Search PubMed⌕ Search

Biomedical subjects

B Berkhout

Publications and source records attributed to B Berkhout.

At least 91 records · Page 5Linked to original sources
PubMed1995

Variable expression of class 1 outer membrane protein in Neisseria meningitidis is caused by variation in the spacing between the -10 and -35 regions of the promoter.

The class 1 outer membrane protein encoded by the porA gene of Neisseria meningitidis is a candidate for a vaccine against meningococcal infection. The expression of class 1 outer membrane protein displays phase variation between three expression levels. Northern (RNA) blot and primer extension analysis revealed that this phase variation is regulated at the transcriptional level. The start site for transcription is located 59 bp upstream of the translational initiation codon. Sequence analysis of the promoter region of the porA gene of a variant without class 1 protein expression revealed nine contiguous guanidine residues between the -10 and -35 domains. Comparison of promoter sequences of different phase variants indicated that the length of the polyguanidine stretch correlated with the expression level of the class 1 outer membrane protein; the presence of 11, 10, or 9 contiguous guanidine residues results in high levels, medium levels, or no expression of class 1 mRNA, respectively. These results suggest that the variable porA expression levels seen in different isolates are modulated by guanidine residue insertion and/or deletion due to slipped-strand mispairing on the polyguanidine stretch within the intervening sequence of the -35 and -10 regions of the promoter. The phase variation of class 1 outer membrane protein may provide a molecular mechanism to evade the host immune defense. Therefore, the protective efficacy of a vaccine based on class 1 outer membrane protein may be questioned.

Bacterial Vaccines↗
PubMed1995

Reduced replication of human immunodeficiency virus type 1 mutants that use reverse transcription primers other than the natural tRNA(3Lys).

Replication of the human immunodeficiency virus type 1 (HIV-1) and other retroviruses involves reverse transcription of the viral RNA genome into a double-stranded DNA. This reaction is primed by the cellular tRNA(3Lys) molecule, which binds to a complementary sequence in the viral genome, referred to as the primer-binding site (PBS). In order to study the specificity of primer usage, we constructed a set of HIV-1 mutants with altered PBS sites corresponding to other tRNA species (tRNA(Ile), tRNA(1,2Lys), tRNA(Phe), tRNA(Pro), tRNA(Trp)). These mutant viruses were able to replicate, although with delayed replication kinetics compared with wild-type HIV-1. Identification of the tRNA species associated with the genomic RNA demonstrated binding of tRNAs complementary to the new PBS sites. However, the occupancy of the mutant PBS sites by these new primers was reduced and correlated well with the replication potential of the mutant viruses. These results suggest that the PBS sequence is not sufficient for annealing of the tRNA primer. Upon prolonged culturing, all mutants reverted to the wild-type PBS(3Lys) sequence. Minor sequence changes in the nucleotides flanking the PBS site indicate that these reversions resulted from annealing of the wild-type tRNA(3Lys) primer onto the mutant PBS sites, followed by copying of part of the tRNA(3Lys) sequence during reverse transcription. Furthermore, the reversion efficiency of the different PBS mutants was found to correlate with their tRNA(Lys)3 binding capacity. A remarkable reversion pathway was observed for the PBSPro variant (PBSPro-->PBSIle-->PBSwt). This pathway can be explained by efficient base pairing of tRNA(Ile) to PBSPro, followed by annealing of tRNA(3Lys) onto the PBSIle intermediate. These results demonstrate that HIV-1 is dedicated to the tRNA(3Lys) primer and that factors other than the PBS sequence determine the selective primer usage of this retrovirus.

Base Sequence↗
PubMed1994

A sensitive promoter assay based on the transcriptional activator Tat of the HIV-1 virus.

We developed a sensitive vector system for the analysis of weak promoter activities. This promoter assay is based on the transcriptional activator protein, Tat, of human immunodeficiency virus type 1 (HIV-1). High-level expression of HIV requires activation in trans by Tat of the promoter in the long terminal repeat (LTR). Here we describe the construction of a promoterless pTat vector. Foreign promoter elements can be inserted upstream from the tat gene, and expression of Tat protein is measured in trans on a co-transfected LTR-CAT reporter plasmid. We show that this binary system is more sensitive than standard pCAT reporter assays.

3T3 Cells↗
PubMed1994

Evolution of a disrupted TAR RNA hairpin structure in the HIV-1 virus.

Human immunodeficiency virus type 1 (HIV-1) RNA contains an extended hairpin structure at the 5' end (the TAR element) that is essential for viral replication. The upper part of the stem-loop structure binds the virally encoded transcriptional activator protein Tat and cellular co-factors, but no clear function for the lower stem region has been established. Here, we report that mutant HIV-1 viruses with base substitutions in the lower stem region are dead, most likely at the level of transcription from an integrated provirus. By using large amounts of these mutant DNA constructs for transfections, revertant viruses with a great variety of genetic changes (point mutations, short deletions) could be isolated in prolonged culture experiments that lasted over 6 months. The pattern and evolution of these changes supported the notion that base-pairing of the lower stem region is essential for optimal HIV-1 replication. The functional and genetic plasticities of this RNA domain and the HIV-1 long terminal repeat promoter are discussed.

Base Composition↗
PubMed1994

The unusual nucleotide content of the HIV RNA genome results in a biased amino acid composition of HIV proteins.

Extremely high frequencies of the A nucleotide are found in the RNA genomes of the lentivirus group of retroviruses. It is presently unknown what molecular force is responsible for this A-pressure. In this manuscript, we demonstrate a correlation between this 'A-pressure' and the amino acid-usage of the lentivirus family. We compared the amino acid composition of the Gag and Pol proteins of the human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2) with that of the second group of human retroviruses; the human T-cell leukemia viruses type I and II (HTLV-I and HTLV-II). Differences in total amino acid content correlate with the preference for A-rich codons in the HIV genome. A pair-wise comparison of homologous amino acid positions in the Pol proteins indicates that both conservative and non-conservative changes can be accounted for by this A-bias. The putative molecular mechanism underlying this A-pressure and the evolutionary consequences are discussed.

Amino Acid Sequence↗
PubMed1994

Intracellular analysis of in vitro modified HIV Tat protein.

Human immunodeficiency viruses HIV-1 and HIV-2 encode a Tat protein that specifically activates transcription from the viral long terminal repeat. To characterize the properties of the Tat proteins, we have expressed them in Escherichia coli. The purified Tat protein was biochemically analyzed and tested for activity upon electroporation into human cell lines. This protein electroporation was used for the intracellular analysis of in vitro modified Tat protein. Our results indicate that the transcriptionally active form of the Tat protein is a monomer. Furthermore, we found that Tat activity is dramatically inhibited by preincubation of the protein with strongly reducing agents. In contrast, no inhibitory effect was measured upon incubation with metal-chelating reagents. These results suggest that the cysteine residues of Tat are involved in the formation of intramolecular disulfide bonds.

Base Sequence↗
PubMed1994

Human immunodeficiency virus uses tRNA(Lys,3) as primer for reverse transcription in HeLa-CD4+ cells.

Significant amounts of different tRNA molecules are present in retroviral particles, but one specific tRNA species functions as primer in reverse transcription. It is generally believed that the HIV-1 virus uses the tRNA(Lys,3) molecule as primer. This is based on sequence complementarity between the 3' end of tRNA(Lys,3) and the primer-binding site (PBS) on HIV-1 genomic RNA. Recent biochemical analyses indicated that tRNA(LYs,3) is indeed incorporated into viral particles. Interestingly, tRNA(Lys,3) could not be detected in virions produced by HeLa-CD4+ cells [(1992) Biochem. Biophys. Res. Commun. 185, 1105-1115]. In order to test whether alternative tRNA molecules can function as primer in HIV replication, we performed a series of experiments based on the observation that tRNA primer sequences are inherited by the viral progeny. We cultured HIV-1 for prolonged periods of time in HeLa-CD4+ cells, but did not detect sequence changes in the PBS region. Furthermore, we found PBS-mutants to be replication-incompetent, again suggesting that HIV-1 solely uses tRNA(Lys,3) as primer. Most importantly, we obtained revertants of one such PBS-mutant, which had restored a wild-type PBS sequence. This tRNA(Lys,3)-mediated repair demonstrates a general requirement for this primer in HIV-1 reverse transcription.

Base Sequence↗
PubMed1994

Premature strand transfer by the HIV-1 reverse transcriptase during strong-stop DNA synthesis.

Reverse transcription of retroviral genomes starts near the 5' end of the viral RNA by use of an associated tRNA primer. According to the current model of reverse transcription, the initial cDNA product, termed minus-strand strong-stop DNA, 'jumps' to a repeated sequence (R region) at the 3' end of the RNA template. The human retroviruses have relatively long R regions (97-247 nucleotides) when compared to murine and avian viruses (16-68 nucleotides). This suggests that the full complement of the R region is not required for strand transfer and that partial cDNA copies of the 5' R can prematurely jump to the 3' R. To test this hypothesis, we generated mutants of the human immunodeficiency virus with R region changes and analyzed whether 5' or 3' R sequences were inherited by the progeny. We found that in most cases, 5' R-encoded sequences are dominant, which is consistent with the model of reverse transcription. Using a selection protocol, however, we were also able to identify progeny viruses with R sequences derived from the original 3' R element. These results suggest that partial strong stop cDNAs can be transferred with R region homologies much shorter than 97 nucleotides.

Base Sequence↗
PubMed1994

Comparison of 5' and 3' long terminal repeat promoter function in human immunodeficiency virus.

The architecture of a retroviral genome presents some unusual features for transcriptional regulation because of duplication of the transcriptional control sequences in the 5' and 3' long terminal repeats (LTRs). We have studied the transcriptional activity of the 5' and 3' LTRs of human immunodeficiency virus type 1 (HIV-1) vectors. Using full-length HIV molecular clones, we demonstrate that both LTRs function as Tat-inducible promoters. However, the absolute levels of transcription were found to be much higher for the 5' LTR than for the 3' LTR promoter. When transcription was assayed for an integrated HIV-1 provirus, we also found that the upstream 5' LTR element was the major transcriptional promoter. 3' LTR transcription, however, can be triggered by inactivation of the 5' LTR promoter. Likewise, 5' LTR transcription is induced in constructs lacking a functional 3' LTR promoter. This phenomenon of promoter suppression may have important implications for the design of HIV-based retrovirus vectors.

Animals↗
PubMed1993

Effects of integration and replication on transcription of the HIV-1 long terminal repeat.

The activity of a promoter is influenced by chromosomal and cell cycle/replication context. We analyzed the influences of integration and replication on transcription of the human immunodeficiency virus (HIV)-1 long terminal repeat (LTR). We found that one requirement for Tat trans-activated expression differed for integrated versus unintegrated HIV-1 LTR. Specifically, the second coding exon of Tat, previously regarded as functionally dispensable, plays a role in the optimal activation of integrated LTRs. We also found that the transcription profile produced from the HIV-1 LTR is influenced by replication. Autonomously replicating vectors that contain the HIV-1 LTR produced patterns of transcription different from counterpart vectors that do not replicate. Both replicating and nonreplicating HIV-1 LTRs remained responsive to Tat.

Antigens, Polyomavirus Transforming↗
PubMed1993

In vivo selection of randomly mutated retroviral genomes.

Darwinian evolution, that is the outgrowth of the fittest variants in a population, usually applies to living organisms over long periods of time. Recently, in vitro selection/amplification techniques have been developed that allow for the rapid evolution of functionally active nucleic acids from a pool of randomized sequences. We now describe a modification of the nucleic acid-evolution protocol in which selection and amplification take place inside living cells by means of a retroviral-based replication system. We have generated a library of HIV-1 DNA genomes with random sequences in particular domains of the TAR element, which is the binding site for the Tat trans-activator protein. This mixture of HIV genomes was transfected into T cells and outgrowth of the fittest viruses was observed within two weeks of viral replication. The results of this in vivo selection analysis are consistent with the notion that primary sequence elements in both TAR bulge and loop domains are critical for Tat-mediated trans-activation and viral replication.

Base Sequence↗
PubMed1993

Secondary structure of the HIV-2 leader RNA comprising the tRNA-primer binding site.

The initiation of reverse transcription of a retroviral RNA genome occurs by a tRNA primer bound near the 5' end of the genomic RNA at a position called the primer-binding site (PBS). To understand the molecular basis for this RNA-RNA interaction, the secondary structure of the leader RNA of the human immunodeficiency virus type 2 (HIV-2) RNA was analyzed. In vitro synthesized HIV-2 RNA was probed with various structure-specific enzymes and chemicals. A computer program was then used to predict the secondary structure consistent with these data. In addition, the nucleotide sequences of different HIV-2 isolates were used to screen for the occurrence of covariation among putative base pairs. The primary sequences have diverged rapidly in some HIV-2 isolates, however, some strikingly conserved secondary structure elements were identified. Most nucleotides in the leader region are involved in base pairing. An exception is the PBS sequence, of which 15 out of 18 nucleotides are exposed in an internal loop. These findings suggest that the overall structure of the HIV-2 genome has evolved to facilitate an optimal interaction with its tRNA primer.

Base Sequence↗
PubMed1993

In vitro dimerization of HIV-2 leader RNA in the absence of PuGGAPuA motifs.

Retroviral particles contain a dimeric genome consisting of two full-length, noncovalently linked RNA molecules. Linkage of the two genomes is thought to be critical for a productive reverse transcription reaction and may increase genetic recombination rates. The molecular nature of the dimer linkage structure (DLS) is poorly understood. It was recently shown that in vitro synthesized retroviral transcripts can dimerize in the absence of protein factors. We studied in vitro dimerization of human immunodeficiency virus type 2 (HIV-2) RNA. Specific dimerization of HIV-2 RNAs was observed upon incubation at 37 degrees C in high-salt buffer. Previously, physical and biochemical studies have mapped dimer linkage structures in retroviral leader RNA close to the gag open reading frame. In this study, we found efficient dimerization of HIV-2 RNAs containing only the 5' terminal 255 nucleotides of the leader RNA. Therefore, it seems likely that multiple dimerization signals are present in retroviral leader RNA. The implications for genome dimerization and genome packaging are discussed.

Genome, Viral↗
PubMed1992

Kinetics of HIV-1 long terminal repeat trans-activation. Use of intragenic ribozyme to assess rate-limiting steps.

We have examined, using self-cleaving ribozymes, the intracellular trans-activation kinetics of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) by viral protein Tat. Experiments were designed to effect a competition (during RNA chain elongation) between cleavage of a nascent RNA containing the Tat-responsive target sequence (TAR) and Tat interaction with the same TAR in the process of LTR-trans-activation. We found that fast self-cleavage of nascent TAR-containing RNA abolished Tat trans-activation. Slowing the cleavage reaction kinetically rescued trans-activation. Based on our results, we conclude that the rate-limiting step in HIV-1 LTR trans-activation is the initial contact made between Tat/TAR/LTR rather than the promoter proximal pausing of RNA polymerases that are tethered to functional TAR.

Base Sequence↗
PubMed1992

Structural features in TAR RNA of human and simian immunodeficiency viruses: a phylogenetic analysis.

A comparative analysis of TAR RNA structures in human and simian immunodeficiency viruses reveals the conservation of certain structural features despite the divergence in sequence. Both the TAR elements of HIV-1 and SIV-chimpanzee can be folded into relatively simple one-stem hairpin structures. Chemical and RNAase probes were used to analyze the more complex structure of HIV-2 TAR RNA, which folds into a branched hairpin structure. A surprisingly similar RNA conformation can be proposed for SIV-mandrill, despite considerable divergence in nucleotide sequence. A third structural presentation of TAR sequences is seen for SIV-african green monkey. These results are generally consistent with the classification of HIV-SIV viruses in four subgroups based on sequence analyses (both nucleotide- and amino acid-sequences). However, some conserved TAR structures were detected for members of different virus subgroups. It is therefore proposed that RNA structure analysis might provide an additional tool for determining phylogenetic relationships among the HIV-SIV viruses.

Base Composition↗
PubMed1992

Natural variants of the HIV-1 long terminal repeat: analysis of promoters with duplicated DNA regulatory motifs.

Sequence variation in the long terminal repeat (LTR) region of HIV-1 was analyzed in viral isolates of 17 infected individuals. Two classes of LTR size variants were found. One HIV-1 variant was detected containing an additional binding site for the transcription factor Sp1. Another LTR size variation was observed in four patients in a region just upstream of the NF-kappa B enhancer. This variation was the result of a duplication of a short DNA sequence (CTG-motif). Cell culture experiments demonstrated that the natural variant with four Sp1 sites had a slightly higher promoter activity and viral replication rate than the isogenic control LTR with three Sp1 sites. No positive effect of the duplicated CTG-motif could be detected. In order to measure small differences in virus production more accurately, equal amounts of a size variant and the wild-type plasmid were cotransfected into T-cells. The virus with four Sp1 sites did outgrow the three Sp1 virus in 35 days of culture and CTG-monomer virus outcompeted the CTG-dimer virus in 42 days. Based on these results we estimate a 5-10% difference in virus production of the LTR variants when compared to that of wild-type.

Base Sequence↗