicated that UL33 and US27 are located at the cell surface, although the majority of both proteins was seen in intracellular organelles located in the perinuclear region of the cell. The intracellular pools of UL33 and US27 showed overlap with markers for endocytic organelles. Antibody-feeding experiments indicated that cell surface US27 undergoes endocytosis. By immunogold labeling of cryosections and electron microscopy, UL33 was seen to localize to multivesicular bodies (MVBs or multivesicular endosomes). Electron microscopy analysis of human cytomegalovirus-infected cells showed that most virus particles wrapped individually into short membrane cisternae, although virus particles were also occasionally seen within and budding into MVBs. Electron microscopy immunolocalization of viral UL33 and US27 on ultrathin cryosections of human cytomegalovirus-infected cells showed gold particles over the membranes into which virions were wrapping, in small membrane tubules and vesicles and in MVBs. Labeling of the human cytomegalovirus glycoproteins gB and gH indicated that these proteins were also present in the same membrane structures. This first electron microscopy analysis of human cytomegalovirus assembly using immunolabeling suggests that the localization of UL33, US27 and US28 to endosomes may allow these proteins to be incorporated into the viral membrane during the final stages of human cytomegalovirus assembly.

The cell biology of receptor-mediated virus entry

Journal of Cell Biology, Nov 28, 2011

Roles for endocytosis in lentiviral replication

Trends in Cell Biology, 1997

Endocytosis of the Viral Chemokine Receptor US28 Does Not Require Beta-Arrestins But Is Dependent on the Clathrin-Mediated Pathway

Traffic, Apr 1, 2003

Arrestins bind phosphorylated G‐protein coupled‐receptors (GPCR) and inhibit agonist‐induced sign... more Arrestins bind phosphorylated G‐protein coupled‐receptors (GPCR) and inhibit agonist‐induced signal transduction by uncoupling the receptors from their cognate G‐proteins. β‐arrestins also act as adaptors that target GPCR to endocytic clathrin‐coated vesicles. Unlike cellular GPCRs, the human cytomegalovirus GPCRs and chemokine receptor, US28, shows constitutive signal transduction activity and undergoes constitutive endocytosis. To determine the role of β‐arrestins in US28 trafficking, we used embryonic fibroblasts derived from β‐arrestin knockout mice. In these cells, the internalization of transfected β2‐adrenergic receptor and of the cellular chemokine receptor CCR5 was impaired. By contrast, US28 distribution was unaffected, and US28‐mediated RANTES internalization was similar in normal and knockout cell lines. To investigate whether a clathrin‐mediated pathway is involved in US28 endocytosis, we developed small interfering RNA against the μ2‐adaptin subunit of the AP‐2 adaptor complex. In cells transfected with μ2 small interfering RNA transferrin endocytosis was severely inhibited. Antibody‐feeding experiments and biochemical analysis showed that US28 internalization was also inhibited. Together, these data indicate that US28 endocytosis occurs via a clathrin‐mediated mechanism but is independent of β‐arrestins.

A Conserved Dileucine Motif Mediates Clathrin and AP-2–dependent Endocytosis of the HIV-1 Envelope Protein

Molecular Biology of the Cell, Feb 1, 2007

During the assembly of enveloped viruses viral and cellular components essential for infectious p... more 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 GYxxØ 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 GYxxØ 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 GYxxØ 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.

Distinct intracellular localization of Lck and Fyn protein tyrosine kinases in human T lymphocytes

Journal of Cell Biology, May 1, 1994

Two src family kinases, lck and fyn, participate in the activation of T lymphocytes. Both of thes... more Two src family kinases, lck and fyn, participate in the activation of T lymphocytes. Both of these protein tyrosine kinases are thought to function via their interaction with cell surface receptors. Thus, lck is associated with CD4, CDS, and Thy-1, whereas fyn is associated with the T cell antigen receptor and Thy-1. In this study, the intracellular localization of these two protein tyrosine kinases in T cells was analyzed by immunofluorescence and confocal microscopy. Lck was present at the plasma membrane, consistent with its proposed role in transmembrane signalling, and was also associated with pericentrosomal vesicles which co-localized with the cationindependent mannose 6-phosphate receptor. Surprisingly, fyn was not detected at the plasma membrane in either Jurkat T cells or T lymphoblasts but was closely associated with the centrosome and to microtubule bundles radiating from the centrosome. In mitotic cells, fyn co-localized with the mitotic spindle and poles. The essentially non-overlapping intracellular distributions of lck and fyn suggest that these kinases may be accessible to distinct regulatory proteins and substrates and, therefore, may regulate different aspects of T cell activation. Anti-phosphotyrosine antibody staining at the plasma membrane increases dra-maticaUy after CD3 cross-lb~king of Jurkat T cells. The localization of lck to the plasma membrane suggests that it may participate in mediating this increase in tyrosine phosphorylation, rather than fyn. Furthermore, the distribution of fyn in mitotic cells raises the possibility that it functions at the M phase of the cell cycle. HE T cell antigen receptor (TcR) ~ comprises the products of six genes (ot/53,~e~'2; . The ¢x/3 heterodimer recognizes an antigenic peptide bound to a major histocompatibiLity complex (MHC) molecule on the surface of an antigen-presenting ceil. The CD3 complex ('r&) and [" chains are involved in the signal transduction function of the TcR. Analysis of chimeric polypeptides containing the cytoplasmic tails of the ~" chain or of the CD3 e chain has characterized a sequence motif which is responsible for the signal transduction capability of these chains . This motif is triplicated in the ~" chain and present as a single copy in each of the CD3 chains and couples the TcR to downstream signalling pathways. Stimulation of the TcR induces the rapid tyrosine phosphorylation of multiple proteins on tyrosine (Hsi et al.,

SFV infection in CHO cells: cell-type specific restrictions to productive virus entry at the cell surface

Journal of Cell Science, 1997

Alphaviruses, such as Semliki Forest virus, normally enter cells by penetration from acidic organ... more Alphaviruses, such as Semliki Forest virus, normally enter cells by penetration from acidic organelles of the endocytic pathway. The virions are internalised intact from the cell surface before undergoing acid-induced fusion in endosomes. To investigate the possibility that endocytosis might play a role in delivering virions to specific sites for replication, we compared SFV infection of baby hamster kidney (BHK) cells and Chinese hamster ovary (CHO) cells following either normal virus fusion in endosomes or experimentally-induced fusion at the cell surface. Whereas baby hamster kidney cells were infected efficiently following fusion in endosomes or at the plasma membrane, Chinese hamster ovary cells were only infected following fusion from endocytic organelles. Virions fused at the plasma membrane of CHO cells failed to initiate viral RNA and protein synthesis. Similar results were observed when CHO cells were challenged with a rhabdovirus, vesicular stomatitis virus. These data suggest that in certain cell types a barrier, other than the plasma membrane, can prevent infection by alpha-and rhabdoviruses fused at the cell surface. Moreover, they suggest the endocytic pathway provides a mechanism for bringing viral particles to a site, or sites, in the cell where replication can proceed.

Taking the Rabs off endocytosis The Rab family of small CTP-binding proteins have been implicated in the regulation of membrane traffic. Recent results suggest that Rab? and Rab5 govern rate-limiting steps in the endocytic pathway

Transport between compartments of the exocytic and en- docytic pathways that is mediated by vesic... more Transport between compartments of the exocytic and en- docytic pathways that is mediated by vesicular carriers, as first proposed by Palade [l], requires the regulation of both vesicle formation and the specificity of subsequent vesicle targetting. This dual regulation maintains the struc- tural and biochemical integrity of each compartment and imparts directionality on the transport process. To date, the molecular mechanisms that ensure transport vesicles dock and fuse with their correct targets have remained ob- scure. The Ras-related Rab family of low molecular weight (21-26kD) GTP-binding proteins appear to have at least some of the credentials for such a role. The Rab proteins - so-called because the first members were identified as Ras-like proteins in a rat brain cDNA library - are the mammalian counterparts of two small GTP-binding proteins, Yptl and Sec4, of Saccharomyces cerevkiae. Work on these yeast proteins provided the first insight into the function of Rabs, when mutations of the WTl and SEC4 genes were found to cause defects in the exocytic pathway. SEC4 mutations are associated with the accumulation of stage-specific vesicular intermediates

Signal Peptide-Binding Drug as a Selective Inhibitor of Co-Translational Protein Translocation

PLOS Biology, Dec 2, 2014

In eukaryotic cells, surface expression of most type I transmembrane proteins requires translatio... more In eukaryotic cells, surface expression of most type I transmembrane proteins requires translation and simultaneous insertion of the precursor protein into the endoplasmic reticulum (ER) membrane for subsequent routing to the cell surface. This co-translational translocation pathway is initiated when a hydrophobic N-terminal signal peptide (SP) on the nascent protein emerges from the ribosome, binds the cytosolic signal recognition particle (SRP), and targets the ribosome-nascent chain complex to the Sec61 translocon, a universally conserved protein-conducting channel in the ER-membrane. Despite their common function in Sec61 targeting and ER translocation, SPs have diverse but unique primary sequences. Thus, drugs that recognise SPs could be exploited to inhibit translocation of specific proteins into the ER. Here, through flow cytometric analysis the small-molecule macrocycle cyclotriazadisulfonamide (CADA) is identified as a highly selective human CD4 (hCD4) down-modulator. We show that CADA inhibits CD4 biogenesis and that this is due to its ability to inhibit cotranslational translocation of CD4 into the lumen of the ER, both in cells as in a cell-free in vitro translation/translocation system. The activity of CADA maps to the cleavable N-terminal SP of hCD4. Moreover, through surface plasmon resonance analysis we were able to show direct binding of CADA to the SP of hCD4 and identify this SP as the target of our drug. Furthermore, CADA locks the SP in the translocon during a post-targeting step, possibly in a folded state, and prevents the translocation of the associated protein into the ER lumen. Instead, the precursor protein is routed to the cytosol for degradation. These findings demonstrate that a synthetic, cell-permeable small-molecule can be developed as a SP-binding drug to selectively inhibit protein translocation and to reversibly regulate the expression of specific target proteins.

Tetherin Can Restrict Cell-Free and Cell-Cell Transmission of HIV from Primary Macrophages to T Cells

PLOS Pathogens, Jul 3, 2014

Bst-2/Tetherin inhibits the release of HIV by tethering newly formed virus particles to the plasm... more Bst-2/Tetherin inhibits the release of HIV by tethering newly formed virus particles to the plasma membrane of infected cells. Although the mechanisms of Tetherin-mediated restriction are increasingly well understood, the biological relevance of this restriction in the natural target cells of HIV is unclear. Moreover, whether Tetherin exerts any restriction on the direct cell-cell spread of HIV across intercellular contacts remains controversial. Here we analyse the restriction endogenous Tetherin imposes on HIV transmission from primary human macrophages, one of the main targets of HIV in vivo. We find that the mRNA and protein levels of Tetherin in macrophages are comparable to those in T cells from the same donors, and are highly upregulated by type I interferons. Improved immunocytochemistry protocols enable us to demonstrate that Tetherin localises to the cell surface, the trans-Golgi network, and the macrophage HIV assembly compartments. Tetherin retains budded virions in the assembly compartments, thereby impeding the release and cell-free spread of HIV, but it is not required for the maintenance of these compartments per se. Notably, using a novel assay to quantify cell-cell spread, we show that Tetherin promotes the transfer of virus clusters from macrophages to T cells and thereby restricts the direct transmission of a dual-tropic HIV-1. Kinetic analyses provide support for the notion that this direct macrophage-T cell spread is mediated, at least in part, by so-called virological synapses. Finally, we demonstrate that the viral Vpu protein efficiently downregulates the cell surface and overall levels of Tetherin, and thereby abrogates this HIV restriction in macrophages. Together, our study shows that Tetherin, one of the most potent HIV restriction factors identified to date, can inhibit virus spread from primary macrophages, regardless of the mode of transmission.

No ESCRTs for Exosomes

Science, Feb 29, 2008

Dendritic cells: New roles for Cdc42 and Rac in antigen uptake?

Current Biology, Oct 1, 2000

In dendritic cells, macropinocytosis is downregulated during maturation and antigen presentation.... more

Clathrin-coated Vesicles and Receptor-mediated Endocytosis

Receptor-mediated endocytosis (RME) is the highly efficient uptake of extracellular molecules int... more Receptor-mediated endocytosis (RME) is the highly efficient uptake of extracellular molecules into cells in membrane-bound vesicles formed by invagination of the plasma membrane. RME frequently occurs through clathrin-coated vesicles. Keywords: clathrin; coated pits; endocytosis; endocytic pathway; adaptors

Trafficking of Viral Membrane Proteins

Springer eBooks, Oct 24, 2005

Many viruses express membrane proteins. For enveloped viruses in particular, membrane proteins ar... more Many viruses express membrane proteins. For enveloped viruses in particular, membrane proteins are frequently structural components of the virus that mediate the essential tasks of receptor recognition and membrane fusion. The functional activities of these proteins require that they are sorted correctly in infected cells. These sorting events often depend on the ability of the virus to mimic cellular protein trafficking signals and to interact with the cellular trafficking machinery. Importantly, loss or modification of these signals can influence virus infectivity and pathogenesis.

The Nef Protein of the Macrophage Tropic HIV-1 Strain AD8 Counteracts Human BST-2/Tetherin

Viruses, Apr 18, 2020

Bone Marrow Stromal Cell Antigen 2 (BST-2)/tetherin inhibits the release of numerous enveloped vi... more Bone Marrow Stromal Cell Antigen 2 (BST-2)/tetherin inhibits the release of numerous enveloped viruses by physically tethering nascent particles to infected cells during the process of viral budding from the cell surface. Tetherin also restricts human immunodeficiency virus (HIV), and pandemic main (M) group HIV type 1s (HIV-1s) are thought to rely exclusively on their Vpu proteins to overcome tetherin-mediated restriction of virus release. However, at least one M group HIV-1 strain, the macrophage-tropic primary AD8 isolate, is unable to express Vpu due to a mutation in its translation initiation codon. Here, using primary monocyte-derived macrophages (MDMs), we show that AD8 Nef protein can compensate for the absence of Vpu and restore virus release to wild type levels. We demonstrate that HIV-1 AD8 Nef reduces endogenous cell surface tetherin levels, physically separating it from the site of viral budding, thus preventing HIV retention. Mechanistically, AD8 Nef enhances internalisation of the long isoform of human tetherin, leading to perinuclear accumulation of the restriction factor. Finally, we show that Nef proteins from other HIV strains also display varying degrees of tetherin antagonism. Overall, we show that M group HIV-1s can use an accessory protein other than Vpu to antagonise human tetherin.