scholarly journals Marking and Gene Expression by a Lentivirus Vector in Transplanted Human and Nonhuman Primate CD34+Cells

2000 ◽  
Vol 74 (3) ◽  
pp. 1286-1295 ◽  
Author(s):  
Dong Sung An ◽  
Robert P. Wersto ◽  
Brian A. Agricola ◽  
Mark E. Metzger ◽  
Stephanie Lu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Human Immunodeficiency Virus ◽  
Animal Model ◽  
Gene Delivery ◽  
Retroviral Vectors ◽  
Model Systems ◽  
Lentivirus Vector ◽  
Alternative Mode ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Recently, gene delivery vectors based on human immunodeficiency virus (HIV) have been developed as an alternative mode of gene delivery. These vectors have a number of advantages, particularly in regard to the ability to infect cells which are not actively dividing. However, the use of vectors based on human immunodeficiency virus raises a number of issues, not the least of which is safety; therefore, further characterization of marking and gene expression in different hematopoietic lineages in primate animal model systems is desirable. We use two animal model systems for gene therapy to test the efficiency of transduction and marking, as well as the safety of these vectors. The first utilizes the rhesus animal model for cytokine-mobilized autologous peripheral blood CD34+ cell transplantation. The second uses the SCID-human (SCID-hu) thymus/liver chimeric graft animal model useful specifically for human T-lymphoid progenitor cell reconstitution. In the rhesus macaques, detectable levels of vector were observed in granulocytes, lymphocytes, monocytes, and, in one animal with the highest levels of marking, erythrocytes and platelets. In transplanted SCID-hu mice, we directly compared marking and gene expression of the lentivirus vector and a murine leukemia virus-derived vector in thymocytes. Marking was observed at comparable levels, but the lentivirus vector bearing an internal cytomegalovirus promoter expressed less efficiently than did the murine retroviral vector expressed from its own long terminal repeats. In assays for infectious HIV type 1 (HIV-1), no replication-competent HIV-1 was detected in either animal model system. Thus, these results indicate that while lentivirus vectors have no apparent deleterious effects and may have advantages over murine retroviral vectors, further study of the requirements for optimal use are warranted.

scholarly journals Bovine leukemia virus protease: comparison with human T-lymphotropic virus and human immunodeficiency virus proteases

2007 ◽  
Vol 88 (7) ◽  
pp. 2052-2063 ◽  
Author(s):  
Tamás Sperka ◽  
Gabriella Miklóssy ◽  
Yunfeng Tie ◽  
Péter Bagossi ◽  
Gábor Zahuczky ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Animal Model ◽  
Amino Acid ◽  
Bovine Leukemia Virus ◽  
Leukemia Virus ◽  
Model Systems ◽  
T Lymphotropic Virus ◽  
Naturally Occurring ◽  
Immunodeficiency Virus ◽  
Hiv 1

Bovine leukemia virus (BLV) is a valuable model system for understanding human T-lymphotropic virus 1 (HTLV-1); the availability of an infectious BLV clone, together with animal-model systems, will help to explore anti-HTLV-1 strategies. Nevertheless, the specificity and inhibitor sensitivity of the BLV protease (PR) have not been characterized in detail. To facilitate such studies, a molecular model for the enzyme was built. The specificity of the BLV PR was studied with a set of oligopeptides representing naturally occurring cleavage sites in various retroviruses. Unlike HTLV-1 PR, but similar to the human immunodeficiency virus 1 (HIV-1) enzyme, BLV PR was able to hydrolyse the majority of the peptides, mostly at the same position as did their respective host PRs, indicating a broad specificity. When amino acid residues of the BLV PR substrate-binding sites were replaced by equivalent ones of the HIV-1 PR, many substitutions resulted in inactive protein, indicating a great sensitivity to mutations, as observed previously for the HTLV-1 PR. The specificity of the enzyme was studied further by using a series of peptides containing amino acid substitutions in a sequence representing a naturally occurring HTLV-1 PR cleavage site. Also, inhibitors of HIV-1 PR, HTLV-1 PR and other retroviral proteases were tested on the BLV PR. Interestingly, the BLV PR was more susceptible than the HTLV-1 PR to the inhibitors tested. Therefore, despite the specificity differences, in terms of mutation intolerance and inhibitor susceptibility of the PR, BLV and the corresponding animal-model systems may provide good models for testing of PR inhibitors that target HTLV-1.

scholarly journals Kinetics of Human Immunodeficiency Virus Type 1 Decay following Entry into Resting CD4+ T Cells

2005 ◽  
Vol 79 (4) ◽  
pp. 2199-2210 ◽  
Author(s):  
Yan Zhou ◽  
Haili Zhang ◽  
Janet D. Siliciano ◽  
Robert F. Siliciano
Keyword(s):  
Gene Expression ◽  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Human Immunodeficiency Virus Type ◽  
Reverse Transcription ◽  
Half Life ◽  
Immunodeficiency Virus ◽  
Kinetics Of ◽  
Hiv 1

ABSTRACT In untreated human immunodeficiency virus type 1 (HIV-1) infection, most viral genomes in resting CD4+ T cells are not integrated into host chromosomes. This unintegrated virus provides an inducible latent reservoir because cellular activation permits integration, virus gene expression, and virus production. It remains controversial whether HIV-1 is stable in this preintegration state. Here, we monitored the fate of HIV-1 in resting CD4+ cells by using a green fluorescent protein (GFP) reporter virus carrying an X4 envelope. After virus entry into resting CD4+ T cells, both rescuable virus gene expression, visualized with GFP, and rescuable virion production, assessed by p24 release, decayed with a half-life of 2 days. In these cells, reverse transcription goes to completion over 2 to 3 days, and 50% of the viruses that have entered undergo functional decay before reverse transcription is complete. We distinguished two distinct but closely related factors contributing to loss of rescuable virus. First, some host cells undergo virus-induced apoptosis upon viral entry, thereby reducing the amount of rescuable virus. Second, decay processes directly affecting the virus both before and after the completion of reverse transcription contribute to the loss of rescuable virus. The functional half-life of full-length, integration-competent reverse transcripts is only 1 day. We propose that rapid intracellular decay processes compete with early steps in viral replication in infected CD4+ T cells. Decay processes dominate in resting CD4+ T cells as a result of the slow kinetics of reverse transcription and blocks at subsequent steps. Therefore, the reservoir of unintegrated HIV-1 in recently infected resting CD4+ T cells is highly labile.

Poly(ADP-ribose) polymerase inhibitors suppress UV-induced human immunodeficiency virus type 1 gene expression at the posttranscriptional level

1991 ◽  
Vol 11 (7) ◽  
pp. 3522-3527
Author(s):  
S Yamagoe ◽  
T Kohda ◽  
M Oishi
Keyword(s):  
Gene Expression ◽  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Specific Gene ◽  
Adp Ribosylation ◽  
Immunodeficiency Virus ◽  
Posttranscriptional Level ◽  
Hiv 1

Gene expression of human immunodeficiency virus type 1 (HIV-1) is induced not only by trans activation mediated through a gene product (tat) encoded by the virus but also by treatment of virus-carrying cells with DNA-damaging agents such as UV light. Employing an artificially constructed DNA in which the chloramphenicol acetyltransferase gene was placed under the control of the HIV-1 long terminal repeat, we analyzed the induction process in HeLa cells and found that inhibitors of poly(ADP-ribose) polymerase suppressed UV-induced HIV-1 gene expression but not tat-mediated expression. We also found that suppression occurs at the posttranscriptional level. These results indicate that HIV-1 gene expression is activated by at least two different mechanisms, one of which involves poly-ADP ribosylation. A possible new role of poly-ADP ribosylation in the regulation of specific gene expression is also discussed.

scholarly journals Evidence for Gene Expression by Unintegrated Human Immunodeficiency Virus Type 1 DNA Species

2004 ◽  
Vol 78 (20) ◽  
pp. 11263-11271 ◽  
Author(s):  
Audrey Brussel ◽  
Pierre Sonigo
Keyword(s):  
Gene Expression ◽  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Viral Gene ◽  
Viral Expression ◽  
Molecular Stability ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The integrated form of human immunodeficiency virus type 1 (HIV-1) DNA is classically considered to be the sole template for viral gene expression. However, several studies have suggested that unintegrated viral DNA species could also support transcription. To determine the contribution of the different species of HIV-1 DNA to viral expression, we first monitored intracellular levels of various HIV-1 DNA and RNA species in a single-round infection assay. We observed that, in comparison to the precocity of HIV-1 DNA synthesis, viral expression was delayed, suggesting that only the HIV-1 DNA species that persist for a sufficient period of time would be transcribed efficiently. We next evaluated the transcriptional activity of the circular forms of HIV-1 DNA bearing two long terminal repeats, since these episomes were reported to exhibit an intrinsic molecular stability. Our results support the notion that these circular species of HIV-1 DNA are naturally transcribed during HIV-1 infection, thereby participating in virus replication.

scholarly journals A Human Immunodeficiency Virus (HIV)–Inducing Factor from the Female Genital Tract Activates HIV‐1 Gene Expression through the κB Enhancer

1998 ◽  
Vol 178 (5) ◽  
pp. 1343-1351 ◽  
Author(s):  
Lena Al‐Harthi ◽  
Gregory T. Spear ◽  
Farhad B. Hashemi ◽  
Alan Landay ◽  
Beverly E. Sha ◽  
...  
Keyword(s):  
Gene Expression ◽  
Human Immunodeficiency Virus ◽  
Genital Tract ◽  
Female Genital Tract ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Female Genital

scholarly journals Rational Design of Drugs That Induce Human Immunodeficiency Virus Replication

2003 ◽  
Vol 77 (19) ◽  
pp. 10227-10236 ◽  
Author(s):  
Dean H. Hamer ◽  
Sven Bocklandt ◽  
Louise McHugh ◽  
Tae-Wook Chun ◽  
Peter M. Blumberg ◽  
...  
Keyword(s):  
Gene Expression ◽  
Human Immunodeficiency Virus ◽  
T Cell Activation ◽  
Rational Design ◽  
Latent Reservoir ◽  
Viral Gene ◽  
Human Immunodeficiency Virus Replication ◽  
Immunodeficiency Virus ◽  
Latently Infected ◽  
Hiv 1

ABSTRACT Drugs that induce human immunodeficiency virus type 1 (HIV-1) replication could be used in combination with highly active antiretroviral therapy (HAART) to reduce the size of the latent reservoir that is in part responsible for viral persistence. Protein kinase C (PKC) is a logical target for such drugs because it activates HIV-1 transcription through multiple mechanisms. Here we show that HIV-1 gene expression can be induced by potent synthetic analogues of the lipid second messenger diacylglycerol (DAG) synthesized on a five-member ring platform that reduces the entropy of binding relative to that of the more flexible DAG template. By varying the alkyl side chains of these synthetic DAG lactones, it was possible to maximize their potency and ability to render latently infected T cells sensitive to killing by an anti-HIV-1 immunotoxin while minimizing the side effects of CD4 and CXCR4 downregulation and tumor necrosis factor alpha upregulation. The two lead compounds, LMC03 and LMC07, regulated a series of PKC-sensitive genes involved in T-cell activation and induced viral gene expression in peripheral blood mononuclear cells from HIV-1-infected individuals. These studies demonstrate the potential for the rational design of agents that, in conjunction with HAART and HIV-specific toxins, can be used to decrease or eliminate the pool of latently infected reservoirs by forcing viral expression.

scholarly journals Multiple Blocks to Human Immunodeficiency Virus Type 1 Replication in Rodent Cells

2000 ◽  
Vol 74 (21) ◽  
pp. 9868-9877 ◽  
Author(s):  
Paul D. Bieniasz ◽  
Bryan R. Cullen
Keyword(s):  
Gene Expression ◽  
Human Immunodeficiency Virus ◽  
Cell Lines ◽  
Infectious Virus ◽  
Human Gene ◽  
Human Cells ◽  
Gene Products ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The recent identification of human gene products that are required for early steps in the human immunodeficiency virus type 1 (HIV-1) life cycle has raised the possibility that rodents might be engineered to support HIV-1 infection. Therefore, we have examined the ability of modified mouse, rat, and hamster cell lines to support productive HIV-1 replication. Rodent cells, engineered to support Tat function by stable expression of a permissive cyclin T1 protein, proved to be able to support reverse transcription, integration, and early gene expression at levels comparable to those observed in human cell lines. Surprisingly, however, levels of CD4- and coreceptor-dependent virus entry were reduced to a variable but significant extent in both mouse and rat fibroblast cell lines. Additional posttranscriptional defects were observed, including a reduced level of unspliced HIV-1 genomic RNA and reduced structural gene expression. Furthermore, the HIV-1 Gag precursor is generally inefficiently processed and is poorly secreted from mouse and rat cells in a largely noninfectious form. These posttranscriptional defects, together, resulted in a dramatically reduced yield of infectious virus (up to 10,000-fold) over a single cycle of HIV-1 replication, as compared to human cells. Interestingly, these defects were less pronounced in one hamster cell line, CHO, which not only was able to produce infectious HIV-1 particles at a level close to that observed in human cells, but also could support transient, low-level HIV-1 replication. Importantly, the blocks to infectious virus production in mouse and rat cells are recessive, since they can be substantially suppressed by fusion with uninfected human cells. These studies imply the existence of one or more human gene products, either lacking or nonfunctional in most rodent cells that are critical for infectious HIV-1 virion morphogenesis.

scholarly journals Dihydroxythiophenes Are Novel Potent Inhibitors of Human Immunodeficiency Virus Integrase with a Diketo Acid-Like Pharmacophore

2006 ◽  
Vol 80 (14) ◽  
pp. 6883-6894 ◽  
Author(s):  
S. Kehlenbeck ◽  
U. Betz ◽  
A. Birkmann ◽  
B. Fast ◽  
A. H. Göller ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Retroviral Vectors ◽  
Cross Resistance ◽  
Binding Motif ◽  
Strand Transfer ◽  
Resistance Mutations ◽  
Immunodeficiency Virus ◽  
Viral Cdna ◽  
Hiv 1

ABSTRACT We have identified dihydroxythiophenes (DHT) as a novel series of human immunodeficiency virus type 1 (HIV-1) integrase inhibitors with broad antiviral activities against different HIV isolates in vitro. DHT were discovered in a biochemical integrase high-throughput screen searching for inhibitors of the strand transfer reaction of HIV-1 integrase. DHT are selective inhibitors of integrase that do not interfere with virus entry, as shown by the inhibition of a vesicular stomatitis virus G-pseudotyped retroviral system. Moreover, in quantitative real-time PCR experiments, no effect on the synthesis of viral cDNA could be detected but rather an increase in the accumulation of 2-long-terminal-repeat cycles was detected. This suggests that the integration of viral cDNA is blocked. Molecular modeling and the structure activity relationship of DHT demonstrate that our compound fits into a two-metal-binding motif that has been suggested as the essential pharmacophore for diketo acid (DKA)-like strand transfer inhibitors (Grobler et al., Proc. Natl. Acad. Sci. USA 99:6661-6666, 2002.). This notion is supported by the profiling of DHT on retroviral vectors carrying published resistance mutations for DKA-like inhibitors where DHT showed partial cross-resistance. This suggests that DHT bind to a common site in the catalytic center of integrase, albeit with an altered binding mode. Taken together, our findings indicate that DHT are novel selective strand transfer inhibitors of integrase with a pharmacophore homologous to DKA-like inhibitors.

scholarly journals RNA Trafficking Signals in Human Immunodeficiency Virus Type 1

2001 ◽  
Vol 21 (6) ◽  
pp. 2133-2143 ◽  
Author(s):  
Andrew J. Mouland ◽  
Hongbin Xu ◽  
Hongyi Cui ◽  
Winfried Krueger ◽  
Trent P. Munro ◽  
...  
Keyword(s):  
Gene Expression ◽  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Rna Transport ◽  
Rna Trafficking ◽  
Trafficking Pathway ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Intracellular trafficking of retroviral RNAs is a potential mechanism to target viral gene expression to specific regions of infected cells. Here we show that the human immunodeficiency virus type 1 (HIV-1) genome contains two sequences similar to the hnRNP A2 response element (A2RE), a cis-acting RNA trafficking sequence that binds to the trans-acting trafficking factor, hnRNP A2, and mediates a specific RNA trafficking pathway characterized extensively in oligodendrocytes. The two HIV-1 sequences, designated A2RE-1, within the major homology region of the gag gene, and A2RE-2, in a region of overlap between the vpr andtat genes, both bind to hnRNP A2 in vitro and are necessary and sufficient for RNA transport in oligodendrocytes in vivo. A single base change (A8G) in either sequence reduces hnRNP A2 binding and, in the case of A2RE-2, inhibits RNA transport. A2RE-mediated RNA transport is microtubule and hnRNP A2 dependent. Differentially labelledgag and vpr RNAs, containing A2RE-1 and A2RE-2, respectively, coassemble into the same RNA trafficking granules and are cotransported to the periphery of the cell. tat RNA, although it contains A2RE-2, is not transported as efficiently asvpr RNA. An A2RE/hnRNP A2-mediated trafficking pathway for HIV RNA is proposed, and the role of RNA trafficking in targeting HIV gene expression is discussed.

scholarly journals Susceptibility of Rat-Derived Cells to Replication by Human Immunodeficiency Virus Type 1

2001 ◽  
Vol 75 (17) ◽  
pp. 8063-8073 ◽  
Author(s):  
Oliver T. Keppler ◽  
Wesley Yonemoto ◽  
Frank J. Welte ◽  
Kathryn S. Patton ◽  
Demetris Iacovides ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Animal Model ◽  
Human Immunodeficiency Virus Type ◽  
Small Animal ◽  
Cell Type ◽  
Small Animal Model ◽  
Immunodeficiency Virus ◽  
Cell Type Specific ◽  
Hiv 1

ABSTRACT Progress in developing a small animal model of human immunodeficiency virus type 1 (HIV-1) disease would greatly facilitate studies of transmission, pathogenesis, host immune responses, and antiviral strategies. In this study, we have explored the potential of rats as a susceptible host. In a single replication cycle, rat cell lines Rat2 and Nb2 produced infectious virus at levels 10- to 60-fold lower than those produced by human cells. Rat-derived cells supported substantial levels of early HIV-1 gene expression, which was further enhanced by overexpression of human cyclin T1. Rat cells displayed quantitative, qualitative, and cell-type-specific limitations in the late phase of the HIV-1 replication cycle including relative expression levels of HIV-1 Gag proteins, intracellular Gag processing, and viral egress. Nb2 cells were rendered permissive to HIV-1 R5 viruses by coexpression of human CD4 and CCR5, indicating that the major restriction on HIV-1 replication was at the level of cellular entry. We also found that primary rat lymphocytes, macrophages, and microglia expressed considerable levels of early HIV-1 gene products following infection with pseudotyped HIV-1. Importantly, primary rat macrophages and microglia, but not lymphocytes, also expressed substantial levels of HIV-1 p24 CA and produced infectious virions. Collectively, these results identify the rat as a promising candidate for a transgenic small animal model of HIV-1 infection and highlight pertinent cell-type-specific restrictions that are features of this species.

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