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At least 973 records · Page 54Linked to original sources
PubMed1996

Maintaining the integrity of human immunodeficiency virus sequence databases.

Human immunodeficiency virus type 1 (HIV-1) sequences are accumulating in the literature at a rapid pace. For this ever-expanding resource to be maximally useful, it is critical that researchers strive to maintain a high level of quality assurance, both in experimental design and conduct and in analyses. Here we present detailed analyses of problematic sets of HIV-1 sequences in the database that include sequence anomalies suggestive of mislabeling or sample contamination problems. These data are examined in the context of currently available HIV-1 sequence information to provide an example of how to identify potentially flawed data. Indicators of potential problems with sequences are (i) sequences that are nearly identical that are supposed to be derived from unlinked individuals and that are markedly distinct from other sequences from the putative source or (ii) sequences that are nearly identical to those of laboratory strains. We provide an outline of methods that researchers can use to perform preliminary laboratory and computational analyses that could help identify problematic data and thus help ensure the integrity of sequence databases.

Base Sequence↗
PubMed2001

Self-deleting suicide vectors (SDSV): selective killing of p53-deficient cancer cells.

A self-deleting retrovirus vector carrying a herpes simplex virus (HSV)-thymidine kinase suicide gene has been developed to selectively kill cancer cells expressing a dysfunctional p53 tumor suppressor protein. When cells containing functional p53 are infected with the virus, the integrated provirus and the HSV-thymidine kinase gene are deleted from the genome by site-specific recombination (Cre/loxP). In contrast, cells without p53 or cells expressing a DNA-binding mutant of p53 retain the provirus and become susceptible to killing by ganciclovir. This strategy provides a new concept for the selective killing of cancer cells that can be adapted to any other dysfunctional transcription factor expressed by different tumors.

Animals↗
PubMed1995

In vivo fate of HIV-1-infected T cells: quantitative analysis of the transition to stable latency.

Although it is presumed that the integration of HIV-1 into the genome of infected CD4+ T lymphocytes allows viral persistence, there has been little direct evidence that CD4+ T cells with integrated provirus function as a latent reservoir for HIV-1 in infected individuals. Using resting CD4+ T-cell populations of extremely high purity and a novel assay that selectively and unambiguously detects integrated HIV-1, we show that resting CD4+ T cells harbouring integrated provirus are present in some infected individuals. However, these cells do not accumulate within the circulating pool of resting CD4+ T cells in the early stages of HIV-1 infection and do not accumulate even after prolonged periods in long-term survivors of HIV-1 infection. These results suggest that because of viral cytopathic effects and/or host effector mechanisms, productively infected CD4+ T cells do not generally survive for long enough to revert to a resting memory state in vivo.

Base Sequence↗
PubMed2001

Differential role of the Mu B protein in phage Mu integration vs. replication: mechanistic insights into two transposition pathways.

The Mu B protein is an ATP-dependent DNA-binding protein and an allosteric activator of the Mu transposase. As a result of these activities, Mu B is instrumental in efficient transposition and target-site choice. We analysed in vivo the role of Mu B in the two different recombination reactions performed by phage Mu: non-replicative transposition, the pathway used during integration, and replicative transposition, the pathway used during lytic growth. Utilizing a sensitive PCR-based assay for Mu transposition, we found that Mu B is not required for integration, but enhances the rate and extent of the process. Furthermore, three different mutant versions of Mu B, Mu BC99Y, Mu BK106A, and Mu B1-294, stimulate integration to a similar level as the wild-type protein. In contrast, these mutant proteins fail to support Mu growth. This deficiency is attributable to a defect in formation of an essential intermediate for replicative transposition. Biochemical analysis of the Mu B mutant proteins reveals common features: the mutants retain the ability to stimulate transposase, but are defective in DNA binding and target DNA delivery. These data indicate that activation of transposase by Mu B is sufficient for robust non-replicative transposition. Efficient replicative transposition, however, demands that the Mu B protein not only activate transposase, but also bind and deliver the target DNA.

Bacteriophage mu↗
PubMed1997

Differential effects of the splice acceptor at nucleotide 3295 of human papillomavirus type 31 on stable and transient viral replication.

In human papillomavirus type 31 (HPV-31), the E1--E4 and E5 open reading frames are expressed from polycistronic mRNAs. The major polycistronic mRNAs which encode E1--E4 and E5 are spliced messages which utilize a splice acceptor at nucleotide (nt) 3295 (SPA3295). Our laboratory recently developed a recombinant system for the synthesis of HPVs following immortalization of primary keratinocytes with cloned HPV-31 genomes (M. G. Frattini et al., Proc. Natl. Acad. Sci. USA 93:3062-3067, 1996). These immortalized cell lines are capable of maintaining HPV-31 DNA as episomes and induce the synthesis of virions in organotypic raft culture. In this study, we used these methods to begin an analysis of the roles of E1--E4 and E5 in HPV pathogenesis by mutating the major splice at nt 3295. Mutation of SPA3295 did not significantly alter the ability of HPV-31 genomes to replicate transiently in keratinocytes, nor did the mutation affect the immortalization potential of HPV-31. However, genomes carrying the SPA3295 mutation were not stably maintained as viral episomes, and the resulting immortalized keratinocyte cell line contained multiple, integrated copies of the mutated HPV-31 DNA. Northern analysis indicated that cell lines immortalized with the mutant HPV-31 expressed transcripts which were similar in size and abundance to wild-type messages, including those transcripts which rely on utilization of SPA3295. RNase protection and reverse transcription-PCR revealed that mutation of SPA3295 resulted in the utilization of a cryptic splice acceptor at nt 3298. These data suggest that the requirements for stable maintenance of HPV genomes are more stringent than those for transient replication and that factors which define these requirement rely on the major splice acceptor at nt 3295.

Alternative Splicing↗
PubMed1996

In vitro synthesis of oncogenic human papillomaviruses requires episomal genomes for differentiation-dependent late expression.

Human papillomavirus (HPV) types 16, 18, 31, and 51 are the etiologic agents of many anogenital cancers including those of the cervix. These "high risk" HPVs specifically target genital squamous epithelia, and their lytic life cycle is closely linked to epithelial differentiation. We have developed a genetic assay for HPV functions during pathogenesis using recircularized cloned HPV 31 genomes that were transfected together with a drug resistance marker into monolayer cultures of normal human foreskin keratinocytes, the natural host cell. After drug selection, cell lines were isolated that stably maintained HPV 31 DNA as episomes and underwent terminal differentiation when grown in organotypic raft cultures. In differentiated rafts, the expression of late viral genes, amplification of viral DNA, and production of viral particles were detected in suprabasal cells. This demonstrated the ability to synthesize HPV 31 virions from transfected DNA templates and allowed an examination of HPV functions during the vegetative viral life cycle. We then used this system to investigate whether an episomal genome was required for the induction of late viral gene expression. When an HPV 31 genome (31E1*) containing a missense mutation in the E1 open reading frame was transfected into normal human keratinocytes, the mutant viral sequences were found to integrate into the host cell chromosomal DNA with both early and late regions intact. While high levels of early viral gene transcription were observed, no late gene expression was detected in rafts of cell lines containing the mutant viral genome despite evidence of terminal differentiation. Therefore, the induction of late viral gene expression required that the viral genomes be maintained as extrachromosomal elements, and terminal differentiation alone was not sufficient. These studies provide the basis for a detailed examination of HPV functions during viral pathogenesis.

Blotting, Northern↗
PubMed1993

Comparison of DNA binding and integration half-site selection by avian myeloblastosis virus integrase.

Insertion of the linear retrovirus DNA genome into the host DNA by the virus-encoded integrase (IN) is essential for efficient replication. We devised an efficient virus-like DNA plasmid integration assay which mimics the standard oligonucleotide assay for integration. It permitted us to study, by electron microscopy and sequence analysis, insertion of a single long terminal repeat terminus (LTR half-site) of one plasmid into another linearized plasmid. The reaction was catalyzed by purified avian myeloblastosis virus IN in the presence of Mg2+. The recombinant molecules were easily visualized and quantitated by agarose gel electrophoresis. Agarose gel-purified recombinants could be genetically selected by transformation of ligated recombinants into Escherichia coli HB101 cells. Electron microscopy also permitted the identification and localization of IN-DNA complexes on the virus-like substrate in the absence of the joining reaction. Intramolecular and intermolecular DNA looping by IN was visualized. Although IN preferentially bound to AT-rich regions in the absence of the joining reaction, there was a bias towards GC-rich regions for the joining reaction. Alignment of 70 target site sequences 5' of the LTR half-site insertions with 68 target sites previously identified for the concerted insertion of both LTR termini (LTR full-site reaction) indicated similar GC inflection patterns with both insertional events. Comparison of the data suggested that IN recognized only half of the target sequences necessary for integration with the LTR half-site reaction.

Avian Myeloblastosis Virus↗
PubMed2002

Molecular indications for in vivo integration of the avian leukosis virus, subgroup J-long terminal repeat into the Marek's disease virus in experimentally dually-infected chickens.

Marek's disease virus, a herpesvirus, and avian leukosis virus, subgroup J, a retrovirus, are oncogenic viruses of poultry. Both viruses may infect the same flock, the same bird and the same cell. In a double-infected cell, the retroviral DNA can integrate into the cellular or the Marek's disease virus (MDV) genome. The retroviral-long terminal repeat (LTR) integration into MDV was first described by Isfort et al., (Proc Natl Acad Sci 89, 991-995, 1992) following tissue culture co-infection. The recombinant virus isolated, RM1, had altered biological properties compared to the parental MDV (Witter R.L., Li D., Jones D., and Kung H.-J., Avian Dis 41, 407-421, 1997) . The issue of retroviral sequence integration into herpesviruses in vivo, in cases of double-virus infection is of wide significance in general virology and veterinary medicine; it also represents a special case of gene transposition. Using the avian system, we aimed to determine occurrence of such integrations in vivo. Chickens were experimentally co-infected with both avian leukosis virus (ALV) subgroup J and with MDV. To demonstrate the presence of the retroviral LTR in the MDV genome we applied the Hot Spot-combined PCR assay (Borenshtain R. and Davidson I., J Virol Meth 82, 119-127, 1999) that consisted of two consecutive steps of amplification. By that HS-cPCR assay, certain MDV genomic sites, defined as HS for integration were specifically amplified, the HS step, and then subjected to screening in an attempt to detect LTR inserts. The screening was achieved by amplification using heterologous primer sets, one for the MDV hot spot and the other for the retroviral LTR, the cPCR step. The products were Southern blotted and hybridized with MDV and ALV-LTR probes. Chimeric molecules were detected and evidenced by an intense signal in 3/10 chickens and weakly in other 3/10 birds. Detection was by LTR amplification, sequencing and multiple alignment to the ALV-J-LTR sequence. The present study indicated that chimeric molecules were produced in vivo.

Animals↗
PubMed1996

A single nucleotide substitution converts potato spindle tuber viroid (PSTVd) from a noninfectious to an infectious RNA for nicotiana tabacum.

Mechanical inoculation of Nicotiana tabacum with the PSTVd isolate KF 440-2 from the host plant tomato resulted in the de novo emergence, replication, and accumulation of a new "tobacco variant," designated PSTVd NT. It produces no symptoms in tobacco but, like PSTV KF 440-2, severe ones in tomato. The sequence analysis of PSTVd NT revealed a single nucleotide substitution from C-->U at position 259. Autonomous viroid replication was also induced in tobacco by genomic integration of oligomeric cDNA copies of PSTVd KF 440-2. Although these cDNAs contained the original tomato-specific C259, the circular PSTVd RNA subsequently accumulating in tobacco also exhibited the C259-->U259 substitution. In the secondary structure of PSTVd, nucleotide 259 is part of an internal loop analogous to loop E of eukaryotic 5S rRNA and presumed to be the only bulged extrahelical nucleotide of this loop. The C259 in PSTVd KF 440-2 and in practically all other isolates and the U259 in PSTVd NT of the loop E-like structure might be involved in protein binding and in viroid processing. The new variant PSTVd NT is genetically stable in both tobacco and tomato.

Base Composition↗
PubMed2003

HIV-1 vectors: fulfillment of expectations, further advancements, and still a way to go.

The ability of lentiviral vectors to transduce and stably integrate their genomes into non-dividing cells was the major reason for the development of the HIV-1 based vector gene delivery system. The first VSV-G pseudotyped lentiviral vectors fulfilled these expectations by ferrying large genetic payloads to non-dividing cells in vitro and in vivo. Here we discuss advances in HIV-1 vector systems which lead to improvement in biosafety, transduction efficiency, longevity and regulation of transgene expression, and vector production. The successful use of the advanced HIV-1 based vector system opened new avenues in establishing transgenic animal models for basic research. Additionally, we describe accomplishments using HIV-1 based vectors to correct pathological courses of incurable diseases in preclinical animal models including Parkinson's disease and beta-thalassemia.

Animals↗
PubMed2004

Efficacy and mode of action of hammerhead and hairpin ribozymes against various HIV-1 target sites.

Ribozymes have been proposed as gene therapy agents against HIV-1, although many fundamental questions about their mechanism of action remain unclear. Few studies have compared directly the potential of different modified ribozyme species against a particular target. Here we compare the relative abilities of hammerhead (HhU5) and hairpin (HpU5) ribozymes directed against a well-studied target RNA that has therapeutic potential, located in the untranslated 5' region (U5), to inhibit HIV-1 replication. The two types of ribozymes showed similar antiviral efficacy after being stably transfected into HUT78 cells and subsequently challenged with HIV-1(SF2), but the HhU5 ribozyme showed faster cleavage kinetics when tested in a cell-free system. In the second part of this study, we examined whether different ribozymes were able to inhibit the integration of proviral DNA in infected HUT78 cells. We found that cell pools stably expressing HpU5 could limit the appearance of integrated provirus, indicating that they could inhibit the infecting viral RNA before reverse transcription. A preintegration effect was also found for cell pools expressing a ribozyme targeting the nef gene (HhNef) or a ribozyme targeting the LTR (HhLTR). However, no discernible preintegration effects were seen for the HhU5 ribozyme or an active ribozyme directed against an RNA target site in the pol gene (HhPol). Thus, the results suggest that the mode of ribozyme action varied between sites and is not dependent solely on inhibiting the infecting viral RNA. Evidence for a preintegration effect is extremely encouraging and indicates that "resistant" cells have some chance to repopulate the immune system through such a selective advantage. We also studied the ability of the different ribozymes to down regulate viral RNA postintegration.

5' Untranslated Regions↗
PubMed1999

Inhibition of replication of HIV-1 at both early and late stages of the viral life cycle by single-chain antibody against viral integrase.

Retroviruses including HIV-1 integrates a DNA copy of their RNA genome into cellular DNA of the infected cell. This reaction, essential and unique to replication of retroviruses, is mediated by the viral enzyme, integrase (IN). We constructed a recombinant gene encoding a single-chain, antigen-binding peptide (scAb2-19), which interacted with a carboxyl terminal part of HIV-1 IN. HeLa CD4 cells expressing scAb2-19 localized in either cytoplasmic or nuclear compartment were resistant to HIV-1 infection at an multiplicity of infection (MOI) of 0.25 or 0.063, but the resistance was overcome when MOI was increased to 1. To determine whether this resistance was due to inhibition of the early events, transduction experiments were performed with a replication-incompetent HIV-1 vector carrying bacterial lacZ driven by an internal Tat-independent cytomegalovirus immediate early promoter. Both cytoplasmic and nuclear expressions of scAb2-19 resulted in decrease in the transduction efficiency on HeLa CD4 cells. This implies that an early step of replication--before or during integration--was affected by the scAb2-19. Furthermore, cytoplasmic expression of scAb2-19 did not affect the viral amount released from the cells transfected with HIV-1 infectious clone DNA (pLAI). However, infectivity relative to reverse transcriptase activity was lower for virions released from the 293T cells cotransfected with pLAI and the cytoplasmic scAb2-19 expression plasmid than for those released from the 293T cells transfected with pLAI alone. This implies that scAb2-19 reduced infectivity of released virions by interfering a late step of the viral replication. The single-chain, antigen-binding peptide molecule may prove useful not only for studies of the functions of IN and its role in the viral life cycle but also for developing a gene therapy strategy against AIDS.

Amino Acid Sequence↗
PubMed2004

Signaling through Toll-like receptors triggers HIV-1 replication in latently infected mast cells.

Evidence that human progenitor mast cells are susceptible to infection with CCR5-tropic strains of HIV-1 and that circulating HIV-1-infected FcepsilonRIalpha(+) cells with a similar progenitor phenotype have been isolated from AIDS patients has led to speculation that mast cells may serve as a potential reservoir for infectious HIV-1. In this study, progenitor mast cells, developed in vitro from CD34(+) cord blood stem cells, were experimentally infected with the CCR5-tropic strain HIV-1Bal after 28 days in culture as they reached their HIV-1-susceptible progenitor stage. HIV-1 p24 Ag levels were readily detectable by day 7 postinfection (PI), peaked at 2-3 wk PI as mature (tryptase/chymase-positive) HIV-1 infection-resistant mast cells emerged, and then steadily declined to below detectable limits by 10 wk PI, at which point integrated HIV-1 proviral DNA was confirmed by PCR quantitation in ( approximately 34% of) latently infected mast cells. Stimulation by ligands for Toll-like receptor (TLR) 2, TLR4, or TLR9 significantly enhanced viral replication in a dose- and time-dependent manner in both HIV-1-infected progenitor and latently infected mature mast cells, without promoting degranulation, apoptosis, cellular proliferation, or dysregulation of TLR agonist-induced cytokine production in infected mast cells. Limiting dilution analysis of TLR activated, latently infected mature mast cells indicated that one in four was capable of establishing productive infections in A301 sentinel cells. Taken together, these results indicate that mast cells may serve both as a viral reservoir and as a model for studying mechanisms of postintegration latency in HIV infection.

Apoptosis↗
PubMed2002

Potassium-dependent folding: a key to intracellular delivery of G-quartet oligonucleotides as HIV inhibitors.

Several groups have demonstrated that G-rich oligonucleotides forming G-quartet structures display activity as potential drugs, such as potent HIV inhibitors. The delivery of G-quartet oligonucleotides to their intracellular targets is a key obstacle to overcome for their clinical success. Here we have developed a novel system to deliver G-rich oligonucleotides into the cell nucleus, e.g., the site of HIV integration. On the basis of the property of potassium-induced formation of G-quartet structure, we explored the difference of K(+) concentrations inside (140 mM) and outside (4 mM) cells to induce the G-rich oligonucleotides to form different structures inside and outside cells. The key steps of this delivery system include the following: (i) First, the G-quartet structure is denatured to form a lipid-DNA complex, so that the molecules can be well delivered into cells. (ii) Then the delivered molecules are induced to form G-quartet structures by potassium inside cells since the G-quartet structure is the primary requirement for inhibition of HIV-1 HIV integrase (IN) activity. The molecules of a novel G-quartet HIV inhibitor, T40214, with the sequence of (GGGC)(4) were successfully delivered into the nuclei of target cells, which significantly decreased HIV-1 replication and increased the probability to target HIV-1 IN in infected cells.

Cell Line↗
PubMed2003

LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1.

We have previously shown correction of X-linked severe combined immunodeficiency [SCID-X1, also known as gamma chain (gamma(c)) deficiency] in 9 out of 10 patients by retrovirus-mediated gamma(c) gene transfer into autologous CD34 bone marrow cells. However, almost 3 years after gene therapy, uncontrolled exponential clonal proliferation of mature T cells (with gammadelta+ or alphabeta+ T cell receptors) has occurred in the two youngest patients. Both patients' clones showed retrovirus vector integration in proximity to the LMO2 proto-oncogene promoter, leading to aberrant transcription and expression of LMO2. Thus, retrovirus vector insertion can trigger deregulated premalignant cell proliferation with unexpected frequency, most likely driven by retrovirus enhancer activity on the LMO2 gene promoter.

Adaptor Proteins, Signal Transducing↗
PubMed2003

[Activating effect of hepatitis B virus preS/S protein on proliferating cell nuclear antigen gene promoter].

BACKGROUND: PreS/S gene was derived from hepatitis B virus (HBV) integration fragment of human hepatocellular carcinoma genome, containing the promoter of preS/S and the C terminal truncated preS/S open reading frame. PreS/S protein may have important roles in processing hepatoma in some HBV-infected patients. The aim of the study was to study the activity of HBV preS/S protein on proliferating cell nuclear antigen (PCNA) promoter and to localize compartment of the preS/S protein in the liver cell line L02. METHODS: The authors studied the effect of the 3 -truncated preS/S on human PCNA promoter by co-transfecting the expression plasmids of luciferase reporter gene, used the immunohistochemical method to localize the preS/S protein. RESULTS: The expression product of the plasmid, pKSH7C-HpaI which contained the 3 -truncated preS/S and the flanking cellular sequences, stimulated the expression of PCNA promoter dose dependently,and its effect was 0.5 folds higher than control. Immunohistochemistry showed that the preS/S protein located in the cytosolic region of the liver cell line L02. CONCLUSIONS: The HBV preS/S protein could stimulate the PCNA promoter of the liver cell, its effect was not direct, which suggests that the effect of preS/S protein on PCNA promoter was probably through the cell signal transduction pathway.

Carcinoma, Hepatocellular↗
PubMed2001

Evidence for a rolling-circle mechanism of phage DNA synthesis from both replicative and integrated forms of CTXphi.

The genes encoding cholera toxin, the principal virulence factor of Vibrio cholerae, are part of the circular single-stranded DNA genome of CTXphi. In toxigenic V. cholerae strains, the CTXphi genome is typically found in integrated arrays of tandemly arranged CTX prophages. Infected cells that lack a chromosomal integration site harbour the CTXphi genome as a plasmid (pCTX). We studied the replication of pCTX and found several indications that this plasmid replicates via a rolling-circle (RC) mechanism. The initiation and termination sites for pCTX plus-strand DNA synthesis were mapped to a 22 bp sequence that contains inverted repeats and a nonanucleotide motif found in the plus-strand origins of several RC replicons. Furthermore, similar to other RC replicons, replication of plasmids containing duplicated pCTX origins resulted in the deletion of sequences between the two origins and the formation of a single chimeric origin. Our previous work revealed that CTX prophage arrays give rise to hybrid CTX virions that contain sequences derived from two adjacent prophages. We now report that the boundaries between the sequences contributed to virions by the upstream and the downstream prophages in an array correspond to the site at which synthesis of plus-strand pCTX DNA is initiated and terminated. These data support the model that plus-strand CTXphi DNA is generated from chromosomal prophages via a novel process analogous to RC replication.

Bacteriophages↗
PubMed1997

Casein kinase II is a selective target of HIV-1 transcriptional inhibitors.

The identification of cellular factors that are required to complete various steps of the HIV-1 life cycle may lead to the development of new therapeutics. One key step, transcription from the integrated provirus, is inhibited by members of two distinct classes of compounds, the flavonoids and the benzothiophenes, via an unknown mechanism, possibly involving a cellular factor. A marked specificity toward inhibiting HIV-1 transcription is evidenced by the ability of drug-treated cells to retain their proliferative and differentiation capabilities. In addition, the compounds do not impede the activation and function of the transcriptional factor NF-kappaB. Here we report on the identification of several cellular proteins that mediate the HIV-1 transcriptional inhibitory property of the flavonoid chrysin. Chemical and immunologic analyses identified these cellular proteins as the individual subunits of casein kinase II (CKII). Though structurally unrelated to chrysin, an HIV-1 inhibitory benzothiophene also bound selectively to CKII. Both chrysin and the benzothiophenes inhibited human recombinant CKII enzymatic activity and showed competitive kinetics with respect to ATP, analogous to the classic CKII inhibitor 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). Moreover, DRB potently inhibited HIV-1 expression in chronically infected cells. CKII may regulate HIV-1 transcription by phosphorylating cellular proteins involved in HIV-1 transactivation that contain multiple CKII phosphorylation consensus sequences.

Anti-HIV Agents↗