Annegret Pelchen-Matthews

Infectious HIV-1 assembles in late endosomes in primary macrophages.

Although human immunodeficiency virus type 1 (HIV-1) is generally thought to assemble at the plasma membrane of infected cells, virions have been observed in intracellular compartments in macrophages. Here, we investigated virus assembly in HIV-1–infected primary human monocyte-derived macrophages (MDM). Electron microscopy of cryosections showed virus particles, identified by their morphology and positive labeling with antibodies to the viral p17, p24, and envelope proteins, in intracellular vacuoles. Immunolabeling demonstrated that these compartments contained the late endosomal marker CD63, which was enriched on vesicles within these structures and incorporated into the envelope of budding virions. The virus-containing vacuoles were also labeled with antibodies against LAMP-1, CD81, and CD82, which were also incorporated into the viral envelope. To assess the cellular source of infectious viruses derived from MDM, virus-containing media from infected cells were precipitated with specific antibodies. Only antibodies against antigens found in late endosomes precipitated infectious virus, whereas antibodies against proteins located primarily on the cell surface did not. Our data indicate that most of the infectious HIV produced by primary macrophages is assembled on late endocytic membranes and acquires antigens characteristic of this compartment. This notion has significant implications for understanding the biology of HIV and its cell–cell transmission.

In macrophages, HIV-1 assembles into an intracellular plasma membrane domain containing the tetraspanins CD81, CD9, and CD53

Deneka et al. 2007. J. Cell Biol. doi:10.1083/jcb.200609050 [OpenUrl][1][Abstract/FREE Full Text][2] [1]: {openurl}?query=rft_id%253Dinfo%253Adoi%252F10.1083%252Fjcb.200609050%26rft_id%253Dinfo%253Apmid%252F17438075%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%

HIV-1 trafficking to the dendritic cell-T-cell infectious synapse uses a pathway of tetraspanin sorting to the immunological synapse

Dendritic cells (DCs) are essential components of the early events of HIV infection. Here, we characterized the trafficking pathways that HIV‐1 follows during its capture by DCs and its subsequent presentation to CD4+ T cells via an infectious synapse. Immunofluorescence microscopy indicates that the virus‐containing compartment in mature DCs (mDCs) co‐labels for the tetraspanins CD81, CD82, and CD9 but contains little CD63 or LAMP‐1. Using ratio imaging of pH‐reporting fluorescent virions in live DCs, we show that HIV‐1 is internalized in an intracellular endocytic compartment with a pH of 6.2. Significantly, we demonstrate that the infectivity of cell‐free virus is more stable at mildly acidic pH than at neutral pH. Using electron microscopy, we confirm that HIV‐1 accumulates in intracellular vacuoles that contain CD81 positive internal membranes but overlaps only partially with CD63. When allowed to contact T cells, HIV‐1‐loaded DCs redistribute CD81, and CD9, as well as internalized HIV‐1, but not the immunological synapse markers MHC‐II and T‐cell receptor to the infectious synapse. Together, our results indicate that HIV‐1 is internalized into a non‐conventional, non‐lysosomal, endocytic compartment in mDCs and further suggest that HIV‐1 is able to selectively subvert components of the intracellular trafficking machinery required for formation of the DC–T‐cell immunological synapse to facilitate its own cell‐to‐cell transfer and propagation.

Simian immunodeficiency virus envelope glycoprotein counteracts tetherin/BST-2/CD317 by intracellular sequestration

Tetherin is an IFN-inducible restriction factor that inhibits HIV-1 particle release in the absence of the HIV-1 countermeasure, viral protein U (Vpu). Although ubiquitous in HIV-1 and simian immunodeficiency viruses from chimpanzees, greater spot nosed monkeys, mustached monkeys, and Mona monkeys, other primate lentiviruses do not encode a Vpu protein. Here we demonstrate that SIV from Tantalus monkeys (SIVtan) encodes an envelope glycoprotein (SIVtan Env) able to counteract tetherin from Tantalus monkeys, rhesus monkeys, sooty mangabeys, and humans, but not from pigs. We show that sensitivity to Vpu but not SIVtan Env can be transferred with the human tetherin transmembrane region. We also identify a mutation in the tetherin extracellular domain, which almost completely abolishes sensitivity of human tetherin to SIVtan Env without compromising antiviral activity or sensitivity to Vpu. SIVtan Env expression results in a reduction of surface tetherin, as well as reduction in tetherin co-localization with mature surface-associated virus. Immuno-electron microscopy reveals co-localization of SIVtan Env with tetherin in intracellular tubulo-vesicular structures, suggesting that tetherin is sequestered away from budding virions at the cell surface. Along with HIV-1 Vpu and SIV Nef, envelope glycoprotein is the third and most broadly active lentiviral-encoded tetherin countermeasure to be described. Our observations emphasize the importance of tetherin in protecting mammals against viral infection and suggest that HIV-1 Vpu inhibitors may select active envelope mutants.

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

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.

Endocytosis in Viral Replication

Although initially recognised as essential for the entry of certain viruses, endocytosis is now known to also play important roles in the replication of, and adaptation to, the host cell of a number of viruses. Here we consider several aspects of this association and recent results that have emerged to support this view.

Chemokine receptor trafficking and viral replication

Summary: Chemokines and chemokine receptors have emerged as crucial factors controlling the development and function of leukocytes. Recent studies have indicated that, in addition to these essential roles, both chemokines and chemokine receptors play critical roles in viral infection and replication. Not only are chemokine receptors key components of the receptor/fusion complexes of primate immunodeficiency viruses, hut chemokines can also influence virus entry and infection. Many viruses, in particular herpesviruses, encode chemokines and chemokine receptors that influence the replication of both the parent virus and other unrelated viruses. The cell surface expression of the chemokine receptors is regulated through their interaction with membrane trafficking pathways, ligands induce receptor internalization and downmodulation through endocytosis, and recycling is regulated within endosomes. Pan of the mechanism through which chemokines protect cells from HIV infection is through ligand‐induced internalization of the specific chemokine receptor co‐receptors. In addition, mechanisms may exist to regulate the trafficking of newly synthesized receptors to the cell surface. Here we discuss aspects of the mechanisms through which chemokine receptors interact with membrane‐trafficking pathways and the influence of these interactions on viral replication.

The Simian Immunodeficiency Virus Envelope Glycoprotein Contains Multiple Signals that Regulate its Cell Surface Expression and Endocytosis

The cell surface expression of the envelope glycoproteins (Envs) of primate immunodeficiency viruses is, at least in part, regulated by endocytosis signal(s) located in the Env cytoplasmic domain. Here, we show that a membrane proximal signal that directs the simian immunodeficiency virus (SIV) Env to clathrin‐coated pits, and is conserved in all SIV and human immunodeficiency virus Envs, conforms to a Yxxø motif (where x can be any amino acid and Ø represents a large hydrophobic residue). This motif is similar to that described for a number of cellular membrane proteins. By surface plasmon resonance we detected a high affinity interaction between peptides containing this membrane proximal signal and both AP1 and AP2 clathrin adaptor complexes. Mutation of the tyrosine in this membrane proximal motif in a SIV Env with a prematurely truncated cytoplasmic domain leads to a ≥25‐fold increase in Env expression on infected cells. By contrast, the same mutation in an Env with a full‐length cytoplasmic domain increases cell surface expression only 4‐fold. We show that this effect results from the presence of additional endocytosis signals in the full‐length cytoplasmic domain. Chimeras containing CD4 ecto‐ and membrane spanning domains and a full‐length SIV Env cytoplasmic domain showed rapid endocytosis even when the membrane proximal tyrosine‐based signal was disrupted. Mapping experiments indicated that at least some of the additional endocytosis information is located between residues 743 and 812 of Env from the SIVmac239 molecular clone. Together, our findings indicate that the cytoplasmic domain of SIV Env contains multiple endocytosis and/or trafficking signals that modulate its surface expression on infected cells, and suggest an important role for this function in pathogenesis.

The ESCRT machinery is not required for human cytomegalovirus envelopment

The human cytomegalovirus (HCMV) has been proposed to complete its final envelopment on cytoplasmic membranes prior to its release to the extracellular medium. The nature of these membranes and the mechanisms involved in virus envelopment and release are poorly understood. Here we show by immunogold‐labelling and electron microscopy that CD63, a marker of multivesicular bodies (MVBs), is incorporated into the viral envelope, supporting the notion that HCMV uses endocytic membranes for its envelopment. We therefore investigated a possible role for the cellular endosomal sorting complex required for transport (ESCRT) machinery in HCMV envelopment. Depletion of tumour suppressor gene 101 and ALIX/AIP1 with small interfering RNAs (siRNAs) in HCMV‐infected cells did not affect virus production. In contrast, siRNAs against the vacuolar protein sorting 4 (VPS4) proteins silenced the expression of VPS4A and VPS4B, inhibited the sorting of epidermal growth factor to lysosomes, the formation of HIV Gag‐derived virus‐like particles and vesicular stomatitis virus infection, but enhanced the number of HCMV viral particles produced. Treatment of infected cells with protease inhibitors also increased viral production. These studies indicate that, in contrast to some enveloped RNA viruses, HCMV does not require the cellular ESCRT machinery to complete its envelopment.

Lack of p56 lck expression correlates with CD4 endocytosis in primary lymphoid and myeloid cells

In cell lines the endocytic properties of CD4 are regulated through its association with the src ‐family tyrosine kinase p56  lck . In lymphoid cell lines expressing p56  lck , CD4 is restricted to the cell surface and undergoes only limited internalization. Phosphorylation of the cytoplasmic domain of CD4 causes p56  lck to dissociate and activates an endocytosis signal leading to the internalization of CD4 through clathrin‐coated pits. In p56  lck ‐negative transfected cell lines CD4 is constitutively internalized, but internalization is inhibited when p56  lck is expressed in these cells. We now demonstrate that these endocytic properties of CD4 determined in transfected cell lines hold true for CD4 naturally expressed on myeloid cell lines (HL‐60 and U937), as well as on primary lymphocytes, monocytes and macrophages isolated from human blood. CD4 showed limited internalization on p56  lck ‐positive lymphocytes, but was rapidly internalized in p56  lck ‐negative monocytes and macrophages. Surprisingly, rapid internalization of CD4 was seen with the lymphocytes from one unidentified donor. In these cells we failed to detect p56  lck expression by Western blotting.