Ben Berkhout

Ben Berkhout
Academisch Medisch Centrum Universiteit van Amsterdam | AMC · Department of Medical Microbiology, Laboratory of Experimental Virology

PhD

About

783
Publications
225,443
Reads
How we measure 'reads'
A 'read' is counted each time someone views a publication summary (such as the title, abstract, and list of authors), clicks on a figure, or views or downloads the full-text. Learn more
34,842
Citations
Additional affiliations
January 2002 - present
Academisch Medisch Centrum Universiteit van Amsterdam
Position
  • Professor of Human Retrovirology, Head of the Laboratory of Experimental Virology
January 1994 - January 2002
Academisch Medisch Centrum Universiteit van Amsterdam
Position
  • Professor (Associate)
January 1991 - January 1993
Academisch Medisch Centrum Universiteit van Amsterdam
Position
  • Professor (Assistant)
Education
January 1979 - January 1986
Leiden University
Field of study

Publications

Publications (783)
Chapter
In recent years, there is an increasing demand for the development of new antiviral strategies due to the prevalence of viral infections such as those caused by the human immunodeficiency (HIV) or the hepatitis B and C viruses (HBV and HCV) and the emergence of a variety of “new” viruses including SARS-CoV-2. The pharmaceutical industry and the sci...
Preprint
Estimating the time since HIV infection (TSI) at population level is essential for tracking changes in the global HIV epidemic. Most methods for determining duration of infection classify samples into recent and non-recent and are unable to give more granular TSI estimates. These binary classifications have a limited recency time window of several...
Article
Full-text available
Background The multiplicity, heterogeneity, and dynamic nature of human immunodeficiency virus type-1 (HIV-1) latency mechanisms are reflected in the current lack of functional cure for HIV-1. Accordingly, all classes of latency-reversing agents (LRAs) have been reported to present variable ex vivo potencies. Here, we investigated the molecular mec...
Article
Full-text available
The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spreads rapidly and harbors many mutations in the spike protein, but the origin of this virus variant remains unclear. We address the role of unusual virus evolution mechanisms such as hypermutation, out-of-frame reading, and recombination. Rather, regular Darwinian...
Preprint
Background CRISPR-Cas technology has revolutionized the use of genome-editing in studies on diverse biological topics. Lentiviral vectors (LVs) have been widely used for the generation of stably transduced cells that durably express the Cas protein and guide RNA (gRNA), the key components of the CRISPR-Cas editing system. This delivery system allow...
Article
Full-text available
Set-point viral load (SPVL), a common measure of human immunodeficiency virus (HIV)-1 virulence, is partially determined by viral genotype. Epidemiological evidence suggests that this viral property has been under stabilising selection, with a typical optimum for the virus between 104 and 105 copies of viral RNA per ml. Here we aimed to detect tran...
Article
Full-text available
The SARS-CoV-2 pandemic has urged the development of protective vaccines and the search for specific antiviral drugs. The modern gene editing toolbox provides alternative methods, such as CRISPR-Cas and RNA interference, that can be adapted as antiviral approaches, and contribute to this search. The unique CRISPR-Cas13d system, with the small crRNA...
Article
We discovered a highly virulent variant of subtype-B HIV-1 in the Netherlands. One hundred nine individuals with this variant had a 0.54 to 0.74 log10 increase (i.e., a ~3.5-fold to 5.5-fold increase) in viral load compared with, and exhibited CD4 cell decline twice as fast as, 6604 individuals with other subtype-B strains. Without treatment, advan...
Article
Full-text available
CRISPR-Cas12a is an alternative class 2 gene-editing tool that may cause less off-target effects than the original Cas9 system. We have previously demonstrated that Cas12a attack with a single CRISPR RNA (crRNA) can neutralize all infectious HIV in an infected T cell line in cell culture. However, we demonstrated that HIV escapes from most crRNAs b...
Article
Full-text available
Novel therapeutic strategies aiming at the permanent inactivation of the HIV-1 reservoir in infected individuals are currently being explored, including approaches based on CRISPR-Cas gene editing. Extinction of all infectious HIV provirus in infected T-cell cultures was previously achieved when cells were transduced with lentiviral vectors for the...
Article
With interest we read the Letter to the Editor of Wei and Sluis-Cremer about the role of human immunodeficiency virus 1 (HIV-1) 3’polypurine tract (3’PPT) mutations in dolutegravir (DTG) resistance 1.…
Article
Full-text available
Adult T-cell leukemia/lymphoma (ATLL) is an aggressive malignancy of CD4+ T-cells associated with HTLV-1 infection. In this study, we used the model of immunodeficient NSG mice reconstituted with a functional human immune system (HIS) to investigate early events in HTLV-1 pathogenesis. Upon infection, human T-cells rapidly increased in the blood an...
Article
Full-text available
Antiretroviral therapy (ART) suppresses HIV-1 replication but does not eradicate the virus. Persistence of HIV-1 latent reservoirs in ART-treated individuals is considered the main obstacle to achieving an HIV-1 cure. However, these HIV-1 reservoirs are not transcriptionally silent, and viral transcripts can be detected in most ART-treated individu...
Article
Full-text available
BACKGROUND: It remains unclear whether combination antiretroviral therapy (ART) regimens differ in their ability to fully suppress HIV replication. Here, we report the results of two cross-sectional studies that compared levels of cell-associated (CA) HIV markers between individuals receiving suppressive ART containing either a non-nucleoside rever...
Article
Full-text available
The highly conserved trans-acting response element (TAR) present in the RNA genome of human immunodeficiency virus 1 (HIV-1) is a stably folded hairpin structure involved in viral replication. However, TAR is also sensed by viral sensors, leading to antiviral immunity. While high variation in the TAR RNA structure renders the virus replication-inco...
Preprint
Full-text available
The multiplicity, heterogeneity and dynamic nature of HIV-1 latency mechanisms are reflected in the current lack of functional cure for HIV-1 and in the various reported ex vivo potencies of latency-reversing agents. Here, we investigated the molecular mechanisms underlying the potency of the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-Azad...
Article
Full-text available
Purpose of review Despite decades of suppressive antiretroviral therapy (ART), HIV-1 reservoirs persist and fuel viral rebound if therapy is interrupted. The persistence of viral reservoirs in infected individuals is the main obstacle to achieving HIV-1 eradication or a long-term remission. Accurate assessment of the viral reservoir size is necess...
Preprint
Full-text available
BACKGROUND It remains unclear whether combination antiretroviral therapy (ART) regimens differ in their ability to fully suppress HIV replication. Here, we report the results of two cross-sectional studies that compared levels of cell-associated (CA) HIV markers between individuals receiving suppressive ART containing either a non-nucleoside revers...
Article
Full-text available
Incomplete restoration of CD4 ⁺ T-cell counts on antiretroviral therapy (ART) is a major predictor of HIV-related morbidity and mortality. To understand the possible mechanisms behind this poor immunological response despite viral suppression, we longitudinally measured more than 50 virological and immunological biomarkers in a cohort of HIV-infect...
Article
Full-text available
Combination antiretroviral therapy (ART) suppresses human immunodeficiency virus (HIV) replication and improves immune function. However, due to the persistence of long-lived HIV reservoirs, therapy interruption almost inevitably leads to a fast viral rebound. A small percentage of individuals who are able to control HIV replication for extended pe...
Chapter
Since the first application of RNA interference (RNAi) in mammalian cells, the expression of short hairpin RNA (shRNA) molecules for targeted gene silencing has become a benchmark technology. Plasmid and viral vector systems can be used to express shRNA precursor transcripts that are processed by the cellular RNAi pathway to trigger sequence-specif...
Chapter
The recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)-Cpf1 system, now reclassified as Cas12a, is a DNA-editing platform analogous to the widely used CRISPR-Cas9 system. The Cas12a system exhibits several distinct features over the CRISPR-Cas9 system, such as increased specificity and a smaller gene size to enco...
Article
Full-text available
The unspliced HIV-1 full-length RNA (HIV-1 RNA) is packaged into virions as the genome and is translated to generate viral structural proteins and enzymes. To serve these functions, HIV-1 RNA must be exported from the nucleus to the cytoplasm. It was recently suggested that export pathways used by HIV-1 RNA could affect its cytoplasmic transport me...
Article
Full-text available
RNA polymerase III promoters such as 7SK, U6 and H1 are widely used for the expression of small non-coding RNAs, including short hairpin RNAs for RNAi experiments and guide RNAs for CRISPR-mediated genome editing. We previously reported dual RNA polymerase activity (Pol II/III) for the human H1 promoter and demonstrated that this promiscuous RNA po...
Article
Full-text available
Studies of vertebrate genomes have indicated that all species contain in their chromosomes stretches of DNA with sequence similarity to viral genomes. How such ‘endogenous’ viral elements (EVEs) ended up in host genomes is usually explained in general terms such as ‘they entered the germ line at some point during evolution’. This seems a correct st...
Article
Full-text available
In adherent individuals, antiretroviral therapy (ART) suppresses HIV replication, restores immune function, and prevents the development of AIDS. However, ART is not curative and has to be followed lifelong. Persistence of viral reservoirs forms the major obstacle to an HIV cure. HIV latent reservoirs persist primarily by cell longevity and prolife...
Article
Full-text available
The CRISPR-Cas9 system has been used for genome editing of various organisms. We reported inhibition of the human immunodeficiency virus (HIV) in cell culture infections with a single guide RNA (gRNA) and subsequent viral escape, but complete inactivation of infectious HIV with certain combinations of two gRNAs. The new RNA-guided endonuclease syst...
Article
Full-text available
Although several studies demonstrated that the HIV proviral DNA can be effectively targeted and inactivated by the CRISPR-Cas9 system, the precise inactivation mechanism has not yet been analyzed. Whereas some studies suggested efficient proviral DNA excision upon dual-gRNA/Cas9 treatment, we previously demonstrated that hypermutation of the target...
Article
Full-text available
Plasma viral load (VL) and CD4+ T-cell count are widely used as biomarkers of HIV-1 replication, pathogenesis, and response to antiretroviral therapy (ART). However, the clinical potential of cell-associated (CA) HIV-1 molecular markers is much less understood. Here, we measured CA HIV-1 RNA and DNA in HIV-infected individuals treated with temporar...
Article
Full-text available
The HIV latent reservoir forms the major hurdle to an HIV cure. The discovery of CD32 as marker of this reservoir has aroused much interest, but subsequent reports have challenged this finding. Here, we observe a positive correlation between the percentages of CD32+ cells among CD4+ T cells of aviremic cART-treated, HIV-infected individuals and the...
Article
Full-text available
Recent studies have shown the potential of broadly neutralizing antibodies (bnAbs) for HIV-1 treatment. One of the candidate antibodies moving into clinical trials is the bnAb PGDM1400. Here, we studied the therapeutic potency and escape pathways of bnAb PGDM1400 during monovalent therapy in human immune system (HIS) mice using the BG505, REJO, MJ4...
Article
Full-text available
Abstract Antiretroviral therapy (ART) can effectively suppress ongoing HIV replication and block disease progression, but the infection is never cured due to the persistence of a small pool of latently infected cells hosting integrated replication-competent HIV proviruses. However, the vast majority of HIV proviruses in ART-treated patients are rep...
Article
Full-text available
Tools based on RNA interference (RNAi) and the recently developed clustered regularly short palindromic repeats (CRISPR) system enable the selective modification of gene expression, which also makes them attractive therapeutic reagents for combating HIV infection and other infectious diseases. Several parallels can be drawn between the RNAi and CRI...
Article
Full-text available
The lack of endogenous RNAi machinery in the malaria parasite Plasmodium hampers gene annotation and hence antimalarial drug and vaccine development. Here, we engineered rodent Plasmodium berghei to express a minimal, non-canonical RNAi machinery that solely requires Argonaute 2 (Ago2) and a modified short hairpin RNA, so-called AgoshRNA. Using thi...
Article
Full-text available
Current antiretroviral drugs can efficiently block HIV replication and prevent transmission, but do not target the HIV provirus residing in cells that constitute the viral reservoir. Because drug therapy interruption will cause viral rebound from this reservoir, HIV-infected individuals face lifelong treatment. Therefore, novel therapeutic strategi...
Article
Full-text available
Combination antiretroviral therapy (ART) suppresses human immunodeficiency virus (HIV) replication and improves immune function, but is unable to eradicate the virus. Therefore, development of an HIV cure has become one of the main priorities of the HIV research field. The main obstacle for an HIV cure is the formation of latent viral reservoirs, w...
Article
The interferon γ-inducible protein 16 (IFI16) is known as immune sensor of retroviral DNA intermediates. We show that IFI16 restricts HIV-1 independently of immune sensing by binding and inhibiting the host transcription factor Sp1 that drives viral gene expression. This antiretroviral activity and ability to bind Sp1 require the N-terminal pyrin d...
Article
The human immunodeficiency virus type-1 (HIV-1) establishes a state of latent infection in a small number of CD4+ T lymphocytes that, nonetheless, represent a major obstacle to viral eradication. We here show that Tripartite Motif-containing protein 22 (TRIM22), an epigenetic inhibitor of Specificity protein 1 (Sp1)-dependent HIV-1 transcription, i...
Article
Full-text available
RNA interference (RNAi) can be triggered by synthetic small interfering RNAs (siRNAs) or transgene-expressed short hairpin RNAs (shRNAs). Recent evidence indicates that shRNA molecules, with a relatively short stem and small loop, are processed by Argonaute 2 protein (Ago2). We named these molecules AgoshRNA as Ago2 is involved in both the processi...
Article
Full-text available
Short hairpin RNAs (shRNAs)can induce gene silencing via the RNA interference (RNAi)mechanism. We designed an alternative shRNA molecule with a relatively short base-paired stem that bypasses Dicer and instead is processed by the Argonaute 2 (Ago2)protein into a single guide RNA strand that effectively induces RNAi. We called these molecules AgoshR...
Article
Full-text available
The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is widely explored for sequence-specific attack on HIV-1 proviral DNA. We recently identified dual-guide RNA (dual-gRNA) combinations that can block HIV-1 replication permanently in infected cell cultures and prevent viral escape. Although the gRNAs were designed to...
Article
Full-text available
Background: The latent HIV-1 reservoir in treated patients primarily consists of resting memory CD4+ T cells. Stimulating the T-cell receptor (TCR), which facilitates transition of resting into effector T cells, is the most effective strategy to purge these latently infected cells. Here we supply evidence that TCR-stimulated effector T cells still...
Article
Full-text available
HIV-1 entry into cells is mediated by the envelope glycoprotein (Env) and represents an attractive target for therapeutic intervention. Two drugs that inhibit HIV entry are approved for clinical use: the membrane fusion inhibitor T20 (Fuzeon, enfuvirtide) and theC-C chemokine receptor type 5 (CCR5) blocker maraviroc (Selzentry). Another class of en...
Article
Full-text available
Cell-associated (CA) HIV RNA has received much attention in recent years as a surrogate measure of the efficiency of HIV latency reversion and because it may provide an estimate of the viral reservoir size. This review provides an update on some recent insights in the biology and clinical utility of this biomarker. We discuss a number of important...
Article
Full-text available
The recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)-Cpf1 system expands the genome editing toolbox. This system exhibits several distinct features compared to the widely used CRISPR-Cas9 system, but has reduced gene editing efficiency. To optimize the CRISPR-Cpf1 (Cas12a) system, we report the inclusion of sel...
Article
Full-text available
The life cycle of HIV-1 requires integration of a DNA copy into the genome of the host cell. Transcription of the viral genes generates RNAs that are exported to the cytoplasm with the contribution of viral and cellular factors to get translated or incorporated in the newly synthesized virions. It has been observed that highly effective antiretrovi...
Article
Full-text available
The RNA-guided endonuclease Cas9 (CRISPR-Cas9) genome editing system has been widely used for biomedical research and holds great potential for therapeutic applications in eukaryotes. The conventional vector-based CRISPR-Cas9 delivery system requires two different RNA polymerase promoters for expression of the guide RNA (gRNA) and Cas9 endonuclease...
Article
Full-text available
Nucleotide skew analysis is a versatile method to study the nucleotide composition of RNA/DNA molecules, in particular to reveal characteristic sequence signatures. For instance, skew analysis of the nucleotide bias of several viral RNA genomes indicated that it is enriched in the unpaired, single-stranded genome regions, thus creating an even more...
Article
Full-text available
Background & Aims Despite high risk behavior, 10‐20% of HCV multiple exposed individuals remain uninfected (MEU), whilst the remainder become infected (MEI). We hypothesize that host factors play a role in HCV susceptibility. We aimed to identify polymorphisms in host genes that encode for proteins involved in viral entry; CD81, Scavenger receptor...
Article
RNA interference (RNAi) was discovered in plants where it functions as the main antiviral pathway and this antiviral role was subsequently extended to invertebrates. But it remained hotly debated whether RNAi fulfils a similar role in mammals that already have a potent innate immune system based on interferon and an elaborate adaptive immune system...
Article
Full-text available
Broadly neutralizing antibodies (bNAbs) such as PGDM1400 show promise as prophylactic and therapeutic agents against HIV-1. Human immune system (HIS) mice were passively immunized with different doses of PGDM1400 and challenged 24 hours later with a high dose of HIV-1JRCSF. We found that PGDM1400 provided protection against HIV-1 challenge in a con...
Article
Full-text available
Studying the evolution of viruses and their molecular epidemiology relies on accurate viral sequence data, so that small differences between similar viruses can be meaningfully interpreted. Despite its higher throughput and more detailed minority variant data, next-generation sequencing has yet to be widely adopted for HIV. The difficulty of accura...
Article
Transcription of the HIV-1 proviral DNA and subsequent processing of the primary transcript results in the production of a large set of unspliced and differentially spliced viral RNAs. The major splice donor site (5'ss) that is located in the untranslated leader of the HIV-1 transcript is used for the production of all spliced RNAs and splicing at...
Article
Full-text available
In eukaryotes, three RNA polymerases (Pol I, II, and III) are responsible for the transcription of distinct subsets of genes. Gene-external type 3 Pol III promoters use defined transcription start and termination sites, and they are, therefore, widely used for small RNA expression, including short hairpin RNAs in RNAi applications and guide RNAs in...
Article
Full-text available
For the production of viral genomic RNA, HIV-1 is dependent on an early viral protein, Tat, which is required for high-level transcription. The quantity of viral RNA detectable in blood of HIV-1 infected individuals varies dramatically, and a factor involved could be the efficiency of Tat protein variants to stimulate RNA transcription. HIV-1 virul...
Article
Full-text available
Jan Svoboda studied aspects of viral latency, in particular with respect to disease induction by avian RNA tumor viruses, which were later renamed as part of the extended retrovirus family. The course of retroviral pathogenesis is intrinsically linked to their unique property of integrating the DNA copy of the retroviral genome into that of the hos...
Chapter
We describe a detailed protocol for the manual workup of blood (plasma/serum) samples from individuals infecte