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Query: UMLS:C0019693 (HIV)
 170,526 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human CXCR4 is the receptor for the CXC chemokine SDF-1alpha and also acts as a coreceptor for T lymphotropic HIV-1 strains. Blocking the surface expression of this receptor via an intrakine approach has recently been shown to efficiently prevent HIV-1 infection of T cells. The CXC-chemokine gene is fused to an endoplasmic reticulum retention signal (KDEL) that retains the newly synthesized chemokine and its receptor within the cell, where both are subsequently degraded. We constructed MoMuLV-based vectors containing the SDF-KDEL construct driven by the "MND" long terminal repeat, using eGFP as a marker gene (MND-SDF-KDEL-IRES-eGFP) and a control vector (MND-X-IRES-eGFP). CEM human T lymphoblastic leukemia cells were transduced with the intrakine vector or the control vector. We detected a marked downregulation of CXCR4 expression in the cells transduced with the intrakine vectors as opposed to the cells transduced with the control vector. However, the eGFP-negative fraction of the cells transduced with the intrakine vector displayed the same CXCR4 downregulation as the eGFP-positive fraction, suggesting an effect in trans. The possibility of this being due to eGFP being silenced while SDF-KDEL was still expressed was excluded by Southern and Northern blot analyses. Upon cultivating the control cells with supernatant of the cells transduced with the intrakine vector, we observed a downregulation of CXCR4 expression on the control cells. Experiments using rhSDF-1alpha showed downregulation by the supernatant to be comparable to that achieved by the exogenous addition of 30 ng/ml SDF-1alpha. To assess the bioactivity of the secreted substance in the supernatant, a chemotaxis assay was performed. The transmigration observed was, once again, within the range of that achieved by the addition of 30 ng/ml SDF-1alpha. We conclude that the intrakine SDF-KDEL, apart from acting within the cell, is also in part secreted and causes the downregulation of the receptor by acting like a secreted chemokine.
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PMID:Intrakines--evidence for a trans-cellular mechanism of action. 1093 27

HIV (human immunodeficiency virus)-1 Env is displayed on the surface of infected cells and subsequently incorporated into virions, which is necessary for the initiation of a viral infection by recognition of the CD4 and the chemokine receptors (such as CCR5 or CXCR4) on the surface of new target cells. As a type 1 integral membrane glycoprotein, Env is cotranslationally translocated into the endoplasmic reticulum. In this report, we characterized the synthesis of Env, which did not occur at a constant rate but by translational/translocational pausing that has not previously been shown with a viral encoded glycoprotein. Overall translation was not impeded by the presence of the reducing agent dithiothreitol in vivo, although this did influence the cleavage of the precursor gp160 into its mature form, gp120. Env interacts transiently with resident components of the endoplasmic reticulum such as calnexin, which had maximal association at a 10-min post-translation. Addition of the glucosidase inhibitor, castanospermine, failed to significantly influence the association of Env with calnexin, consistent with the notion that calnexin recognizes components other than alpha-terminal glucose. Moreover, castanospermine treatment failed to affect the infectivity of virions. Taken together, this report demonstrates the existence of translational/translocational pausing for a viral glycoprotein and suggests that trimming of glucose from HIV-1 Env is not essential for the initiation of virus infection.
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PMID:Characterization of the biosynthesis of human immunodeficiency virus type 1 Env from infected T-cells and the effects of glucose trimming of Env on virion infectivity. 1105 27

Cytosolic degradation of endogenously synthesized proteins by the proteasome and translocation of processed peptides to the endoplasmic reticulum by the transporters associated with antigen presentation constitutes the classical route for antigen presentation by MHC class I proteins. We have previously defined an alternative pathway in the secretory route involving proteolytic maturation of precursor proproteins for chimeric hepatitis B virus secretory core protein HBe containing a class I epitope at its carboxy-terminus. We extend those results by demonstrating that intracellular delivery of the trans-Golgi network protease furin increases both proteolytic maturation and antigen presentation of the chimeric HBe proteins. An additional class I epitope from the HIV envelope gp160 protein was inserted into this COOH-terminal region of two different chimeric HBe proteins. This epitope was also presented to CTL in a transporter-independent manner involving furin, and protein maturation and antigen presentation were also enhanced by furin over-expression. Presentation of this second epitope was restricted by a different class I allele, thus suggesting that antigen presentation by this new pathway may apply to any antigenic epitope and class I molecule. These results define the furin proteolytic maturation pathway of HBe in the secretory route as a general antigen processing route for MHC class I presentation.
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PMID:Generation of MHC class I peptide antigens by protein processing in the secretory route by furin. 1120 52

Studies were carried out to analyse the ultrastructural changes and the distribution of hepatitis A virus (HAV)/antigens at subcellular level in buffalo green monkey kidney (BGMK) cells persistently infected with HM-175 strain of HAV. HAV infected BGMK cells showed distinct abnormalities in the endoplasmic reticulum and cytoplasmic membrane as compared to uninfected cells. The abnormalities were characterized by wavy arrays, structures like myelin, annulate lamellae, cytoplasmic inclusion bodies and vesicles. The wavy arrays within the cytoplasm of the host cells appeared to represent degenerating membranes. A complex myelin like body was found in close association with a group of virus like particles. Annulate lamellae like structures involving single paired membrane were detected infrequently whereas the cytoplasmic vesicles were numerous in these cells. An indirect immunogold technique was utilized to localize the HAV antigenin infected cells. A high density immunogold label for HIV like particles was predominantly detected in cytoplasmic vesicles. These results suggest a strong association of membrane substructure in vesicle forms with the compartmentalized replication of HAV within persistently infected host cells.
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PMID:Electron microscopy of buffalo green monkey kidney cells persistently infected with hepatitis A virus and immunolocalization of HAV antigens. 1134 3

CD8+ cytolytic T lymphocytes (CTL) are almost certainly an important component of a potentially protective immune response to HIV. To test the ability of pertussis toxin (PT) to deliver an HIV-derived major histocompatibility complex (MHC) class I peptide for CTL stimulation, we constructed a fusion of the gp120 P18-I10 CTL epitope with a genetically detoxified derivative of PT (PT9K/129G) and assayed this fusion for its ability to stimulate a gp120-specific CTL response in vitro and in vivo. Antigen-presenting cells incubated with this fusion protein were lysed by P18-I10-specific CTL in vitro and this activity was shown to be MHC class I restricted. The activity was inhibited by brefeldin A but was not inhibited by proteasome inhibitors, possibly because PT undergoes retrograde intracellular transport through the Golgi apparatus to the endoplasmic reticulum and delivers epitopes directly to nascent class I molecules. Mice immunized intraperitoneally with a single dose of the fusion protein without adjuvant raised a strong gp120-specific CTL response in the spleen. This CTL response was dependent on (1) the dose of fusion administered, (2) the fusion of the epitope with the toxin (since coadministration of peptide and toxin gave no response), and (3) the activity of CD8+ cells. These data demonstrate that this detoxified derivative to PT, which is already a component of a licensed vaccine for humans, could represent a useful vaccine vector molecule for stimulation of HIV-specific CTL responses.
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PMID:Stimulation of HIV gp120-specific cytolytic T lymphocyte responses in vitro and in vivo using a detoxified pertussis toxin vector. 1142 23

The dissemination of T cell hybridomas to multiple nonhematopoietic tissues is blocked by pertussis toxin, suggesting the involvement of a chemokine. To study whether this chemokine is SDF-1, we employed a strategy proposed previously for gene therapy of AIDS, whereby the SDF-1 receptor CXCR4 (also a coreceptor for HIV) is retained in the endoplasmic reticulum (ER) and fails to reach the cell surface. We transfected SDF-1, carrying an ER retention sequence, into a T cell hybridoma. This altered chemokine is retained in the ER, where it binds CXCR4 and prevents the latter protein from reaching the surface. These cells failed to migrate toward SDF-1 or to invade fibroblast monolayers, although they could still migrate toward thymus and activation-regulated chemokine (TARC) and invade TARC-treated monolayers. Furthermore, the ability of the transfected cells to disseminate to multiple organs upon intravenous injection into mice was abolished. This dissemination reflects the in vivo migration patterns of activated and memory T cells into nonhematopoietic tissues, which is thus likely to depend on CXCR4. Attempts to block CXCR4 function as a therapy for AIDS may affect this migration with consequences for T cell function. Our results also suggest a decisive role for CXCR4 in the dissemination of hematopoietic malignancies expressing this receptor.
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PMID:Retention of CXCR4 in the endoplasmic reticulum blocks dissemination of a T cell hybridoma. 1145 80

The membrane-spanning domain (MSD) of a number of retroviral transmembrane (TM) glycoproteins, including those from the human and simian immunodeficiency viruses (HIV and SIV), have been predicted to contain a charged arginine residue. The wild-type SIV TM glycoprotein is 354 amino acids long. The entire putative cytoplasmic domain of SIV (amino acids 193 to 354) is dispensable for virus replication in vitro, and such truncation-containing viruses are capable of reaching wild-type titers after a short delay. We show here that further truncation of eight additional amino acids to TM185 results in a protein that lacks fusogenicity but is, nevertheless, efficiently incorporated into budding virions. By analyzing a series of nonsense mutations between amino acids 193 and 185 in Env expression vectors and in the SIVmac239 proviral clone, a region of the SIV TM that contains the minimum requirement for glycoprotein-mediated cell-to-cell fusion and that for virus replication was identified. Virus entry and infectivity were evident in truncations to a minimum of 189 amino acids, whereas cell-cell fusion was observed for a protein of only 187 amino acids. Glycoprotein was efficiently incorporated into budding virions in truncations up to 185 amino acids, indicating that such proteins are membrane anchored and are transported to the cell surface. However, truncation of the TM to 180 amino acids resulted in a protein that displays a transport defect and may be retained in the endoplasmic reticulum. Based on our analyses of these mutants, an alternative model for the MSD of SIV is proposed. Our model suggests that membrane-imbedded charged residues can be neutralized by side-chain interactions with lipid polar head groups. As a consequence, the membrane-spanning region can be reduced by more than a helical turn. This new model accounts for the ability of truncations within the predicted MSD to remain membrane anchored and maintain biological activity.
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PMID:Mutations within the putative membrane-spanning domain of the simian immunodeficiency virus transmembrane glycoprotein define the minimal requirements for fusion, incorporation, and infectivity. 1155 92

The human immunodeficiency virus, type 1 (HIV-1) entry process is triggered by interaction between the viral envelope and a seven membrane-spanning domain receptor at the cell surface, usually the CCR5 chemokine receptor. Different naturally occurring mutations in the CCR5 gene abolish receptor function, the most frequent being a 32-nucleotide deletion resulting in a truncated protein (Delta32) lacking the last three transmembrane domains (TM5-7). This mutant is retained in the endoplasmic reticulum and exerts a trans-dominant negative (TDN) effect on the wild type, preventing its exit from this compartment. This TDN effect is often considered as evidence for the oligomerization of CCR5 during transport to the cell surface. Here we use a genetic approach to define the structural determinants of the TDN effect of the Delta32 mutant. It was abolished by certain deletions and by mutations of cysteine residues preventing formation of a disulfide link between the first and second extracellular loops, suggesting that conformation of Delta32 is important for its interaction with CCR5. To circumvent this problem, we used chimeric forms of the Delta32 and wild type CCR5, consisting in substitutions with homologous domains from the mouse CCR5. All chimeric full-length receptors were expressed at the cell surface and were functional for interaction with HIV-1 or with a chemokine ligand, when assayed. The TDN effect was only observed if both the TM3 domain in CCR5 and the TM4 domain in Delta32 were from human origin, whereas the rest of the proteins could be from either origin. This suggests that the TDN effect involves some form of interaction between these transmembrane domains. Alternatively, but less likely to us, substitutions in TM4 could affect the conformation of CCR5 in the endoplasmic reticulum but not at the cell surface. However that may be, it seems that the TDN effect of the Delta32 mutant has no bearing to the issue of CCR5 dimerization and to its possible role in the processing of the receptor to the cell surface.
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PMID:Determinants of the trans-dominant negative effect of truncated forms of the CCR5 chemokine receptor. 1160 Apr 94

The envelope proteins (env) of simian immunodeficiency virus (SIV) and HIV type 1 assemble to form noncovalently associated oligomers in the endoplasmic reticulum. After cleavage in a Golgi compartment, oligomeric env complexes are transported to the surface of infected cells, where incorporation into budding virions can occur. Difficulties in obtaining adequate quantities of virions retaining env, as well as the unstable nature and hydrophobicity of the oligomer, may account for the absence of previous biophysical studies to determine the oligomeric valency of membrane-associated env. The aim of this study was to evaluate the oligomeric state of SIV env before membrane-fusion activation. Virion-associated env, obtained by crosslinking and detergent extraction, and non-crosslinked secreted env ectodomain (recombinant gp140) were purified by lentil-lectin chromatography and gel filtration as single predominant species. Sedimentation equilibrium-derived mass values for both forms of SIV env were close to those predicted for trimeric assemblies. Determination of the mass of individual molecules by scanning transmission electron microscopy confirmed that SIV virion-associated env and gp140 formed largely homogeneous populations of trimers. Furthermore, a triangular or tri-lobed morphology was clearly visualized in a subset of the trimers.
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PMID:Oligomeric structure of virion-associated and soluble forms of the simian immunodeficiency virus envelope protein in the prefusion activated conformation. 1175 36

The Pr55gag gene product of human immunodeficiency virus type 1 (HIV-1) is sufficient to direct the formation of retrovirus-like particles (RVLPs). Recent biochemical evidence has indicated the presence of Gag intermediates in the cytoplasm; however, the Gag assembly process into RVLPs remains incompletely defined. The authors present here the subcellular localization of Gag mutant proteins in BSC40 and Jurkat cells by immunoelectron microscopy (IEM). The full Gag/Pol and Gag precursors, a C-terminal deletion mutant lacking a portion of nucleocapsid (NC), and all p6Gag gave rise to similar levels of RVLPs at the cell surface. A C-terminal deletion of all NC and p6Gag abrogated particle formation, whereas p24 was found in patches at the cell surface. Deletion of matrix (MA) sequences from Gag resulted in intracellular particles, and myristylation was not required for particle formation in the context of the MA deletion. Matrix expression was enhanced with Gag/Pol or Env coexpression as determined by semiquantitative IEM. p24 protein was targeted at vacuolar and mitochondrial membranes, but not at Golgi cisternae. In addition, aggregations of Gag intermediates and RVLPs in the cytoplasm, rough endoplasmic reticulum, cisternae, and mitochondria were noted. These results provide defined in situ evidence that HIV-1 particle assembly occurs in the cytosol in addition to budding at most intracellular membranes.
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PMID:Tracking the assembly pathway of human immunodeficiency virus type 1 Gag deletion mutants by immunogold labeling. 1175 66


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