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Biomedical subjects

R W Doms

Publications and source records attributed to R W Doms.

At least 73 records · Page 4Linked to original sources
PubMed1999

Functional dissection of CCR5 coreceptor function through the use of CD4-independent simian immunodeficiency virus strains.

With rare exceptions, all simian immunodeficiency virus (SIV) strains can use CCR5 as a coreceptor along with CD4 for viral infection. In addition, many SIV strains are capable of using CCR5 as a primary receptor to infect CD4-negative cells such as rhesus brain capillary endothelial cells. By using coupled fluorescence-activated cell sorter (FACS) and infection assays, we found that even very low levels of CCR5 expression could support CD4-independent virus infection. CD4-independent viruses represent valuable tools for finely dissecting interactions between Env and CCR5 which may otherwise be masked due to the stabilization of these contacts by Env-CD4 binding. Based on the ability of SIV Env to bind to and mediate infection of cells expressing CCR5 chimeras and mutants, we identified the N terminus of CCR5 as a critical domain for direct Env binding and for supporting CD4-independent virus infection. However, the activity of N-terminal domain CCR5 mutants could be rescued by the presence of CD4, indicating that other regions of CCR5 are important for post-binding events that lead to viral entry. Rhesus CCR5 supported CD4-independent infection and direct Env binding more efficiently than did human CCR5 due to a single amino acid difference in the N terminus. Interestingly, uncleaved, oligomeric SIV Env protein bound to both CD4 and CCR5 less efficiently than did monomeric gp120. Finally, several mutations present in chronically infected monkey populations are shown to decrease the ability of CCR5 to serve as a primary viral receptor for the SIV isolates examined.

Amino Acid Sequence↗
PubMed1998

Genetic acceleration of AIDS progression by a promoter variant of CCR5.

The CCR5 gene encodes a cell surface chemokine receptor molecule that serves as the principal coreceptor, with CD4, for macrophage-tropic (R5) strains of human immunodeficiency virus-type 1 (HIV-1). Genetic association analysis of five cohorts of people with acquired immunodeficiency syndrome (AIDS) revealed that infected individuals homozygous for a multisite haplotype of the CCR5 regulatory region containing the promoter allele, CCR5P1, progress to AIDS more rapidly than those with other CCR5 promoter genotypes, particularly in the early years after infection. Composite genetic epidemiologic analyses of genotypes bearing CCR5P1, CCR5-Delta32, CCR2-64I, and SDF1-3'A affirmed distinct regulatory influences for each gene on AIDS progression. An estimated 10 to 17 percent of patients who develop AIDS within 3.5 years of HIV-1 infection do so because they are homozygous for CCR5P1/P1, and 7 to 13 percent of all people carry this susceptible genotype. The cumulative and interactive influence of these AIDS restriction genes illustrates the multigenic nature of host factors limiting AIDS disease progression.

Acquired Immunodeficiency Syndrome↗
PubMed1998

Use of GPR1, GPR15, and STRL33 as coreceptors by diverse human immunodeficiency virus type 1 and simian immunodeficiency virus envelope proteins.

Human and simian immunodeficiency viruses (HIV and SIV, respectively) use chemokine receptors as coreceptors along with CD4 to mediate viral entry. Several orphan receptors, including GPR1, GPR15, and STRL33, can also serve as coreceptors for a more limited number of HIV and SIV isolates. We investigated whether these orphan receptors could function as efficient coreceptors for a diverse group of HIV and SIV envelopes (Envs) in comparison with the principal coreceptors CCR5 and CXCR4. We found that a limited number of HIV-1 isolates could mediate inefficient cell-cell fusion with the orphan receptors relative to CCR5 and CXCR4; however, none of the orphan receptors tested could support pseudotype virus infection despite robust infection via CCR5 or CXCR4. All except one of the SIV Envs tested mediated some degree of cell-cell fusion and pseudotype infection, with target cells expressing at least one of these orphan receptors, although CCR5 proved to be the most efficient coreceptor for infection. Only one SIV Env protein, BK28, could mediate infection using GPR1 as a coreceptor, albeit much less efficiently than with CCR5. In addition, use of these coreceptors did not correlate with the published tropism of the SIV clones and was strictly CD4 dependent for both SIV and HIV. We also examined the expression of these molecules in cell lines and primary cells widely used for virus propagation and as targets for infection. All cells examined expressed STRL33, a more limited number expressed GPR15, and GPR1 was much more restricted in its expression pattern. Taken together, our results indicate that GPR15 and STRL33 are rarely used by HIV-1 but are more frequently used by SIV strains, although not in a manner that correlates with SIV tropism.

Animals↗
PubMed1998

HIV type I envelope determinants for use of the CCR2b, CCR3, STRL33, and APJ coreceptors.

The envelope (Env) proteins of primate lentiviruses interact sequentially with CD4 and a coreceptor to infect cells. Changes in coreceptor use strongly influence viral tropism and pathogenesis. We followed the evolution of coreceptor use in pig-tailed macaques that developed severe CD4 T-cell loss during the derivation of a pathogenic simian HIV (SHIV) that contained the tat, rev, vpu, and env genes of the HXBc2 strain of HIV-1 in a genetic background of SIVmac239. The Env from the parental virus as well as one derived from the first macaque to develop AIDS exclusively used CXCR4 as a coreceptor, indicating that CXCR4 can function as a coreceptor in macaques even though it is rarely used by simian immunodeficiency viruses. One Env (Pnb5), obtained from a macrophage-tropic virus isolated from the cerebral spinal fluid, did not use CCR5 or CXCR4. Instead, it used CCR2b and to a lesser extent CCR3, STRL33, and APJ to infect cells. Chimeras between Pnb5 and the parental X4 Env indicated that the V3 loop is the major determinant of CXCR4 use, with other regions of Env influencing the efficiency with which this coreceptor was used. In contrast, the Pnb5 V1/2 and V3 regions in combination were both necessary and sufficient to confer full use of CCR2b, CCR3, STRL33, and APJ to the parental X4 Env protein. These results are consistent with a single, conserved binding site in Env that interacts with multiple coreceptors in conjunction with the V1/2 and V3 loops, and suggest that the V1/2 region plays a more important role in governing the use of CCR2b, CCR3, STRL33, and APJ than for CXCR4.

Animals↗
PubMed1998

Detection of a novel intraneuronal pool of insoluble amyloid beta protein that accumulates with time in culture.

The amyloid-beta peptide (Abeta) is produced at several sites within cultured human NT2N neurons with Abeta1-42 specifically generated in the endoplasmic reticulum/intermediate compartment. Since Abeta is found as insoluble deposits in senile plaques of the AD brain, and the Abeta peptide can polymerize into insoluble fibrils in vitro, we examined the possibility that Abeta1-40, and particularly the more highly amyloidogenic Abeta1-42, accumulate in an insoluble pool within NT2N neurons. Remarkably, we found that formic acid extraction of the NT2N cells solubilized a pool of previously undetectable Abeta that accounted for over half of the total intracellular Abeta. Abeta1-42 was more abundant than Abeta1-40 in this pool, and most of the insoluble Abeta1-42 was generated in the endoplasmic reticulum/intermediate compartment pathway. High levels of insoluble Abeta were also detected in several nonneuronal cell lines engineered to overexpress the amyloid-beta precursor protein. This insoluble intracellular pool of Abeta was exceptionally stable, and accumulated in NT2N neurons in a time-dependent manner, increasing 12-fold over a 7-wk period in culture. These novel findings suggest that Abeta amyloidogenesis may be initiated within living neurons rather than in the extracellular space. Thus, the data presented here require a reexamination of the prevailing view about the pathogenesis of Abeta deposition in the AD brain.

Alzheimer Disease↗
PubMed1998

The CC chemokine I-309 inhibits CCR8-dependent infection by diverse HIV-1 strains.

Using a chemokine receptor model based on known receptor sequences, we identified several members of the seven transmembrane domain G-protein superfamily as potential chemokine receptors. The orphan receptor ChemR1, which has recently been shown to be a receptor for the CC chemokine I-309, scored very high in our model. We have confirmed that I-309, but not a number of other chemokines, can induce a transient Ca2+ flux in cells expressing CCR8. In addition, the human erythroleukemic cell line K562 responded chemotactically in a dose-responsive manner to this chemokine. Since several chemokine receptors have been shown to be required as coreceptors for HIV-1 infection, we asked whether human immunodeficiency virus type 1 (HIV-1) could efficiently utilize CCR8. Here we show that the CCR8 receptor can serve as a coreceptor for diverse T-cell tropic, dual-tropic, and macrophage-tropic HIV-1 strains and that I-309 was a potent inhibitor of HIV-1 envelope-mediated cell-cell fusion and virus infection. Furthermore, we show by flow cytometry and immunohistochemistry that antibodies generated against the CCR8 receptor amino-terminal peptide cross-reacted with U-87 MG cells stably expressing CCR8, THP-1 cells, HL-60 cells, and human monocytes, a target cell for HIV-1 infectivity in vivo.

Amino Acid Sequence↗
PubMed1998

ChemR23, a putative chemoattractant receptor, is expressed in monocyte-derived dendritic cells and macrophages and is a coreceptor for SIV and some primary HIV-1 strains.

Leukocyte chemoattractants act through a rapidly growing subfamily of G protein-coupled receptors. We report the cloning of a novel human gene encoding an orphan receptor (ChemR23) related to the C3a, C5a and formyl Met-Leu-Phe receptors, and more distantly to the subfamilies of chemokine receptors. ChemR23 transcripts were found to be abundant in monocyte-derived dendritic cells and macrophages, treated or not with LPS. Low expression could also be detected by reverse transcription-PCR in CD4+ T lymphocytes. The gene encoding ChemR23 was assigned by radiation hybrid mapping to the q21.2-21.3 region of human chromosome 12, outside the gene clusters identified so far for chemoattractant receptors. Given the increasing number of chemoattractant receptors used by HIV-1, HIV-2 and SIV as coreceptors, ChemR23 was tested in fusion assays for potential coreceptor activity by a range of viral strains. None of the tested HIV-2 strains made use of ChemR23 as a coreceptor, but several SIV strains (SIVmac316, SIVmac239, SIVmacl7E-Fr and SIVsm62A), as well as a primary HIV-1 strain (92UG024-2) used it efficiently. ChemR23 therefore appears as a coreceptor for immunodeficiency viruses that does not belong to the chemokine receptor family. It is also a putative chemoattractant receptor relatively specific for antigen-presenting cells, and it could play an important role in the recruitment or trafficking of these cell populations. Future work will be required to identify the ligand(s) of this new G protein-coupled receptor and to define its precise role in the physiology of dendritic cells and macrophages.

Amino Acid Sequence↗
PubMed1998

An intricate Web: chemokine receptors, HIV-1 and hematopoiesis.

Cellular infection by the human immunodeficiency virus type 1 (HIV-1) requires interaction of the viral envelope protein with CD4 and at least one additional cell surface molecule, termed a "cofactor" or "coreceptor." Recent discoveries have determined that macrophage-tropic strains of HIV-1 which are largely responsible for sexual transmission require the beta-chemokine receptor CCR5 in addition to CD4, while the T cell tropic viruses that emerge later after infection use the alpha-chemokine receptor CXCR4. Thus, both CD4 and the appropriate chemokine receptor must be expressed on the cell surface in order for HIV-1 to enter the cell and establish an infection. The in vivo importance of CCR5 for HIV-1 is demonstrated by the finding that individuals homozygous for a 32 bp deletion (delta 32) in the CCR5 gene that renders them effectively CCR5-negative are highly resistant to virus infection. In this review, the structure-function correlates of the chemokine receptors that serve as major coreceptors for HIV-1 and simian immunodeficiency virus entry will be reviewed. Since certain chemokines have been implicated as stem cell inhibitory factors, the biological consequences of chemokine receptor expression as it relates to HIV-1-associated hematodyspoiesis will also be discussed.

Animals↗
PubMed1998

Structure-function studies of the HIV-1 coreceptors.

Ten seven-transmembrane-domain G protein-coupled receptors have been identified that are functional HIV-1, HIV-2 and SIV coreceptors. However, the specific structures these receptors have in common that enable them to mediate HIV entry are unknown. Structure-function analyses have revealed that the determinants of coreceptor activity are distinct for each coreceptor, coreceptor activity is dependent on multiple extracellular domains, and various envelope proteins may interact differently with the same coreceptor. G protein coupling and receptor internalization are not required for fusion and infection of established cell lines, or for inhibition of infection by chemokines. The structure-function studies have also helped determine the mechanism by which previously described small molecules inhibit HIV-1 entry. Finally, these studies have led to a hypothesis as to how coreceptor utilization evolves during the course of an infection.

Amino Acid Sequence↗
PubMed1998

CD4-independent utilization of the CXCR4 chemokine receptor by HIV-1 and HIV-2.

HIV entry is mediated by an interaction between CD4 and members of the chemokine receptor family of proteins. It is likely that CD4 induces conformational changes in the viral envelope glycoproteins that facilitate a subsequent interaction with the chemokine receptor. To understand these events, variants of HIV-2 and HIV-1 have been derived that are able to interact directly with CXCR4 in the absence of CD4. One HIV-2 variant. termed HIV-2/vcp, has an expanded host range that includes CXCR4+/CD4- lymphoid and nonlymphoid cell lines. In contrast to T-tropic isolates of HIV-1, HIV-2/vcp was shown to induce > 95% downregulation of CXCR4 on chronically infected cells and was able to superinfect HIV-1-infected cells. A variant of HIV-1/IIIB termed HIV-1/IIIBx was also derived that is both replication competent and fusogenic for a CD4-negative subclone of SupT1 cells, termed BC7. Infection of BC7 cells by HIV-1/IIIBx was resistant to anti-CD4 monoclonal antibodies but inhibited by the anti-CXCR4 mAb, 12G5. HIV-1/IIIBx was highly fusogenic on 3T3 cells expressing CXCR4 in the absence of CD4. In contrast to HIV-2/vcp, the host range of HIV-1/IIIBx was highly restricted and replication in several CD4+/CXCR4+ lymphoid cell lines was reduced compared to HIV-1/IIIB. In addition, HIV-1/IIIBx failed to downregulate CXCR4 on chronically infected cells. These studies indicate that HIV-1 and HIV-2 variants can be derived in vitro that utilize CXCR4 in the absence of CD4. Although the mechanism(s) for these changes remain unclear, possibilities include an increased avidity of the viral envelope glycoprotein for CXCR4 and/or the increased exposure of the chemokine receptor binding site. Further biochemical and molecular analysis of the envelope glycoproteins from these viruses should be helpful in addressing these and other possibilities.

Animals↗
PubMed1998

An orphan seven-transmembrane domain receptor expressed widely in the brain functions as a coreceptor for human immunodeficiency virus type 1 and simian immunodeficiency virus.

Both CD4 and an appropriate coreceptor are necessary for infection of cells by human immunodeficiency virus type 1 (HIV-1) and most strains of HIV-2. The chemokine receptors CCR5 and CXCR4 are the major HIV-1 coreceptors, although some virus strains can also utilize alternative coreceptors such as CCR3 to infect cells. In contrast, most if not all simian immunodeficiency virus (SIV) strains use CCR5 as a coreceptor, and many SIV strains can use CCR5 independently of CD4. In addition, several orphan seven-transmembrane receptors which can serve as HIV-1 and SIV coreceptors have been identified. Here we report that APJ, an orphan seven-transmembrane domain receptor with homology to the angiotensin receptor family, functions as a coreceptor for a number of HIV-1 and SIV strains. APJ was expressed widely in the human brain and in NT2N neurons. APJ transcripts were also detected by reverse transcription-PCR in the CD4-positive T-cell line C8166, but not in peripheral blood leukocytes, microglia, phytohemagglutinin (PHA)- or PHA/interleukin-2-stimulated peripheral blood mononuclear cells, monocytes, or monocyte-derived macrophages. The widespread distribution of APJ in the central nervous system coupled with its use as a coreceptor by some HIV-1 strains indicates that it may play a role in neuropathogenesis.

Animals↗
PubMed1998

Identification of determinants on a dualtropic human immunodeficiency virus type 1 envelope glycoprotein that confer usage of CXCR4.

The chemokine receptors CCR5 and CXCR4, in combination with CD4, mediate cellular entry of macrophage-tropic (M-tropic) and T-cell-tropic strains of human immunodeficiency virus type 1 (HIV-1), respectively, while dualtropic viruses can use either receptor. We have constructed a panel of chimeric viruses and envelope glycoproteins in which various domains of the dualtropic HIV-1(DH12) gp160 were introduced into the genetic background of an M-tropic HIV-1 isolate, HIV-1(AD8). These constructs were employed in cell fusion and virus infectivity assays using peripheral blood mononuclear cells, MT4 T cells, primary monocyte-derived macrophages, or HOS-CD4 cell lines, expressing various chemokine receptors, to assess the contributions of different gp120 subdomains in coreceptor usage and cellular tropism. As expected, the dualtropic HIV-1(DH12) gp120 utilized either CCR3, CCR5, or CXCR4, whereas HIV-1(AD8) gp120 was able to use only CCR3 or CCR5. We found that either the V1/V2 or the V3 region of HIV-1(DH12) gp120 individually conferred on HIV-1(AD8) the ability to use CXCR4, while the combination of both the V1/V2 and V3 regions increased the efficiency of CXCR4 use. In addition, while the V4 or the V5 region of HIV-1(DH12) gp120 failed to confer the capacity to utilize CXCR4 on HIV-1(AD8), these regions were required in conjunction with regions V1 to V3 of HIV-1(DH12) gp120 for efficient utilization of CXCR4. Comparison of virus infectivity analyses with various cell types and cell fusion assays revealed assay-dependent discrepancies and indicated that events occurring at the cell surface during infection are complex and cannot always be predicted by any one assay.

Animals↗
PubMed1998

Chemokine receptor utilization by human immunodeficiency virus type 1 isolates that replicate in microglia.

The role of human immunodeficiency virus (HIV) strain variability remains a key unanswered question in HIV dementia, a condition affecting around 20% of infected individuals. Several groups have shown that viruses within the central nervous system (CNS) of infected patients constitute an independently evolving subset of HIV strains. A potential explanation for the replication and sequestration of viruses within the CNS is the preferential use of certain chemokine receptors present in microglia. To determine the role of specific chemokine coreceptors in infection of adult microglial cells, we obtained a small panel of HIV type 1 brain isolates, as well as other HIV strains that replicate well in cultured microglial cells. These viruses and molecular clones of their envelopes were used in infections, in cell-to-cell fusion assays, and in the construction of pseudotypes. The results demonstrate the predominant use of CCR5, at least among the major coreceptors, with minor use of CCR3 and CXCR4 by some of the isolates or their envelope clones.

Adult↗
PubMed1998

Influence of the CCR2-V64I polymorphism on human immunodeficiency virus type 1 coreceptor activity and on chemokine receptor function of CCR2b, CCR3, CCR5, and CXCR4.

The chemokine receptors CCR5 and CXCR4 are used by human immunodeficiency virus type 1 (HIV-1) in conjunction with CD4 to infect cells. In addition, some virus strains can use alternative chemokine receptors, including CCR2b and CCR3, for infection. A polymorphism in CCR2 (CCR2-V64I) is associated with a 2- to 4-year delay in the progression to AIDS. To investigate the mechanism of this protective effect, we studied the expression of CCR2b and CCR2b-V64I, their chemokine and HIV-1 coreceptor activities, and their effects on the expression and receptor activities of the major HIV-1 coreceptors. CCR2b and CCR2b-V64I were expressed at similar levels, and neither molecule affected the expression or coreceptor activity of CCR3, CCR5, or CXCR4 in cotransfected cell lines. Peripheral blood mononuclear cells (PBMCs) from CCR2-V64I heterozygotes had normal levels of CCR2b and CCR5 but slightly reduced levels of CXCR4. CCR2b and CCR2b-V64I functioned equally well as HIV-1 coreceptors, and CCR2-V64I PBMCs were permissive for HIV-1 infection regardless of viral tropism. The MCP-1-induced calcium mobilization mediated by CCR2b signaling was unaffected by the polymorphism, but MCP-1 signaling mediated by either CCR2b- or CCR2-V64I-encoded receptors resulted in heterologous desensitization (i.e., limiting the signal response of other receptors) of both CCR5 and CXCR4. The heterologous desensitization of CCR5 and CXCR4 signaling by both CCR2 allele receptor types provides a mechanistic link that might help explain the in vivo effects of CCR2 gene variants on progression to AIDS as well as the reported antiviral activity of natural CCR2 ligands.

Cell Line, Transformed↗
PubMed1997

CD4-independent, CCR5-dependent infection of brain capillary endothelial cells by a neurovirulent simian immunodeficiency virus strain.

Brain capillary endothelial cells (BCECs) are targets of CD4-independent infection by HIV-1 and simian immunodeficiency virus (SIV) strains in vitro and in vivo. Infection of BCECs may provide a portal of entry for the virus into the central nervous system and could disrupt blood-brain barrier function, contributing to the development of AIDS dementia. We found that rhesus macaque BCECs express chemokine receptors involved in HIV and SIV entry including CCR5, CCR3, CXCR4, and STRL33, but not CCR2b, GPR1, or GPR15. Infection of BCECs by the neurovirulent strain SIV/17E-Fr was completely inhibited by aminooxypentane regulation upon activation, normal T cell expression and secretion in the presence or absence of ligands, but not by eotaxin or antibodies to CD4. We found that the envelope (env) proteins from SIV/17E-Fr and several additional SIV strains mediated cell-cell fusion and virus infection with CD4-negative, CCR5-positive cells. In contrast, fusion with cells expressing the coreceptors STRL33, GPR1, and GPR15 was CD4-dependent. These results show that CCR5 can serve as a primary receptor for SIV in BCECs and suggest a possible CD4-independent mechanism for blood-brain barrier disruption and viral entry into the central nervous system.

Animals↗