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Biomedical subjects

Ben Berkhout

Publications and source records attributed to Ben Berkhout.

At least 73 records · Page 4Linked to original sources
PubMed2005

Alternative tRNA priming of human immunodeficiency virus type 1 reverse transcription explains sequence variation in the primer-binding site that has been attributed to APOBEC3G activity.

It is generally assumed that human immunodeficiency virus type 1 (HIV-1) uses exclusively the cellular tRNA(3)(Lys) molecule as a primer for reverse transcription. We demonstrate that HIV-1 uses not only tRNA(3)(Lys) but also an alternative tRNA primer. This tRNA was termed tRNA(5)(Lys), and the near completion of the human genome project has allowed the identification of four tRNA(5)(Lys)encoding genes. Priming with tRNA(5)(Lys) results in a single nucleotide polymorphism in the viral primer-binding site that is present in multiple natural and laboratory HIV isolates. This sequence variation was recently attributed to APOBEC3G activity. However, our results show that alternative tRNA priming can cause this mutation in the absence of APOBEC3G.

APOBEC-3G Deaminase↗
PubMed2005

Increased multinucleoside drug resistance and decreased replicative capacity of a human immunodeficiency virus type 1 variant with an 8-amino-Acid insert in the reverse transcriptase.

Resistance to antiretroviral drugs is generally conferred by specific amino acid substitutions, rather than insertions or deletions, in reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). The exception to these findings is the amino acid insertions found in the beta3-beta4 loop of the RT enzyme in response to treatment with nucleoside reverse transcriptase inhibitors. This insert consists most commonly of two amino acids, but we describe in detail the evolution of a variant with an 8-amino-acid (aa) insert in a patient treated with zidovudine (ZDV) and 2'-3'-dideoxycytidine (ddC). The 24-nucleotide insert is a partial duplication of local sequences but also contains a sequence segment of unknown origin. Extensive sequence analysis of longitudinal patient samples indicated that the HIV-1 population prior to the start of therapy contained not the wild-type amino acid 215T in RT but a mixture with 215D and 215C. Treatment with ZDV and subsequent ZDV-ddC combination therapy resulted in the evolution of an HIV-1 variant with a typical ZDV resistance genotype (41L, 44D, 67N, 69D, 210W, 215Y), which was slowly replaced by the insert-containing variant (41L, 44D, insert at position 69, 70R, 210W, 215Y). The latter variant demonstrated increased resistance to a wide range of drugs, indicating that the 8-aa insert augments nucleoside analogue resistance. The gain in drug resistance of the insert variant came at the expense of a reduction in replication capacity when assayed in the absence of drugs. We compared these data with the resistance and replication properties of 133 insert-containing sequences of different individuals present in the ViroLogic database and found that the size and actual sequence of the insert at position 69 influence the level of resistance to nucleoside analogues.

Adult↗
PubMed2005

Improving the safety of a conditional-live human immunodeficiency virus type 1 vaccine by controlling both gene expression and cell entry.

Live attenuated human immunodeficiency virus type 1 (HIV-1) vaccines are considered unsafe because faster-replicating pathogenic virus variants may evolve after vaccination. We previously presented a conditional-live HIV-1 variant of which replication can be switched off as an alternative vaccination strategy. To improve the safety of such a vaccine, we constructed a new HIV-1 variant that depends not only on doxycycline for gene expression but also on the T20 peptide for cell entry. Replication of this virus can be limited to the level required to induce the immune system by transient administration of doxycycline and T20. Subsequent withdrawal of these inducers efficiently blocks viral replication and evolution.

AIDS Vaccines↗
PubMed2005

High plasma HIV load in the CRF01-AE outbreak among injecting drug users in Finland.

An explosive outbreak of HIV-1 caused by the recombinant subtype AE (CRF01-AE) was detected in 1998 among Finnish injecting drug users (IDUs). These IDUs were compared with IDUs from the Amsterdam Cohort Study (ACS) infected with subtype B, to detect possible differences between 2 western IDU cohorts infected with different subtypes. Markers for progression (viral load and CD4+lymphocyte count) were compared between 93 IDUs with CRF01-AE and 63 IDUs with subtype B. Only persons with a seroconversion interval =2 y were included. During 48 months of follow-up, both cohorts were similar in CD4+ cell decline, but the Finnish IDUs had 0.34-0.94 log10 copies/ml higher viral loads. The Amsterdam IDUs had a low viral load (<1000 copies/ml) significantly more often than the Finnish IDUs. The difference could not be explained by the use of antiretrovirals. The higher viral load may have contributed to the rapid spread of the recombinant virus in the Finnish outbreak.

Adolescent↗
PubMed2004

Genome structure and transcriptional regulation of human coronavirus NL63.

BACKGROUND: Two human coronaviruses are known since the 1960s: HCoV-229E and HCoV-OC43. SARS-CoV was discovered in the early spring of 2003, followed by the identification of HCoV-NL63, the fourth member of the coronaviridae family that infects humans. In this study, we describe the genome structure and the transcription strategy of HCoV-NL63 by experimental analysis of the viral subgenomic mRNAs. RESULTS: The genome of HCoV-NL63 has the following gene order: 1a-1b-S-ORF3-E-M-N. The GC content of the HCoV-NL63 genome is extremely low (34%) compared to other coronaviruses, and we therefore performed additional analysis of the nucleotide composition. Overall, the RNA genome is very low in C and high in U, and this is also reflected in the codon usage. Inspection of the nucleotide composition along the genome indicates that the C-count increases significantly in the last one-third of the genome at the expense of U and G. We document the production of subgenomic (sg) mRNAs coding for the S, ORF3, E, M and N proteins. We did not detect any additional sg mRNA. Furthermore, we sequenced the 5' end of all sg mRNAs, confirming the presence of an identical leader sequence in each sg mRNA. Northern blot analysis indicated that the expression level among the sg mRNAs differs significantly, with the sg mRNA encoding nucleocapsid (N) being the most abundant. CONCLUSIONS: The presented data give insight into the viral evolution and mutational patterns in coronaviral genome. Furthermore our data show that HCoV-NL63 employs the discontinuous replication strategy with generation of subgenomic mRNAs during the (-) strand synthesis. Because HCoV-NL63 has a low pathogenicity and is able to grow easily in cell culture, this virus can be a powerful tool to study SARS coronavirus pathogenesis.

Amino Acid Sequence↗
PubMed2004

SIVdrl detection in captive mandrills: are mandrills infected with a third strain of simian immunodeficiency virus?

A pol-fragment of simian immunodeficiency virus (SIV) that is highly related to SIVdrl-pol from drill monkeys (Mandrillus leucophaeus) was detected in two mandrills (Mandrillus sphinx) from Amsterdam Zoo. These captivity-born mandrills had never been in contact with drill monkeys, and were unlikely to be hybrids. Their mitochondrial haplotype suggested that they descended from founder animals in Cameroon or northern Gabon, close to the habitat of the drill. SIVdrl has once before been found in a wild-caught mandrill from the same region, indicating that mandrills are naturally infected with a SIVdrl-like virus. This suggests that mandrills are the first primate species to be infected with three strains of SIV: SIVmnd1, SIVmnd2, and SIVdrl.

Animals↗
PubMed2004

Depletion of CD4 T lymphocytes in human lymphoid tissue infected ex vivo with doxycycline-dependent HIV-1.

We investigated whether CD4+ T cells that do not produce HIV-1 are killed in HIV-infected human lymphoid tissue. Tissue blocks were inoculated with high amount of doxycycline-dependent HIV-rtTA. Doxycycline triggered productive infection and loss of CD4+ T cells in these tissues, whereas without doxycycline, neither productive infection nor CD4+ T cell depletion was detected in spite of the massive presence of virions in the tissue and of viral DNA in the cells. Thus, HIV-1 alone is sufficient to deplete productively infected CD4+ T cells but is not sufficient to cause the death of uninfected or latently infected CD4+ T cells.

Anti-Infective Agents↗
PubMed2004

T-cell activation leads to poor activation of the HIV-1 clade E long terminal repeat and weak association of nuclear factor-kappaB and NFAT with its enhancer region.

The enhancer region in the human immunodeficiency virus type 1 (HIV-1) 5'-long terminal repeat (LTR) is very important for viral transcription. This promoter sequence binds both nuclear factor-kappaB and NFAT, two important modulators of HIV-1 gene expression. Previous studies have indicated that the enhancer regions of the different HIV-1 clade LTRs differ in their number of NF-kappaB-binding sites. In this study, we have compared the activation potential of the different HIV-1 clade and HIV-2 LTRs and assessed their interaction with NFAT and NF-kappaB. In T-cell lines and primary CD4(+) T-cells, the results showed that the HIV-1 clade E LTR (with a single NF-kappaB-binding site) was the weakest LTR regardless of the tested activators, whereas the HIV-2 LTR was the most responsive LTR. The clade E enhancer region was also demonstrated to be the weakest enhancer region in transfection experiments with luciferase reporter-based vectors. Electrophoretic mobility shift assays with extracts from activated CD4(+) T-cells indicated that, although NF-kappaB and NFAT bound all enhancers, HIV-1 clade E and HIV-2 LTR enhancers were poor binding targets for these two factors. Weak NFAT binding to clade E enhancers was also confirmed using NFAT1-expressing 293T cells in competition experiments. We have also shown the absence of interaction of NF-kappaB or NFAT with the third NF-kappaB repeat present in clade C. However, the clade C enhancer bound NFAT more efficiently than all other enhancer regions tested. Our results hence demonstrate for the first time that differences in the binding of NF-kappaB and NFAT to the enhancer regions could be responsible for some of the observed variation in HIV-1 clade LTR activation, whereas HIV-2 LTR activation seems mostly independent of these interactions.

Binding Sites↗
PubMed2004

Declining trend in transmission of drug-resistant HIV-1 in Amsterdam.

OBJECTIVE: Symptomatic primary HIV infections are over-represented in the mainly hospital-based studies on transmission of resistant HIV-1. We examined a more general population for the prevalence of resistant HIV-1 strains among primary infections. DESIGN: From 1994 to 2002 primary infections were identified within the Amsterdam Cohort Studies (ACS) among homosexual men and drug users, and at the Academic Medical Center (AMC). Whereas primary HIV-1-infected AMC patients, often presented with symptoms of acute retroviral syndrome, ACS participants largely seroconverted during follow-up and thus brought also asymptomatic primary infections to our study. METHODS: Reverse transcriptase (RT) and protease sequences were obtained by population-based nucleotide sequence analysis of the first HIV RNA-positive sample available. Subtypes were identified by phylogenetic analysis. Mutations were identified based on the IAS-USA resistance table. RESULTS: A total of 100 primary HIV-1 infections were identified (32 AMC and 68 ACS). Transmission of drug-resistant strains decreased over calendar time, with 20% [95% confidence interval (CI), 10-34%] of infections bearing drug-resistant mutations before 1998 versus only 6% (95% CI, 1-17%) after 1998. No multi-drug resistance pattern was observed. The median plasma HIV-1 RNA level of the first RNA positive sample was significantly lower for the individuals infected with a resistant strain versus those infected with wild-type, suggesting a fitness-cost to resistance. Four of seven non-B subtypes corresponded with the prevalent subtype in the presumed country of infection, and none showed resistance mutations. CONCLUSIONS: The transmission of drug-resistant HIV-1 strains in Amsterdam has decreased over time. Monitoring should be continued as this trend might change.

Adult↗
PubMed2004

Adenovirus types 5 and 35 seroprevalence in AIDS risk groups supports type 35 as a vaccine vector.

The seroprevalence of adenovirus types 5 (Ad5) and 35 (Ad35) was investigated in patients at risk of AIDS. The seroprevalence of Ad5 was higher than Ad35 in HIV-infected patients from The Netherlands (60% versus 7%) and sub-Saharan Africa (90% versus 20%). The seroprevalence was similar among HIV-infected and uninfected individuals, and remained constant during progression to AIDS. Ad35 is less prone to neutralization than Ad5, encouraging the further development of Ad35 for vaccination against HIV.

Acquired Immunodeficiency Syndrome↗
PubMed2004

Identification of a new human coronavirus.

Three human coronaviruses are known to exist: human coronavirus 229E (HCoV-229E), HCoV-OC43 and severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV). Here we report the identification of a fourth human coronavirus, HCoV-NL63, using a new method of virus discovery. The virus was isolated from a 7-month-old child suffering from bronchiolitis and conjunctivitis. The complete genome sequence indicates that this virus is not a recombinant, but rather a new group 1 coronavirus. The in vitro host cell range of HCoV-NL63 is notable because it replicates on tertiary monkey kidney cells and the monkey kidney LLC-MK2 cell line. The viral genome contains distinctive features, including a unique N-terminal fragment within the spike protein. Screening of clinical specimens from individuals suffering from respiratory illness identified seven additional HCoV-NL63-infected individuals, indicating that the virus was widely spread within the human population.

Adult↗
PubMed2004

Evolution of the HIV-1 envelope glycoproteins with a disulfide bond between gp120 and gp41.

BACKGROUND: We previously described the construction of an HIV-1 envelope glycoprotein complex (Env) that is stabilized by an engineered intermolecular disulfide bond (SOS) between gp120 and gp41. The modified Env protein antigenically mimics the functional wild-type Env complex. Here, we explore the effects of the covalent gp120 - gp41 interaction on virus replication and evolution. RESULTS: An HIV-1 molecular clone containing the SOS Env gene was only minimally replication competent, suggesting that the engineered disulfide bond substantially impaired Env function. However, virus evolution occurred in cell culture infections, and it eventually always led to elimination of the intermolecular disulfide bond. In the course of these evolution studies, we identified additional and unusual second-site reversions within gp41. CONCLUSIONS: These evolution paths highlight residues that play an important role in the interaction between gp120 and gp41. Furthermore, our results suggest that a covalent gp120 - gp41 interaction is incompatible with HIV-1 Env function, probably because this impedes conformational changes that are necessary for fusion to occur, which may involve the complete dissociation of gp120 from gp41.

Amino Acid Substitution↗
PubMed2004

Probing alternative foldings of the HIV-1 leader RNA by antisense oligonucleotide scanning arrays.

Scanning arrays of antisense DNA oligonucleotides provide a novel and systematic means to study structural features within an RNA molecule. We used this approach to probe the structure of the untranslated leader of the human immunodeficiency virus type 1 (HIV-1) RNA genome. This 335 nt RNA encodes multiple important replication signals and adopts two mutually exclusive conformations. The poly(A) and the dimer initiation signal (DIS) sequences of the leader RNA are base-paired in the long-distance interaction (LDI) conformation, but both domains form distinct hairpins in the branched multiple hairpins (BMH) conformation. An RNA switch mechanism has been proposed to regulate the activity of the DIS dimerization signal that is masked in one, yet exposed in the other conformation. The two RNA conformations demonstrate discrete differences in the array-based hybridization patterns. LDI shows increased hybridization in the poly(A) region and decreased hybridization in the DIS region when compared with BMH. These results provide additional evidence for the structure models of the two alternative leader RNA conformations. We also found a correlation between the efficiency of oligonucleotide hybridization and the accessibility of the RNA structure as determined by chemical and enzymatic probing in previous studies. The array approach therefore provides a very sensitive method to detect structural differences in related transcripts.

Base Pairing↗
PubMed2004

Viral evolution as a tool to improve the tetracycline-regulated gene expression system.

We present viral evolution as a novel and powerful method to optimize non-viral proteins. We used this approach to optimize the tetracycline (Tc)-regulated gene expression system (Tet system) for its function in mammalian cells. The components of the Tet system were incorporated in the human immunodeficiency virus (HIV)-1 virus such that viral replication is controlled by this regulatory system. Upon long term replication of this HIV-rtTA virus in human T cells, we obtained a virus variant with an enhanced replication potential resulting from an improved rtTA component of the introduced Tet system. We identified a single amino acid exchange, F86Y, which enhances the transcriptional activity and doxycycline (dox) sensitivity of rtTA. We generated a new rtTA variant that is 5-fold more active at high dox levels than the initial rtTA, and 25-fold more sensitive to dox, whereas the background activity in the absence of dox is not increased. This new rtTA variant will be very useful in biological applications that require a more sensitive or active Tet system. Our results demonstrate that the viral evolution strategy can be used to improve the activity of genes by making them an integral and essential part of the virus.

Biotechnology↗