Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

CCR5 and CXCR4 are the two major coreceptors that have been identified for human immunodeficiency virus (HIV) entry. We have modified several beta-galactosidase-based HIV indicator cell lines to express CCR5 and/or CXCR4. Using these new reagents, we have been able to detect all primary isolates tested using one or both of these cell lines. However, there is large variation in the absolute viral infectivity among primary strains. Furthermore, all HIV strains are capable of causing syncytia in the indicator cells when the coreceptor is present regardless of whether they had previously been characterized as "syncytia-inducing" or "non-syncytium-inducing."
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PMID:Indicator cell lines for detection of primary strains of human and simian immunodeficiency viruses. 920 Dec 29

HIV-2 GH-1 is a molecular clone derived from an AIDS patient from Ghana. In contrast to the prototypic molecular clone ROD, GH-1 exhibits a narrow range of target cell specificity. By an infectious assay using HeLa-CD4 cells stably transfected with an HIV-1 LTR-beta-galactosidase reporter gene and transiently expressing various cloned chemokine receptors, we have examined the coreceptor usage of GH-1. In contrast to ROD, which uses principally CXCR4, GH-1 was found to use mainly if not exclusively CCR5 but not CXCR4. The distinct coreceptor usage of these two molecular clones allowed us to further map the region of gp120 that is important for the coreceptor specificity. By constructing a series of chimeric viruses between GH-1 and ROD, we have demonstrated that the C-terminal half of the V3 loop region of gp120 determines the differential coreceptor usage between GH-1 and ROD, and only a few amino acid differences in this region appear to be able to shift the specificity between CCR5 and CXCR4. Notably, the shift in the coreceptor usage from CCR5 to CXCR4 is associated with an increase in the net positive charge in the V3 region.
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PMID:Small amino acid changes in the V3 loop of human immunodeficiency virus type 2 determines the coreceptor usage for CXCR4 and CCR5. 1054 50

A highly desirable approach to prevention of human immunodeficiency virus type 1 (HIV-1) transmission during sexual intercourse is the development of nontoxic, topical, broad spectrum microbicides effective against transmission of cell-associated and cell-free virus. Toward this end, the HIV-1 inactivation potential of surface active agents C31G and an alkyl sulfate, sodium dodecyl sulfate (SDS) was assessed. Because of its extensive use as a microbicidal agent, nonoxynol-9 (N-9) was used as a reference against which C31G and SDS were compared. Viral inactivation was measured using HIV-1 LTR-beta-galactosidase indicator cells (expressing CD4 or CD4/CCR5) derived from HeLa cells, a cell line of human cervical adenocarcinoma origin. In experiments which examined inactivation of cell-free HIV-1, C31G was generally more effective than N-9. Viral inactivation by SDS occurred at twice the concentration necessary to achieve similar levels of inactivation using either N-9 or C31G. Using HeLa and HeLa-derived cells in cytotoxicity studies, it was demonstrated that SDS is as much as 11 and five times less cytotoxic than N-9 or C31G, respectively, during 48 h of continuous exposure. SDS (unlike C31G and N-9) can inactivate non-enveloped viruses such as human papillomavirus (HPV) [Howett, M.K., Neely, E.B., Christensen, N.D., Wigdahl, B., Krebs, F.C., Malamud, D., Patrick, S.D., Pickel, M.D., Welsh, P.A., Reed, C.A., Ward, M.G., Budgeon, L.R., Kreider, J.W., 1999. A broad-spectrum microbicide with virucidal activity against sexually transmitted viruses. Antimicrob. Agents Chemother. 43(2), 314-321]. Since addition of SDS to C31G or N-9 may make the resulting microbicidal mixtures broadly effective against both enveloped and non-enveloped viruses, several surface active agent combinations were evaluated for their abilities to inactivate HIV-1. Addition of SDS to either C31G or N-9 resulted in mixtures that were only slightly less effective than equivalent concentrations of C31G or N-9 alone. To investigate inactivation of cell-associated infectivity, HIV-1 IIIB-infected SupT1 cells were treated with N-9, C31G, or SDS. Inactivation of cell-associated infectivity required higher microbicide concentrations than were needed for inactivation of cell-free virus. However, the relative activities of N-9, C31G, or SDS were similar to those seen in assays of inactivation using cell-free virus. These studies suggest that C31G and SDS may be attractive candidates for human trials as topical microbicides effective against HIV-1 transmission since both function at concentrations that provide effective viral inactivation with low levels of cytotoxicity. SDS microbicides (used alone or with other microbicides) may provide the added advantage of protection from HPV infection.
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PMID:Inactivation of human immunodeficiency virus type 1 by nonoxynol-9, C31G, or an alkyl sulfate, sodium dodecyl sulfate. 1055 74

To determine whether C-C chemokines play an important role in the phenotype switch of human immunodeficiency virus (HIV) from CCR5 to CXCR4 usage during the course of an infection in vivo, macrophage inflammatory protein (MIP)-1alpha-resistant variants were isolated from CCR5-tropic (R5) HIV-1 in vitro. The selected variants displayed reduced sensitivities to MIP-1alpha (fourfold) through CCR5-expressing CD4-HeLa/long terminal repeat-beta-galactosidase (MAGI/CCR5) cells. The variants were also resistant to other natural ligands for CCR5, namely, MIP-1beta (>4-fold) and RANTES (regulated upon activation, normal T-cell expressed and secreted) (6-fold). The env sequence analyses revealed that the variants had amino acid substitutions in V2 (valine 166 to methionine) and V3 (serine 303 to glycine), although the same V3 substitution appeared in virus passaged without MIP-1alpha. A single-round replication assay using a luciferase reporter HIV-1 strain pseudotyped with mutant envelopes confirmed that mutations in both V2 and V3 were necessary to confer the reduced sensitivity to MIP-1alpha, MIP-1beta, and RANTES. However, the double mutant did not switch its chemokine receptor usage from CCR5 to CXCR4, indicating the altered recognition of CCR5 by this mutant. These results indicated that V2 combined with the V3 region of the CCR5-tropic HIV-1 envelope modulates the sensitivity of HIV-1 to C-C chemokines without altering the ability to use chemokine receptors.
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PMID:Involvement of both the V2 and V3 regions of the CCR5-tropic human immunodeficiency virus type 1 envelope in reduced sensitivity to macrophage inflammatory protein 1alpha. 1064 51

Phenotypic drug susceptibility assays of human immunodeficiency virus type 1 (HIV-1) isolates generally use time-consuming, expensive assays with peripheral blood mononuclear cells. A new HIV-1 indicator cell line, MAGI-CCR5, has been developed and applied for this purpose. This cell line expresses human CD4, the two major HIV-1 coreceptors, CCR5 and CXCR4, the reporter gene beta-galactosidase driven by the HIV-1 LTR, and quantitates infection within 48 h. A panel of reference strains and primary HIV-1 isolates were all found to infect this cell line. Susceptibility assays with a nucleoside (zidovudine, ZDV) and a non-nucleoside reverse transcriptase inhibitor (nevirapine, NVP) were performed with reference and primary isolates. The assay was modified into two steps for protease inhibitor (indivinavir, IDV and ritonavir, RTV) susceptibility assays. Primary isolates derived from drug naive patients displayed mean baseline 50% effective concentrations (EC50) of 0.14 microM for ZDV, 0.33 microM for NVP, and 0.02 microM for IDV. Isolates derived from patients under treatment displayed increased EC50 concentrations. The MAGI-CCR5 cell line offers a rapid, efficient, and reproducible method of testing a wide range of HIV-1 isolates for drug susceptibility.
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PMID:Rapid phenotypic drug susceptibility assay for HIV-1 with a CCR5 expressing indicator cell line. 1071 48

The first step in cellular entry of HIV involves binding of the viral envelope glycoprotein complex (gp120/gp41) to specific receptor molecules on the target cells. The cell-cell fusion (syncytium formation) between env expressing cells and CD4+ cells mimics the viral infection of the host cells. To search for anti-HIV substances preventing this process, we constructed the recombinant cell lines, HeLa/CD4/Lac-Z and HeLa/T-env/Tat for T-cell tropic (HIV-1(NL4-3)) system, and HOS/CD4/CCR5/Lac-Z and HeLa/M-env/Tat for macrophage tropic (HIV-1(SF162)) system. When each pair of cells were co-incubated for 20 hours, the multinuclear giant cells (syncytia) were formed and beta-galactosidase was expressed. These systems are less biohazardous because no infectious virus particles are used. Their validity in screening for anti-HIV substances which inhibit syncytium formation was confirmed using various known HIV entry inhibitors.
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PMID:A simple screening system for anti-HIV drugs: syncytium formation assay using T-cell line tropic and macrophage tropic HIV env expressing cell lines--establishment and validation. 1177 37

Neutralizing antibody (NAb) is a critical component of an immune system that can potentially provide sterilizing protection against human immunodeficiency virus type 1 (HIV-1). Therefore, an in vitro assay that can rapidly, safely, and accurately evaluate the NAb response vaccine candidates elicit, especially against a large number of HIV-1 variants, would be highly valuable. It has been demonstrated that HIV-1 envelope glycoprotein lacking the cytoplasmic domain can pseudotype murine leukemia virus encoding the beta-galactosidase gene and that this pseudovirus can specifically infect CD4(+) cells (Schnierle BS, Stitz J, Bosch V, et al.: Proc Natl Acad Sci USA 1997;94:8640-8645). Because the pseudovirus is not biohazardous and because the infection can be quantitatively determined within 2 days, we examined the feasibility of using the pseudovirus for high-throughput neutralization assays for HIV-1. We have generated viruses pseudotyped with gp140 of six different HIV-1 isolates (LAI, RF, Bal, AD8, 89.6, and DH12). All six pseudoviruses were infectious and exhibited expected coreceptor usage phenotype in HOS-CD4 cells expressing either CCR5 or CXCR4. More importantly, the neutralization sensitivity profile of these pseudoviruses was virtually identical to that observed from more conventional neutralization assays using either HIV-1 or SHIV. All pseudoviruses could be neutralized by broadly reactive human monoclonal antibody IgG1 b12. Our results indicate that the pseudoviruses are ideal for high-throughput evaluation of immune sera for their capacity to broadly neutralize a large number of HIV-1 isolates.
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PMID:Development of a safe and rapid neutralization assay using murine leukemia virus pseudotyped with HIV type 1 envelope glycoprotein lacking the cytoplasmic domain. 1178 23

The fusion reaction mediated by viral envelope glycoproteins proceeds through an ordered series of conformational changes in the envelope glycoprotein. Fusion inhibitors have been developed that target glycoprotein subunits, arresting the reaction at different points in the process. We report the development of a novel method for detecting viral glycoprotein-mediated fusion that is based on the principle of alpha-complementation of beta-galactosidase. The method is simple, accurate, has a high signal-to-noise ratio, is suited for high-throughput screening, and does not require new transcription or protein synthesis. Cells expressing a viral envelope glycoprotein and the N-terminal alpha fragment of beta-galactosidase were mixed with cells expressing the C-terminal beta-galactosidase fragment, CD4, CCR5, or CXCR4. Fusion was detected after 30 min and continued to increase to very high levels for more than 5 h. The assay was used to examine the temperature dependence of fusion and the effect of coreceptor and glycoprotein density on inhibitor activity.
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PMID:Alpha-complementation assay for HIV envelope glycoprotein-mediated fusion. 1498 Apr 94

The ability of certain theta-defensins, including retrocyclin-1, to protect human cells from infection by HIV-1 marks them as potentially useful molecules. Theta-defensins composed of L-amino acids are likely to be unstable in environments that contain host and microbial proteases. This study compared the properties of two enantiomeric theta-defensins, retrocyclin-1, and RC-112. Although these peptides have identical sequences, RC-112 is composed exclusively of D-amino acids, whereas retrocyclin-1 contains only L-amino acids. We compared the ability of these peptides to protect JC53-BL human cells from infection by 30 primary HIV-1 isolates. JC53-BL cells are modified HeLa cells that express surface CD4, CXCR4, and CCR5. They also contain reporter cassettes that are driven by the HIV-1 LTR, and express beta-galactosidase and luciferase. The HIV-1 isolates varied in co-receptor specificity and included subtypes A, B, C, D, CRF01-AE, and G. RC-112 was several fold more potent than retrocyclin-1 across the entire HIV-1 panel. Although RC-112 bound immobilized gp120 and CD4 with lower affinity than did retrocyclin-1, surface plasmon resonance experiments performed with 1 microg/mL of RC-112 and retrocyclin-1 revealed that both glycoproteins were bound to a similar extent. The superior antiviral performance of RC-112 most likely reflected its resistance to degradation by surface-associated or secreted proteases of the JC53-BL target cells. Theta-defensins composed exclusively of D-amino acids merit consideration as starting points for designing microbicides for topical application to the vagina or rectum.
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PMID:A theta-defensin composed exclusively of D-amino acids is active against HIV-1. 1517 19

This article describes the automation of an in vitro cell-based fusion assay for the identification of novel inhibitors of receptor mediated HIV-1 entry. The assay utilises two stable cell lines: one expressing CD4, CCR5 and an LTR-promoter/beta-galactosidase reporter construct, and the other expressing gp160 and tat. Accumulation of beta-galactosidase can only occur following fusion of these two cell lines via the gp160 and receptor mediators, as this event facilitates the transfer of the tat transcription factor between the two cell types. Although similar cell fusion systems have been described previously, they have not met the requirements for HTS due to complexity, throughput and reagent cost. The assay described in this article provides significant advantage, as (a) no transfection/infection events are required prior to the assay, reducing the potential for variability, (b) cells are mixed in solution, enhancing fusion efficiency compared to adherent cells, (c) miniaturization to low volume enables screening in 384-well plates; and (d) online cell dispensing facilitates automated screening. This assay has been employed to screen approximately 650,000 compounds in a singleton format. The data demonstrate that the assay is robust, with a Z' consistently above 0.6, which compares favourably with less complex biochemical assays.
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PMID:Development and automation of a 384-well cell fusion assay to identify inhibitors of CCR5/CD4-mediated HIV virus entry. 1545 38


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