scholarly journals Heterocyclic Compounds That Inhibit Rev-RRE Function and Human Immunodeficiency Virus Type 1 Replication

2008 ◽  
Vol 52 (9) ◽  
pp. 3169-3179 ◽  
Author(s):  
Deidra Shuck-Lee ◽  
Fei Fei Chen ◽  
Ryan Willard ◽  
Sharmila Raman ◽  
Roger Ptak ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Life Cycle ◽  
Cell Line ◽  
Particle Production ◽  
Screening Assay ◽  
Rev Response Element ◽  
Immunodeficiency Virus ◽  
A Cell ◽  
Viruslike Particles

ABSTRACT A cell-based screening assay was performed to identify compounds that inhibited the postintegration stage of the human immunodeficiency virus (HIV) life cycle. This assay utilized a cell line that contains the HIV gag and pol genes expressed in a Rev-dependent fashion. The cell line produces about 10 to 15 ng of p24 per milliliter of medium over a 24-h period in the form of viruslike particles. Any compound that inhibits a postintegration step in the HIV life cycle scores in this assay by decreasing particle production. Forty thousand compounds were screened, and 192 compounds were selected from the original screen because they showed more than 50% inhibition at a 10 μM concentration. The cumulative evidence presented in this study strongly suggests that 2 of the 192 compounds work as inhibitors of HIV Rev function. This was determined by a variety of cell-based assays, although the compounds do not interfere with Rev-RRE (Rev response element) binding in vitro. Both compounds inhibit replication of the lab isolate NL4-3 as well as an HIV primary isolate from Brazil (93BR021) and thus are promising leads as therapeutic candidates that target HIV replication through inhibition of Rev function.

scholarly journals Transforming growth factor beta suppresses human immunodeficiency virus expression and replication in infected cells of the monocyte/macrophage lineage.

1991 ◽  
Vol 173 (3) ◽  
pp. 589-597 ◽  
Author(s):  
G Poli ◽  
A L Kinter ◽  
J S Justement ◽  
P Bressler ◽  
J H Kehrl ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cell ◽  
Growth Factor ◽  
Cell Line ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Tgf Beta ◽  
Immunodeficiency Virus ◽  
Growth Factor Beta

The pleiotropic immunoregulatory cytokine transforming growth factor beta (TGF-beta) potently suppresses production of the human immunodeficiency virus (HIV), the causative agent of the acquired immunodeficiency syndrome, in the chronically infected promonocytic cell line U1. TGF-beta significantly (50-90%) inhibited HIV reverse transcriptase production and synthesis of viral proteins in U1 cells stimulated with phorbol myristate acetate (PMA) or interleukin 6 (IL-6). Furthermore, TGF-beta suppressed PMA induction of HIV transcription in U1 cells. In contrast, TGF-beta did not significantly affect the expression of HIV induced by tumor necrosis factor alpha (TNF-alpha). These suppressive effects were not mediated via the induction of interferon alpha (IFN-alpha). TGF-beta also suppressed HIV replication in primary monocyte-derived macrophages infected in vitro, both in the absence of exogenous cytokines and in IL-6-stimulated cultures. In contrast, no significant effects of TGF-beta were observed in either a chronically infected T cell line (ACH-2) or in primary T cell blasts infected in vitro. Therefore, TGF-beta may play a potentially important role as a negative regulator of HIV expression in infected monocytes or tissue macrophages in infected individuals.

scholarly journals Topotecan inhibits human immunodeficiency virus type 1 infection through a topoisomerase-independent mechanism in a cell line with altered topoisomerase I.

1997 ◽  
Vol 41 (5) ◽  
pp. 977-981 ◽  
Author(s):  
J L Zhang ◽  
P L Sharma ◽  
C J Li ◽  
B J Dezube ◽  
A B Pardee ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Cell Line ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Topoisomerase I ◽  
Immunodeficiency Virus ◽  
Latently Infected ◽  
A Cell ◽  
Hiv 1

Topotecan (TPT), a known inhibitor of topoisomerase I, has previously been shown to inhibit the replication of several viruses. The mechanism of inhibition was proposed to be the inhibition of topoisomerase I. We report that TPT decreased replication of human immunodeficiency virus type 1 (HIV-1) in CPT-K5, a cell line with a topoisomerase I mutation. TPT inhibited production of HIV-1 RNA and p24 in CPT-K5 and wild-type cells equally effectively. The antiviral effects of TPT were observed not only in the topoisomerase-mutated CPT-K5 line but also in peripheral blood mononuclear cells (PBMC) acutely infected with clinical isolates and in OM10.1 cells latently infected with HIV and activated by tumor necrosis factor alpha. Little toxicity from TPT was noted in HIV-1-infected PBMC and in CPT-K5 and OM10.1 cells as measured by cell growth and proliferation assays. These observations suggest that TPT targets factors in virus replication other than cellular topoisomerase I and inhibits cytokine-mediated activation in latently infected cells by means other than cytotoxicity. These results suggest a potential for TPT and for other camptothecins in anti-HIV therapy alone and in combination with other antiretroviral drugs.

scholarly journals Altered Gag Polyprotein Cleavage Specificity of Feline Immunodeficiency Virus/Human Immunodeficiency Virus Mutant Proteases as Demonstrated in a Cell-Based Expression System

2006 ◽  
Vol 80 (16) ◽  
pp. 7832-7843 ◽  
Author(s):  
Ying-Chuan Lin ◽  
Ashraf Brik ◽  
Aymeric de Parseval ◽  
Karen Tam ◽  
Bruce E. Torbett ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Feline Immunodeficiency Virus ◽  
Ex Vivo ◽  
Expression System ◽  
Immunodeficiency Virus ◽  
A Cell ◽  
Virus Mutant ◽  
Substrate Inhibitor ◽  
Hiv 1

ABSTRACT We have used feline immunodeficiency virus (FIV) protease (PR) as a mutational system to study the molecular basis of substrate-inhibitor specificity for lentivirus PRs, with a focus on human immunodeficiency virus type 1 (HIV-1) PR. Our previous mutagenesis studies demonstrated that discrete substitutions in the active site of FIV PR with structurally equivalent residues of HIV-1 PR dramatically altered the specificity of the mutant PRs in in vitro analyses. Here, we have expanded these studies to analyze the specificity changes in each mutant FIV PR expressed in the context of the natural Gag-Pol polyprotein ex vivo. Expression mutants were prepared in which 4 to 12 HIV-1-equivalent substitutions were made in FIV PR, and cleavage of each Gag-Pol polyprotein was then assessed in pseudovirions from transduced cells. The findings demonstrated that, as with in vitro analyses, inhibitor specificities of the mutants showed increased HIV-1 PR character when analyzed against the natural substrate. In addition, all of the mutant PRs still processed the FIV polyprotein but the apparent order of processing was altered relative to that observed with wild-type FIV PR. Given the importance of the order in which Gag-Pol is processed, these findings likely explain the failure to produce infectious FIVs bearing these mutations.

scholarly journals Development of a Cell-Based Assay Probing the Specific Interaction between the Human Immunodeficiency Virus Type 1 Nucleocapsid and Psi RNA In Vivo

2007 ◽  
Vol 81 (11) ◽  
pp. 6151-6155 ◽  
Author(s):  
Soo In Jang ◽  
Young Ho Kim ◽  
Soon Young Paik ◽  
Ji Chang You
Keyword(s):  
Human Immunodeficiency Virus ◽  
Specific Interaction ◽  
Deletion Mapping ◽  
Packaging Signal ◽  
Immunodeficiency Virus ◽  
A Cell ◽  
First Time

ABSTRACT Here, we describe a cell-based in vivo assay that probes the specific interaction between nucleocapsid (NC) protein and Psi (Ψ) RNA, the human immunodeficiency virus (HIV) packaging signal. The results demonstrate for the first time a specific NC-Ψ interaction within living cells. The specificity and applicability of the assay were confirmed by mutational studies of NC and deletion-mapping analyses of Ψ-RNA as well as by testing the in vivo NC-binding effects of NC-aptamer RNAs identified previously in vitro. This assay system would facilitate further detailed studies of the NC-Ψ interaction in vivo and the screening of various anti-HIV molecules targeting NC and the specific interaction.

scholarly journals A Cell Line-Based Neutralization Assay for Primary Human Immunodeficiency Virus Type 1 Isolates That Use either the CCR5 or the CXCR4 Coreceptor

1999 ◽  
Vol 73 (11) ◽  
pp. 8966-8974 ◽  
Author(s):  
Alexandra Trkola ◽  
Jamie Matthews ◽  
Cynthia Gordon ◽  
Tom Ketas ◽  
John P. Moore
Keyword(s):  
Human Immunodeficiency Virus ◽  
Cell Line ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Neutralization Assay ◽  
Immunodeficiency Virus ◽  
A Cell ◽  
Cxcr4 Coreceptor ◽  
Hiv 1

ABSTRACT We describe here a cell line-based assay for the evaluation of human immunodeficiency virus type 1 (HIV-1) neutralization. The assay is based on CEM.NKR cells, transfected to express the HIV-1 coreceptor CCR5 to supplement the endogenous expression of CD4 and the CXCR4 coreceptor. The resulting CEM.NKR-CCR5 cells efficiently replicate primary HIV-1 isolates of both R5 and X4 phenotypes. A comparison of the CEM.NKR-CCR5 cells with mitogen-activated peripheral blood mononuclear cells (PBMC) in neutralization assays with sera from HIV-1-infected individuals or specific anti-HIV-1 monoclonal antibodies shows that the sensitivity of HIV-1 neutralization is similar in the two cell types. The CEM.NKR-CCR5 cell assay, however, is more convenient to perform and eliminates the donor-to-donor variation in HIV-1 replication efficiency, which is one of the principal drawbacks of the PBMC-based neutralization assay. We suggest that this new assay is suitable for the general measurement of HIV-1 neutralization by antibodies.

scholarly journals The 5′ and 3′ TAR Elements of Human Immunodeficiency Virus Exert Effects at Several Points in the Virus Life Cycle

1998 ◽  
Vol 72 (11) ◽  
pp. 9217-9223 ◽  
Author(s):  
Atze T. Das ◽  
Bep Klaver ◽  
Ben Berkhout
Keyword(s):  
Human Immunodeficiency Virus ◽  
Life Cycle ◽  
Reverse Transcription ◽  
Wild Type ◽  
Rna Packaging ◽  
Virus Life Cycle ◽  
Activation Of Transcription ◽  
Immunodeficiency Virus

ABSTRACT The human immunodeficiency virus type 1 RNA genome contains a terminal repeat (R) sequence that encodes the TAR hairpin motif, which has been implicated in Tat-mediated activation of transcription. More recently, a variety of other functions have been proposed for this structured RNA element. To determine the replicative roles of the 5′ and 3′ TAR hairpins, we analyzed multiple steps in the life cycle of wild-type and mutant viruses. A structure-destabilizing mutation was introduced in either the 5′, the 3′, or both TAR motifs of the proviral genome. As expected, opening of the 5′ TAR hairpin caused a transcription defect. Because the level of protein expression was not similarly reduced, the translation of this mRNA was improved. No effect of the 3′ hairpin on transcription and translation was measured. Mutations of the 5′ and 3′ hairpin structures reduced the efficiency of RNA packaging to similar extents, and RNA packaging was further reduced in the 5′ and 3′ TAR double mutant. Upon infection of cells with these virions, a reduced amount of reverse transcription products was synthesized by the TAR mutant. However, no net reverse transcription defect was observed after correction for the reduced level of virion RNA. This result was confirmed in in vitro reverse transcription assays. These data indicate that the 5′ and 3′ TAR motifs play important roles in several steps of the replication cycle, but these structures have no significant effect on the mechanism of reverse transcription.

scholarly journals Functional Surfaces of the Human Immunodeficiency Virus Type 1 Capsid Protein

2003 ◽  
Vol 77 (9) ◽  
pp. 5439-5450 ◽  
Author(s):  
Uta K. von Schwedler ◽  
Kirsten M. Stray ◽  
Jennifer E. Garrus ◽  
Wesley I. Sundquist
Keyword(s):  
Human Immunodeficiency Virus ◽  
Life Cycle ◽  
Capsid Protein ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Particle Production ◽  
Viral Assembly ◽  
Immunodeficiency Virus ◽  
Immature Particle

ABSTRACT The human immunodeficiency virus type 1 initially assembles and buds as an immature particle that is organized by the viral Gag polyprotein. Gag is then proteolyzed to produce the smaller capsid protein CA, which forms the central conical capsid that surrounds the RNA genome in the mature, infectious virus. To define CA surfaces that function at different stages of the viral life cycle, a total of 48 different alanine-scanning surface mutations in CA were tested for their effects on Gag protein expression, processing, particle production and morphology, capsid assembly, and infectivity. The 27 detrimental mutations fall into three classes: 13 mutations significantly diminished or altered particle production, 9 mutations failed to assemble normal capsids, and 5 mutations supported normal viral assembly but were nevertheless reduced more than 20-fold in infectivity. The locations of the assembly-defective mutations implicate three different CA surfaces in immature particle assembly: one surface encompasses helices 4 to 6 in the CA N-terminal domain (NTD), a second surrounds the crystallographically defined CA dimer interface in the C-terminal domain (CTD), and a third surrounds the loop preceding helix 8 at the base of the CTD. Mature capsid formation required a distinct surface encompassing helices 1 to 3 in the NTD, in good agreement with a recent structural model for the viral capsid. Finally, the identification of replication-defective mutants with normal viral assembly phenotypes indicates that CA also performs important nonstructural functions at early stages of the viral life cycle.

scholarly journals An Orphan G Protein-Coupled Receptor, GPR1, Acts as a Coreceptor To Allow Replication of Human Immunodeficiency Virus Types 1 and 2 in Brain-Derived Cells

1999 ◽  
Vol 73 (6) ◽  
pp. 5231-5239 ◽  
Author(s):  
Nobuaki Shimizu ◽  
Yasushi Soda ◽  
Katsuaki Kanbe ◽  
Hui-Yu Liu ◽  
Atsushi Jinno ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Cell Line ◽  
G Protein ◽  
Chemokine Receptors ◽  
Single Point ◽  
G Protein Coupled Receptor ◽  
Immunodeficiency Virus ◽  
G Protein Coupled ◽  
Hiv 1

ABSTRACT Twelve G protein-coupled receptors, including chemokine receptors, act as coreceptors and determinants for the cell tropisms of human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIV). We isolated HIV-1 variants from T-cell-line (T)- and macrophage (M)-tropic (i.e., dualtropic) (R5-R3-X4) HIV-1 strains and also produced six HIV-1 mutants carrying single-point amino acid substitutions at the tip of the V3 region of the Env protein of HIV-1. These variants and three mutants infected brain-derived CD4-positive cells that are resistant to M-, T-, or dualtropic (R5, X4, or R5-X4) HIV-1 strains. However, a factor that determines this cell tropism has not been identified. This study shows that primary brain-derived fibroblast-like cell strains, BT-3 and BT-20/N, as well as a CD4-transduced glioma cell line, U87/CD4, which were susceptible to these HIV-1 variants and mutants and the HIV-2ROD strain, expressed mRNA of an orphan G protein-coupled receptor (GPCR), GPR1. When a CD4-positive cell line which was strictly resistant to infection with diverse HIV-1 and HIV-2 strains was transduced with GPR1, the cell line became susceptible to these HIV-1 variants and mutants and to an HIV-2 strain but not to T- or dualtropic HIV-1 strains, and numerous syncytia formed after infection. These results indicate that GPR1 functions as a coreceptor for the HIV-1 variants and mutants and for the HIV-2ROD strain in vitro.

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