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Ben Berkhout

Publications and source records attributed to Ben Berkhout.

At least 19 recordsLinked to original sources
PubMed2025

HIV-phyloTSI: subtype-independent estimation of time since HIV-1 infection for cross-sectional measures of population incidence using deep sequence data.

BACKGROUND: Estimating the time since HIV infection (TSI) at population level is essential for tracking changes in the global HIV epidemic. Most methods for determining TSI give a binary classification of infections as recent or non-recent within a window of several months, and cannot assess the cumulative impact of an intervention. RESULTS: We developed a Random Forest Regression model, HIV-phyloTSI, which combines measures of within-host diversity and divergence to generate continuous TSI estimates directly from viral deep-sequencing data, with no need for additional variables. HIV-phyloTSI provides a continuous measure of TSI up to 9 years, with a mean absolute error of less than 12 months overall and less than 5 months for infections with a TSI of up to a year. It performs equally well for all major HIV subtypes based on data from African and European cohorts. CONCLUSIONS: We demonstrate how HIV-phyloTSI can be used for incidence estimates on a population level.

HIV Infections↗
PubMed2007

In HIV-1 pathogenesis the die is cast during primary infection.

The chronic stage of HIV-1 infection has been extensively described as a slowly evolving phase, in which the virus induces T-cell death slightly faster than the human body is able to recover. In contrast, T-cell and viral replication dynamics during primary infection have been less well studied. Recent studies in the SIV-macaque model and in HIV-positive patients during the acute infection period have highlighted the massive and irreversible depletion of CD4 memory T cells in the mucosa, particularly in the gut. Hence, gut-associated lymphoid tissue (GALT) plays a central role in the early stages of HIV-1 pathogenesis. Due to its particular cytokine expression pattern, GALT may favour the differential replication of certain HIV-1 subtypes during primary infection, particularly of subtype C. This could enhance the chance of a successful transmission. Moreover, these early events taking place in GALT during primary infection have major implications for therapy and vaccine design.

Acute Disease↗
PubMed2006

Human coronavirus 229E encodes a single ORF4 protein between the spike and the envelope genes.

BACKGROUND: The genome of coronaviruses contains structural and non-structural genes, including several so-called accessory genes. All group 1b coronaviruses encode a single accessory protein between the spike and envelope genes, except for human coronavirus (HCoV) 229E. The prototype virus has a split gene, encoding the putative ORF4a and ORF4b proteins. To determine whether primary HCoV-229E isolates exhibit this unusual genome organization, we analyzed the ORF4a/b region of five current clinical isolates from The Netherlands and three early isolates collected at the Common Cold Unit (CCU) in Salisbury, UK. RESULTS: All Dutch isolates were identical in the ORF4a/b region at amino acid level. All CCU isolates are only 98% identical to the Dutch isolates at the nucleotide level, but more closely related to the prototype HCoV-229E (>98%). Remarkably, our analyses revealed that the laboratory adapted, prototype HCoV-229E has a 2-nucleotide deletion in the ORF4a/b region, whereas all clinical isolates carry a single ORF, 660 nt in size, encoding a single protein of 219 amino acids, which is a homologue of the ORF3 proteins encoded by HCoV-NL63 and PEDV. CONCLUSION: Thus, the genome organization of the group 1b coronaviruses HCoV-NL63, PEDV and HCoV-229E is identical. It is possible that extensive culturing of the HCoV-229E laboratory strain resulted in truncation of ORF4. This may indicate that the protein is not essential in cell culture, but the highly conserved amino acid sequence of the ORF4 protein among clinical isolates suggests that the protein plays an important role in vivo.

Aged↗
PubMed2006

Host-related nucleotide composition and codon usage as driving forces in the recent evolution of the Astroviridae.

The evolutionary history of the Astroviridae comprises the ancient separation between avian and mammalian astrovirus lineages followed by diversification among mammalian astroviruses. The latter process included several cross-species transmissions. We found that the recent, but not the ancient, evolution of astroviruses was associated with a switch in nucleotide composition and codon usage among non-human mammalian versus human/avian astroviruses. Virus and hosts phylogenies based on codon usage agreed with each other and matched the hosts' evolutionary emergence order. This recent switch in driving forces acting at the synonymous level points to the adaptation of codon usage by viruses to that of their hosts after cross-species transmissions. This is the first demonstration of nucleotide composition and codon usage being active driving forces during the recent evolutionary history of a virus group in the host-parasite system.

Animals↗
PubMed2006

Core structure of S2 from the human coronavirus NL63 spike glycoprotein.

Human coronavirus NL63 (HCoV-NL63) has recently been identified as a causative agent of acute respiratory tract illnesses in infants and young children. The HCoV-NL63 spike (S) protein mediates virion attachment to cells and subsequent fusion of the viral and cellular membranes. This viral entry process is a primary target for vaccine and drug development. HCoV-NL63 S is expressed as a single-chain glycoprotein and consists of an N-terminal receptor-binding domain (S1) and a C-terminal transmembrane fusion domain (S2). The latter contains two highly conserved heptad-repeat (HR) sequences that are each extended by 14 amino acids relative to those of the SARS coronavirus or the prototypic murine coronavirus, mouse hepatitis virus. Limited proteolysis studies of the HCoV-NL63 S2 fusion core identify an alpha-helical domain composed of a trimer of the HR segments N57 and C42. The crystal structure of this complex reveals three C42 helices entwined in an oblique and antiparallel manner around a central triple-stranded coiled coil formed by three N57 helices. The overall geometry comprises distinctive high-affinity conformations of interacting cross-sectional layers of the six helices. As a result, this structure is unusually stable, with an apparent melting temperature of 78 degrees C in the presence of the denaturant guanidine hydrochloride at 5 M concentration. The extended HR regions may therefore be required to prime the group 1 S glycoproteins for their fusion-activating conformational changes during viral entry. Our results provide an initial basis for understanding an intriguing interplay between the presence or absence of proteolytic maturation among the coronavirus groups and the membrane fusion activity of their S glycoproteins. This study also suggests a potential strategy for the development of improved HCoV-NL63 fusion inhibitors.

Amino Acid Sequence↗
PubMed2006

Second site escape of a T20-dependent HIV-1 variant by a single amino acid change in the CD4 binding region of the envelope glycoprotein.

BACKGROUND: We previously described the selection of a T20-dependent human immunodeficiency virus type-1 (HIV-1) variant in a patient on T20 therapy. The fusion inhibitor T20 targets the viral envelope (Env) protein by blocking a conformational switch that is critical for viral entry into the host cell. T20-dependent viral entry is the result of 2 mutations in Env (GIA-SKY), creating a protein that undergoes a premature conformational switch, and the presence of T20 prevents this premature switch and rescues viral entry. In the present study, we performed 6 independent evolution experiments with the T20-dependent HIV-1 variant in the absence of T20, with the aim to identify second site compensatory changes, which may provide new mechanistic insights into Env function and the T20-dependence mechanism. RESULTS: Escape variants with improved replication capacity appeared within 42 days in 5 evolution cultures. Strikingly, 3 cultures revealed the same single amino acid change in the CD4 binding region of Env (glycine at position 431 substituted for arginine: G431R). This mutation was sufficient to abolish the T20-dependence phenotype and restore viral replication in the absence of T20. The GIA-SKY-G431R escape variant produces an Env protein that exhibits reduced syncytia formation and reduced cell-cell fusion activity. The escape variant was more sensitive to an antibody acting on an early gp41 intermediate, suggesting that the G431R mutation helps preserve a pre-fusion Env conformation, similar to T20 action. The escape variant was also less sensitive to soluble CD4, suggesting a reduced CD4 receptor affinity. CONCLUSION: The forced evolution experiments indicate that the premature conformational switch of the T20-dependent HIV-1 Env variant (GIA-SKY) can be corrected by a second site mutation in Env (GIA-SKY-G431R) that affects the interaction with the CD4 receptor.

Amino Acid Sequence↗
PubMed2006

Modification of the Tet-On regulatory system prevents the conditional-live HIV-1 variant from losing doxycycline-control.

BACKGROUND: We have previously constructed a doxycycline (dox)-dependent HIV-1 variant by incorporating the Tet-On gene regulatory system into the viral genome. Replication of this HIV-rtTA virus is driven by the dox-inducible transactivator protein rtTA, and can be switched on and off at will. We proposed this conditional-live virus as a novel vaccine approach against HIV-1. Upon vaccination, replication of HIV-rtTA can be temporarily activated by transient dox administration and controlled to the extent needed for optimal induction of the immune system. However, subsequent dox-withdrawal may impose a selection for virus variants with reduced dox-dependence. RESULTS: We simulated this on/off switching of virus replication in multiple, independent cultures and could indeed select for HIV-rtTA variants that replicated without dox. Nearly all evolved variants had acquired a typical amino acid substitution at position 56 in the rtTA protein. We developed a novel rtTA variant that blocks this undesired evolutionary route and thus prevents HIV-rtTA from losing dox-control. CONCLUSION: The loss of dox-control observed upon evolution of the dox-dependent HIV-1 variant was effectively blocked by modification of the Tet-On regulatory system.

Binding Sites↗
PubMed2006

Dendritic cell-mediated HIV-1 transmission to T cells of LAD-1 patients is impaired due to the defect in LFA-1.

BACKGROUND: Dendritic cells (DC) have been proposed to mediate sexual HIV-1 transmission by capturing the virus in the mucosa and subsequently presenting it to CD4+ T cells. We have demonstrated before that DC subsets expressing higher levels of intercellular adhesion molecule-1 (ICAM-1) are better HIV-1 transmitters. ICAM-1 binds leukocyte function-associated molecule-1 (LFA-1) on T cells, an integrin responsible for adhesion and signaling at the immunological synapse. To corroborate the importance of the ICAM-1- LFA-1 interaction, we performed transmission experiments to LFA-1 negative leukocytes from Leukocyte Adhesion Deficiency type 1 (LAD-1) patients. RESULTS: We clearly show that DC-mediated HIV-1 transmission to LAD-1 T cells is impaired in comparison to healthy controls. Furthermore, HIV-1 transmission to T cells from a unique LAD-1 patient with a well characterized LFA-1 activation defect was impaired as well, demonstrating that activation of LFA-1 is crucial for efficient transmission. Decreased cell adhesion between DC and LAD-1 T cells could also be illustrated by significantly smaller DC-T cell clusters after HIV-1 transmission. CONCLUSION: By making use of LFA-1 defect cells from unique patients, this study provides more insight into the mechanism of HIV-1 transmission by DC. This may offer new treatment options to reduce sexual transmission of HIV-1.

Adolescent↗
PubMed2006

Mosaic structure of human coronavirus NL63, one thousand years of evolution.

Before the SARS outbreak only two human coronaviruses (HCoV) were known: HCoV-OC43 and HCoV-229E. With the discovery of SARS-CoV in 2003, a third family member was identified. Soon thereafter, we described the fourth human coronavirus (HCoV-NL63), a virus that has spread worldwide and is associated with croup in children. We report here the complete genome sequence of two HCoV-NL63 clinical isolates, designated Amsterdam 57 and Amsterdam 496. The genomes are 27,538 and 27,550 nucleotides long, respectively, and share the same genome organization. We identified two variable regions, one within the 1a and one within the S gene, whereas the 1b and N genes were most conserved. Phylogenetic analysis revealed that HCoV-NL63 genomes have a mosaic structure with multiple recombination sites. Additionally, employing three different algorithms, we assessed the evolutionary rate for the S gene of group Ib coronaviruses to be approximately 3 x 10(-4) substitutions per site per year. Using this evolutionary rate we determined that HCoV-NL63 diverged in the 11th century from its closest relative HCoV-229E.

Algorithms↗
PubMed2006

Construction of doxycyline-dependent mini-HIV-1 variants for the development of a virotherapy against leukemias.

T-cell acute lymphoblastic leukemia (T-ALL) is a high-risk type of blood-cell cancer. We describe the improvement of a candidate therapeutic virus for virotherapy of leukemic cells. Virotherapy is based on the exclusive replication of a virus in leukemic cells, leading to the selective removal of these malignant cells. To improve the safety of such a virus, we constructed an HIV-1 variant that replicates exclusively in the presence 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. This HIV-rtTA virus replicates in a strictly dox-dependent manner. In this virus, additional deletions and/or inactivating mutations were introduced in the genes for accessory proteins. These proteins are essential for virus replication in untransformed cells, but dispensable in leukemic T cells. These minimized HIV-rtTA variants contain up to 7 deletions/inactivating mutations (TAR, Tat, vif, vpR, vpU, nef and U3) and replicate efficiently in the leukemic SupT1 T cell line, but do not replicate in normal peripheral blood mononuclear cells. These virus variants are also able to efficiently remove leukemic cells from a mixed culture with untransformed cells. The therapeutic viruses use CD4 and CXCR4 for cell entry and could potentially be used against CXCR4 expressing malignancies such as T-lymphoblastic leukemia/lymphoma, NK leukemia and some myeloid leukemias.

Anti-Bacterial Agents↗
PubMed2006

Silencing of HIV-1 with RNA interference: a multiple shRNA approach.

Double-stranded RNA can induce gene silencing via a process known as RNA interference (RNAi). Previously, we have shown that stable expression of a single shRNA targeting the HIV-1 Nef gene strongly inhibits HIV-1 replication. However, this was not sufficient to maintain inhibition. One of the hallmarks of RNAi, its sequence specificity, presented a way out for the virus, as single nucleotide substitutions in the target region abolished inhibition. For the development of a durable gene therapy that prevents viral escape, we proposed to combine multiple shRNAs against conserved HIV-1 regions. Therefore, we screened 86 different shRNAs targeting highly conserved regions. We identified multiple shRNAs that act as potent inhibitors of virus replication. We show, for the first time, that expression of three different shRNAs from a single lentiviral vector results in similar levels of inhibition per shRNA compared to single shRNA vectors. Thus, their combined expression results in a much stronger inhibition of virus production. Moreover, when we infected cells transduced with a double shRNA viral vector, virus escape was delayed. These results confirm that RNAi has great potential as an antiviral gene therapy approach and support our efforts to develop this strategy for treatment of HIV-1-infected individuals.

Base Sequence↗
PubMed2006

The virion-associated incoming HIV-1 RNA genome is not targeted by RNA interference.

BACKGROUND: RNA interference (RNAi) has proven to be a powerful tool to suppress gene expression and can be used as a therapeutic strategy against human pathogenic viruses such as human immunodeficiency virus type 1 (HIV-1). Theoretically, RNAi-mediated inhibition can occur at two points in the replication cycle, upon viral entry before reverse transcription of the RNA genome, and on the newly transcribed viral RNA transcripts. There have been conflicting results on whether RNAi can target the RNA genome of infecting HIV-1 particles. We have addressed this issue with HIV-1-based lentiviral vectors. RESULTS: We determined the transduction efficiency of a lentiviral vector, as measured by GFP expressing cells, which reflects the number of successful integration events in a cell line stably expressing shNef. We did not observe a difference in the transduction efficiency comparing lentiviral vectors with or without the Nef target sequence in their genome. The results were similar with particles pseudotyped with either the VSV-G or HIV-1 envelope. Additionally, no reduced transduction efficiencies were observed with multiple other shRNAs targeting the vector genome or with synthetic siNef when transiently transfected prior to transduction. CONCLUSION: Our findings indicate that the incoming HIV-1 RNA genome is not targeted by RNAi, probably due to inaccessibility to the RNAi machinery. Thus, therapeutic RNAi strategies aimed at preventing proviral integration should be targeting cellular receptors or co-factors involved in pre-integration events.

Cloning, Molecular↗
PubMed2006

Differential susceptibility of naïve, central memory and effector memory T cells to dendritic cell-mediated HIV-1 transmission.

BACKGROUND: Dendritic cells (DC) have been proposed to facilitate sexual transmission of HIV-1 by capture of the virus in the mucosa and subsequent transmission to CD4+ T cells. Several T cell subsets can be identified in humans: naïve T cells (TN) that initiate an immune response to new antigens, and memory T cells that respond to previously encountered pathogens. The memory T cell pool comprises central memory (TCM) and effector memory cells (TEM), which are characterized by distinct homing and effector functions. The TEM cell subset, which can be further divided into effector Th1 and Th2 cells, has been shown to be the prime target for viral replication after HIV-1 infection, and is abundantly present in mucosal tissues. RESULTS: We determined the susceptibility of TN, TCM and TEM cells to DC-mediated HIV-1 transmission and found that co-receptor expression on the respective T cell subsets is a decisive factor for transmission. Accordingly, CCR5-using (R5) HIV-1 was most efficiently transmitted to TEM cells, and CXCR4-using (X4) HIV-1 was preferentially transmitted to TN cells. CONCLUSION: The highly efficient R5 transfer to TEM cells suggests that mucosal T cells are an important target for DC-mediated transmission. This may contribute to the initial burst of virus replication that is observed in these cells. TN cells, which are the prime target for DC-mediated X4 virus transmission in our study, are considered to inefficiently support HIV-1 replication. Our results thus indicate that DC may play a decisive role in the susceptibility of TN cells to X4 tropic HIV-1.

CD4-Positive T-Lymphocytes↗
PubMed2006

Identification of alternative amino acid substitutions in drug-resistant variants of the HIV-1 reverse transcriptase.

OBJECTIVE/DESIGN: To identify new drug-resistance-associated mutations in the HIV-1 reverse transcriptase (RT) protein, we screened the RT sequence database of our hospital for alternative amino acid substitutions at known RT drug-resistance positions. METHOD: The genotypic database used for this analysis contained 1322 RT sequences from 1015 patients. We analysed this RT database with a focus on alternative mutations at RT positions known to be involved in drug resistance. The patterns of drug resistance associated with these alternative mutations were investigated in a separate database containing genotype and drug-susceptibility results. RESULTS: We identified multiple alternative resistance-associated mutations at amino acid positions 44, 62, 67, 69, 70, 74, 75, 103, 181, 190, 210, and 219 in RT. Phenotypic analysis indicated that drug-resistance properties of the alternative Y181V and L74I mutants are similar, but not identical, to that of the well-known Y181C and L74V mutations. CONCLUSION: This initial survey indicates that many resistance-associated phenomena can be distilled from existing data. These findings endorse a more extensive analysis by computerized methods.

Anti-HIV Agents↗
PubMed2006

Opposing roles of blood myeloid and plasmacytoid dendritic cells in HIV-1 infection of T cells: transmission facilitation versus replication inhibition.

CD11c(+) myeloid dendritic cells (MDCs) and CD11c(-) CD123(+) plasmacytoid DCs (PDCs) have been identified as main human DC subsets. MDCs are professional antigen-presenting cells for T cells, and include Langerhans cells, dermal DCs, and interstitial DCs. They have been associated with HIV-1 capture and sexual transmission, whereas PDCs play an important role in the innate immune responses to different types of viruses, including HIV-1. To compare the influence of MDCs and PDCs on HIV-1 infection of T cells, we isolated donor-matched MDCs and PDCs from peripheral blood, activated them by adding different maturation-inducing compounds, and cocultured them with T cells and HIV-1. We found that MDCs enhance HIV-1 infection through capture of the virus and subsequent transmission to T cells, and that differently matured MDC subsets have different HIV-1 transmission efficiencies. These differences were not due to soluble factors, viral capture differences, or the expression of integrins ICAM-1, -2, -3, or LFA-1. In contrast, regardless of their state of maturation, PDCs inhibit HIV-1 replication in T cells through the secretion of IFNalpha and an additional, unidentified small molecule. This study shows that the 2 main types of DCs have opposing roles in HIV-1 infection of T cells.

Cell Line↗
PubMed2006

A conditionally replicating HIV-based vector that stably expresses an antiviral shRNA against HIV-1 replication.

Human pathogenic viruses can be targeted by therapeutic strategies based on RNA interference. Whereas the administration of synthetic short interfering RNAs (siRNAs) may transiently inhibit viral replication, long-term inhibition may be achieved through stable intracellular expression of siRNAs or short hairpin RNAs (shRNAs). Both approaches face serious problems with delivery to the right cells in an infected individual. We explored the potential of a replicating HIV-based vector to deliver an antiviral shRNA cassette into HIV-1-susceptible target cells to block chronic HIV-1 infection. The vector is based on a doxycycline (dox)-dependent HIV-1 variant that we previously proposed as a conditional-live HIV-1 vaccine. With dox, this virus spreads efficiently to all HIV-susceptible cells. Subsequent dox withdrawal generates cells with a transcriptionally silent integrated provirus, but with an active shRNA expression cassette. Because the shRNA targets viral sequences that are removed from the vector construct, there is no self-targeting, yet there is specific shutdown of HIV-1 replication.

Cell Line, Tumor↗