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B Berkhout

Publications and source records attributed to B Berkhout.

At least 19 recordsLinked to original sources
PubMed2006

Optimization of the Tet-On system for regulated gene expression through viral evolution.

The ability to control (trans)gene expression is important both for basic biological research and applications such as gene therapy. In vivo use of the inducible tetracycline (Tc)-regulated gene expression system (Tet-On system) is limited by its low sensitivity for the effector doxycycline (dox). We used viral evolution to optimize this Escherichia coli-derived regulatory system for its function in mammalian cells. The components of the Tet-On system (the transcriptional activator rtTA and its tetO DNA binding site) were incorporated into the human immunodeficiency virus (HIV)-1 genome to control viral replication. Prolonged culturing of this HIV-rtTA virus resulted in virus variants that acquired mutations in the rtTA gene. Some of these mutations enhance the transcriptional activity and dox-sensitivity of the rtTA protein. This improvement was observed with different tetO-containing promoters and was independent of the episomal or chromosomal status of the target gene. Combination of these beneficial mutations resulted in greatly improved rtTA variants that are seven-fold more active and 100-fold more dox-sensitive than the original Tet-On system. Furthermore, some of the new Tet-On systems are responsive to Tc and minocycline. Importantly, these rtTA variants show no activity in the absence of dox. The optimized rtTA variants are particularly useful for in vivo applications that require a more sensitive or more active Tet-On system.

Anti-Bacterial Agents↗
PubMed2006

Inhibition of human immunodeficiency virus type 1 by RNA interference using long-hairpin RNA.

Inhibition of virus replication by means of RNA interference has been reported for several important human pathogens, including human immunodeficiency virus type 1 (HIV-1). RNA interference against these pathogens has been accomplished by introduction of virus-specific synthetic small interfering RNAs (siRNAs) or DNA constructs encoding short-hairpin RNAs (shRNAs). Their use as therapeutic antiviral against HIV-1 is limited, because of the emergence of viral escape mutants. In order to solve this durability problem, we tested DNA constructs encoding virus-specific long-hairpin RNAs (lhRNAs) for their ability to inhibit HIV-1 production. Expression of lhRNAs in mammalian cells may result in the synthesis of many siRNAs targeting different viral sequences, thus providing more potent inhibition and reducing the chance of viral escape. The lhRNA constructs were compared with in vitro diced double-stranded RNA and a DNA construct encoding an effective nef-specific shRNA for their ability to inhibit HIV-1 production in cells. Our results show that DNA constructs encoding virus-specific lhRNAs are capable of inhibiting HIV-1 production in a sequence-specific manner, without inducing the class I interferon genes.

Animals↗
PubMed2006

RNA interference: its use as antiviral therapy.

RNA interference (RNAi) is a sequence-specific gene-silencing mechanism that has been proposed to function as a defence mechanism of eukaryotic cells against viruses and transposons. RNAi was first observed in plants in the form of a mysterious immune response to viral pathogens. But RNAi is more than just a response to exogenous genetic material. Small RNAs termed microRNA (miRNA) regulate cellular gene expression programs to control diverse steps in cell development and physiology. The discovery that exogenously delivered short interfering RNA (siRNA) can trigger RNAi in mammalian cells has made it into a powerful technique for generating genetic knock-outs. It also raises the possibility to use RNAi technology as a therapeutic tool against pathogenic viruses. Indeed, inhibition of virus replication has been reported for several human pathogens including human immunodeficiency virus, the hepatitis B and C viruses and influenza virus. We reviewed the field of antiviral RNAi research in 2003 (Haasnoot et al. 2003), but many new studies have recently been published. In this review, we present a complete listing of all antiviral strategies published up to and including December 2004. The latest developments in the RNAi field and their antiviral application are described.

Animals↗
PubMed2006

Immunogenicity and protection of a recombinant human adenovirus serotype 35-based malaria vaccine against Plasmodium yoelii in mice.

Given the promise of recombinant adenovirus type 5 (rAd5) as a malaria vaccine carrier in preclinical models, we evaluated the potency of rAd35 coding for Plasmodium yoelii circumsporozoite protein (rAd35PyCS). We chose rAd35 since a survey with serum samples from African subjects demonstrated that human Ad35 has a much lower seroprevalence of 20% and a much lower geometric mean neutralizing antibody titer (GMT) of 48 compared to Ad5 (seroprevalence, 85%; GMT, 1,261) in countries with a high malaria incidence. We also demonstrated that immunization with rAd35PyCS induced a dose-dependent and potent, CS-specific CD8(+) cellular and humoral immune response and conferred significant inhibition (92 to 94%) of liver infection upon high-dose sporozoite challenge. Furthermore, we showed that in mice carrying neutralizing antibody activity against Ad5, mimicking a human situation, CS-specific T- and B-cell responses were significantly dampened after rAd5PyCS vaccination, resulting in loss of inhibition of liver infection upon sporozoite challenge. In contrast, rAd35 vaccine was as potent in naive mice as in Ad5-preimmunized mice. Finally, we showed that heterologous rAd35-rAd5 prime-boost regimens were more potent than rAd35-rAd35 because of induction of anti-Ad35 antibodies after rAd35 priming. The latter data provide a further rationale for developing rAd prime-boost regimens but indicate that priming and boosting Ad vectors must be immunologically distinct and also should be distinct from Ad5. Collectively, the data presented warrant further development of rAd35-based vaccines against human malaria.

Adenoviruses, Human↗
PubMed2005

Human parechovirus infections in Dutch children and the association between serotype and disease severity.

BACKGROUND: Human parechoviruses (HPeVs) are members of the family Picornaviridae and are classified into 3 known serotypes: HPeV1, HPeV2, and the recently identified HPeV3. HPeV1 and HPeV2 infections are most commonly associated with mild respiratory or gastrointestinal symptoms and occasionally with severe disease conditions, such as flaccid paralysis and encephalitis. HPeV3 infection has been associated with transient paralysis and neonatal infection and has until now only been reported in Japan and Canada. METHODS: Culture isolates considered to be enterovirus on the basis of cell culture but that were found to be enterovirus negative by 5' untranslated region reverse-transcriptase polymerase chain reaction (5'UTR RT-PCR) during the period December 2000 through January 2005 were selected. Isolates were tested by HPeV 5'UTR RT-PCR and were genotyped by sequencing the VP1 region. Phylogenetic analysis was performed, and the association with clinical symptoms was established. RESULTS: Thirty-seven (12%) of the 303 isolates that tested positive for enterovirus by cell culture were in fact HPeV. The majority of the HPeV-positive isolates (n = 27) could be identified as HPeV1. The remaining 10 isolates, which were grown from samples obtained in 2001, 2002, and 2004, could be typed as the recently identified HPeV3. HPeV was exclusively detected in children aged < 3 years. Children infected with HPeV3 were significantly younger than children infected with HPeV1, and sepsis-like illness and central nervous system involvement were more frequently reported in children infected with HPeV3. CONCLUSIONS: We report HPeV infections in young children during the period of 2000-2005 and show an association between HPeV3 infection and sepsis-like illness and central nervous system involvement in neonates.

Central Nervous System Infections↗
PubMed2003

Inhibiting HIV-1 entry with fusion inhibitors.

In recent years, tremendous progress has been made in understanding the HIV-1 entry process in which the viral and cellular membranes are fused, resulting in the subsequent delivery of the viral genome into the host cell. The mechanistic insight gained from these studies has led to the formulation of exciting new approaches for therapeutic intervention. One of the first and clinically most advanced drugs to emerge from this effort is the fusion inhibitor T20. T20 acts by freezing a transient structural intermediate of the HIV-1 fusion process, thus blocking an essential step in viral entry. With phase III clinical trials already well underway, the success of T20 indicates that targeting of the viral entry process will soon be an important component of antiretroviral therapy. This review addresses this rapidly developing area of HIV research, with a focus on the mechanistic role of fusion inhibitors targeted to the HIV-1 gp41 transmembrane glycoprotein. We will review the results of recent clinical trials with T20 and discuss possible mechanisms of viral escape through the evolution of drug-resistant HIV-1 variants. We will also discuss ongoing research on fusion inhibitor susceptibility testing and the development of new improved fusion inhibitors.

Clinical Trials as Topic↗
PubMed2001

The hepatitis B virus X protein induces HIV-1 replication and transcription in synergy with T-cell activation signals: functional roles of NF-kappaB/NF-AT and SP1-binding sites in the HIV-1 long terminal repeat promoter.

Co-infection with hepatitis B virus (HBV) and human immunodeficiency virus type-1 (HIV-1) is relatively common. However, the impact of this co-infection on the clinical outcome of HIV infection has not been elucidated. We herein demonstrate that the HBV X protein (HBx) superinduces ongoing HIV-1 replication and HIV-1 long terminal repeat (LTR) transcription by synergizing with Tat protein and with T-cell activation signals. Although HBx cooperated with mitogenic stimuli in the induction of reporter plasmids harboring the HIV-1 kappaB enhancer, in both a NF-kappaB-dependent manner and a NF-AT-dependent manner, deletion of this element from the LTR did not affect the HBx-mediated up-regulation in the presence of Tat and/or mitogens. In contrast, mutation of the proximal LTR Sp1-binding sites abolished the HBx-mediated synergistic activation, but only when it was accompanied by deletion of the kappaB enhancer. When HBx was targeted to the nucleus, its ability to synergize with cellular activation stimuli was maintained. Furthermore, mutations of HBx affecting its interaction with the basal transcription machinery abrogated the synergistic activation by HBx, suggesting that this protein exerts its function by acting as a nuclear co-activator. These results indicate that HBx could contribute to a faster progression to AIDS in HBV-HIV co-infected individuals.

Base Sequence↗
PubMed2001

Mutations in the TAR hairpin affect the equilibrium between alternative conformations of the HIV-1 leader RNA.

The HIV-1 untranslated leader RNA can adopt two mutually exclusive conformations that represent alternative secondary structures. This leader RNA can fold either an extended duplex through long-distance base pairing or a branched conformation in which the RNA locally folds into hairpin structures. Both leader RNA conformations have the TAR hairpin in common, which forms the extreme 5' end of all HIV-1 transcripts. We report that truncation of the TAR hairpin shifts the equilibrium between the two RNA conformations away from the thermodynamically favored long-distance interaction. However, the equilibrium is partially restored in response to the cations Na(+) and Mg(2+). The transcripts with mutant TAR structures allowed us to investigate conditions affecting the competition between the alternative conformations of the HIV-1 leader RNA. We also demonstrate that the change in conformation of the leader RNA due to TAR truncations severely affects formation of the HIV-1 RNA dimer.

Base Pairing↗
PubMed2001

The dimer initiation site hairpin mediates dimerization of the human immunodeficiency virus, type 2 RNA genome.

The untranslated leader of retroviral RNA genomes encodes multiple structural signals that are critical for virus replication. In the human immunodeficiency virus, type 1 (HIV-1) leader, a hairpin structure with a palindrome-containing loop is termed the dimer initiation site (DIS), because it triggers in vitro RNA dimerization through base pairing of the loop-exposed palindromes (kissing loops). Controversy remains regarding the region responsible for HIV-2 RNA dimerization. Different studies have suggested the involvement of the transactivation region, the primer binding site, and a hairpin structure that is the equivalent of the HIV-1 DIS hairpin. We have performed a detailed mutational analysis of the HIV-2 leader RNA, and we also used antisense oligonucleotides to probe the regions involved in dimerization. Our results unequivocally demonstrate that the DIS hairpin is the main determinant for HIV-2 RNA dimerization. The 6-mer palindrome sequence in the DIS loop is essential for dimer formation. Although the sequence can be replaced by other 6-mer palindromes, motifs that form more than two A/U base pairs do not dimerize efficiently. The inability to form stable kissing-loop complexes precludes formation of dimers with more extended base pairing. Structure probing of the DIS hairpin in the context of the complete HIV-2 leader RNA suggests a 5-base pair elongation of the DIS stem as it is proposed in current RNA secondary structure models. This structure is supported by phylogenetic analysis of leader RNA sequences from different viral isolates, indicating that RNA genome dimerization occurs by a similar mechanism for all members of the human and simian immunodeficiency viruses.

Base Sequence↗
PubMed2001

Initiation of HIV-1 reverse transcription is regulated by a primer activation signal.

Reverse transcription of the human immunodeficiency virus type 1 (HIV-1) RNA genome appears to be strictly regulated at the level of initiation. The primer binding site (PBS), at which the tRNA(3)(Lys) molecule anneals and reverse transcription is initiated, is present in a highly structured region of the untranslated leader RNA. Detailed mutational analysis of the U5 leader stem identified a sequence motif in the U5 region that is critical for activation of the PBS-bound tRNA(3)(Lys) primer. This U5 motif, termed the primer activation signal (PAS), may interact with the TPsiC arm of the tRNA(3)(Lys) primer, similar to the additional interaction proposed for the genome of Rous sarcoma virus and its tRNA(Trp) primer. This suggests that reverse transcription is regulated by a common mechanism in all retroviruses. In HIV-1, the PAS is masked through base pairing in the U5 leader stem. This provides a mechanism for positive and negative regulation of reverse transcription. Based on structure probing of the mutant and wild-type RNAs, an RNA secondary structure model is proposed that juxtaposes the critical PAS and PBS motifs.

5' Untranslated Regions↗
PubMed2001

In vitro evolution of a highly replicating, doxycycline-dependent HIV for applications in vaccine studies.

A major concern associated with the use of vaccines based on live-attenuated viruses is the possible and well documented reversion to pathogenic phenotypes. In the case of HIV, genomic deletions or mutations introduced to attenuate viral pathogenicity can be repaired by selection of compensating mutations. These events lead to increased virus replication rates and, eventually, disease progression. Because replication competence and degree of protection appear to be directly correlated, further attenuation of a vaccine virus may compromise the ability to elicit a protective immune response. Here, we describe an approach toward a safe attenuated HIV vaccine. The system is not based on permanent reduction of infectivity by alteration of important viral genomic sequences, but on strict control of replication through the insertion of the tetracycline (Tet) system in the HIV genome. Furthermore, extensive in vitro evolution was applied to the prototype Tet-controlled HIV to select for variants with optimized rather than diminished replication capacity. The final product of evolution has properties uniquely suited for use as a vaccine strain. The evolved virus is highly infectious, as opposed to a canonically attenuated virus. It replicates efficiently in T cell lines and in activated and unstimulated peripheral blood mononuclear cells. Most importantly, replication is strictly dependent on the nontoxic Tetanalogue doxycycline and can be turned on and off. These results suggest that this in vitro evolved, doxycycline-dependent HIV might represent a useful tool toward the development of a safer, live-attenuated HIV vaccine.

AIDS Vaccines↗
PubMed2001

Evolution of AZT resistance in HIV-1: the 41-70 intermediate that is not observed in vivo has a replication defect.

The human immunodeficiency virus type 1 (HIV-1) is notorious for its ability to evolve drug-resistance in patients treated with potent antivirals. Resistance to inhibitors of the viral reverse transcriptase (RT) enzyme is frequently mediated by a single amino acid substitution within RT. Resistance against the nucleoside analogue AZT is remarkable in that multiple amino acid changes accumulate over time to yield virus variants with high-level drug resistance. We now report that in addition to drug-resistance properties, the relative replication capacity of the virus variants affects the evolution of AZT resistance. Some of the typical AZT-resistance mutations have a negative impact on virus replication, and the 41-70 double mutant was found to represent a particularly poor virus. Furthermore, introduction of additional AZT-resistance mutations (41-70-215) leads to nearly complete restoration of virus replication. These results may explain the absence of the 41-70 double mutant in clinical samples and indicate that the evolution of AZT resistance is also influenced by virus replication parameters. Prolonged passage of the replication-impaired 41-70 virus in the absence of AZT yielded several fast-replicating variants. These revertants have compensatory changes in the RT polymerase, some of which have been observed previously in AZT-treated patients. Because we could select for these changes without drug pressure, these changes are likely to improve the RT enzyme function and the HIV-1 replication capacity.

Anti-HIV Agents↗
PubMed2001

Two alternating structures of the HIV-1 leader RNA.

In this study we demonstrate that the HIV-1 leader RNA exists in two alternative conformations, a branched structure consisting of several well-known hairpin motifs and a more stable structure that is formed by extensive long-distance base pairing. The latter conformation was first identified as a compactly folded RNA that migrates unusually fast in nondenaturing gels. The minimally required domains for formation of this conformer were determined by mutational analysis. The poly(A) and DIS regions of the leader are the major determinants of this RNA conformation. Further biochemical characterization of this conformer revealed that both hairpins are disrupted to allow extensive long-distance base pairing. As the DIS hairpin is known to be instrumental for formation of the HIV-1 RNA dimer, the interplay between formation of the conformer and dimerization was addressed. Formation of the conformer and the RNA dimer are mutually exclusive. Consequently, the conformer must rearrange into a branched structure that exposes the dimer initiation signal (DIS) hairpin, thus triggering formation of the RNA dimer. This structural rearrangement is facilitated by the viral nucleocapsid protein NC. We propose that this structural polymorphism of the HIV-1 leader RNA acts as a molecular switch in the viral replication cycle.

Alternative Splicing↗
PubMed2001

Structural features in the HIV-1 repeat region facilitate strand transfer during reverse transcription.

Two obligatory DNA strand transfers take place during reverse transcription of a retroviral RNA genome. The first strand transfer is facilitated by terminal repeat (R) elements in the viral genome. This strand-transfer reaction depends on base pairing between the cDNA of the 5'R and the 3'R. There is accumulating evidence that retroviral R regions contain features other than sequence complementarity that stimulate this critical nucleic acid hybridization step. The R region of the human immunodeficiency virus type 1 (HIV-1) is relatively extended (97 nt) and encodes two well-conserved stem-loop structures, the TAR and poly(A) hairpins. The role of these motifs was studied in an in vitro strand-transfer assay with two separate templates, the 5'R donor and the 3'R acceptor, and mutants thereof. The results indicate that the upper part of the TAR hairpin structure in the 5'R donor is critical for efficient strand transfer. This seems to pose a paradox, as the 5'R template is degraded by RNase H before strand transfer occurs. We propose that it is not the RNA hairpin motif in the 5'R donor, but rather the antisense motif in the ssDNA copy, which can also fold a hairpin structure, that is critical for strand transfer. Mutation of the loop sequence in the TAR hairpin of the donor RNA, which is copied in the loop of the cDNA hairpin, reduces the transfer efficiency more than fivefold. It is proposed that the natural strand-transfer reaction is enhanced by interaction of the anti-TAR ssDNA hairpin with the TAR hairpin in the 3'R acceptor. Base pairing can occur between the complementary loops ("loop-loop kissing"), and strand transfer is completed by the subsequent formation of an extended RNA-cDNA duplex.

Base Sequence↗
PubMed2001

Strict control of human immunodeficiency virus type 1 replication by a genetic switch: Tet for Tat.

Live-attenuated human immunodeficiency virus type 1 (HIV-1) variants have shown great promise as AIDS vaccines, but continued replication can lead to the selection of faster-replicating variants that are pathogenic. We therefore designed HIV-1 genomes that replicate exclusively upon addition of the nontoxic effector doxycycline (dox). This was achieved by replacement of the viral TAR-Tat system for transcriptional activation by the Escherichia coli-derived Tet system for inducible gene expression. These designer "HIV-rtTA" viruses replicate in a strictly dox-dependent manner both in a T-cell line and in primary blood cells, and the rate of replication can be fine-tuned by simple variation of the dox concentration. These HIV-rtTA viruses provide a tool to perform genetics, e.g., selection and optimization experiments, with the E. coli-derived Tet reagents in a eukaryotic background. Furthermore, such viruses may represent improved vaccine candidates because their replication can be turned on and off at will.

Bacterial Proteins↗
PubMed2001

Repair of a Rev-minus human immunodeficiency virus type 1 mutant by activation of a cryptic splice site.

We isolated a revertant virus after prolonged culturing of a replication-impaired human immunodeficiency virus type 1 (HIV-1) mutant of which the Rev open reading frame was inactivated by mutation of the AUG translation initiation codon. Sequencing of the tat-rev region of this revertant virus identified a second-site mutation in tat that restored virus replication in the mutant background. This mutation activated a cryptic 5' splice site (ss) that, when used in conjunction with the regular HIV 3' ss #5, fuses the tat and rev reading frames to encode a novel T-Rev fusion protein that rescues Rev function. We also demonstrate an alternative route to indirectly activate this cryptic 5' ss by mutational inactivation of an adjacent exon splicing silencer element.

Animals↗