HIV-1 nucleocapsid protein and replication protein A influence the strand displacement DNA synthesis of lentiviral reverse transcriptase.
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Animals↗
Biomedical subjects
M Hottiger
Publications and source records attributed to M Hottiger.
Differential discrimination of DNA polymerase for variants of the non-standard nucleobase pair between xanthosine and 2,4-diaminopyrimidine, two components of an expanded genetic alphabet.
Mammalian DNA polymerases alpha and epsilon, the Klenow fragment of Escherichia coli DNA polymerase I and HIV-1 reverse transcriptase (RT) were examined for their ability to incorporate components of an expanded genetic alphabet in different forms. Experiments were performed with templates containing 2'-deoxyxanthosine (dX) or 2'-deoxy-7-deazaxanthosine (c7dX), both able to adopt a hydrogen bonding acceptor-donor-acceptor pattern on a purine nucleus (puADA). Thus these heterocycles are able to form a non-standard nucleobase pair with 2,4-diaminopyrimidine (pyDAD) that fits the Watson-Crick geometry, but is joined by a non-standard hydrogen bonding pattern. HIV-1 RT incorporated d(pyDAD)TP opposite dX with a high efficiency that was largely independent of pH. Specific incorporation opposite c7dX was significantly lower and also independent of pH. Mammalian DNA polymerases alpha and epsilon from calf thymus and the Klenow fragment from E. coli DNA polymerase I failed to incorporate d(pyDAD)TP opposite c7dX.
Animals↗
[Quantification of proviral FIV DNA using competitive PCR].
Quantification of provirus copies is important in the context of different biological questions. The most reliable approach for DNA quantification is a PCR based on coamplification of two templates of similar length, the target sequence and the reference template, sharing the same primer recognition sequences. During the amplification, the two templates compete for the same primer set (competitive PCR, or cPCR) AND consequently amplify at the same rate independently of the number of cycles. The amplified products can be distingushed by their different lengths. After densitometrical analysis, the proviral copy number of experimentally feline immunodeficiency virus infected cats could be calculated, since a known amount of reference template was used. The method described here proved to be very sensitive (10 copies for the competitor-DNA) and was used to quantitate the proviral load during several experiments in which the influence of periodical immunestimulations and the effect of vaccines on the virus load was studied.
Animals↗
Human Immunodeficiency Virus type 1 reverse transcriptase.
The Human Immunodeficiency Virus type 1 (HIV-1) is a retrovirus and a causative agent of the Acquired Immuno Deficiency Syndrome (AIDS). Retroviruses are distinct from other viruses in their ability to encode an enzyme called reverse transcriptase (RT). The RT is the enzyme mainly involved in replication. It performs RNA- as well as DNA-dependent DNA synthesis in order to convert the single-stranded viral RNA genome into double-stranded DNA. The double-stranded DNA is stably integrated into the host cell genome and is used as a template for the production of a new viral generation. The HIV-1 RT is partially encoded by the POL open reading frame of the HIV-1 genome and consists of two subunits of 66 kDa (p66) and 51 kDa (p51). The p66 polypeptide encodes the reverse transcriptase and the RNase H domain. Half of the p66 molecules are further processed to generate the p51 protein with an identical N-terminus, but lacking the C-terminus which encodes the RNase H domain. In vivo both polypeptides are found in equimolar amounts thus forming a heterodimer. This dimerization is critical for the enzymatic activity. In this review we summarize (i) the replication cycle of HIV-1, (ii) the enzymatic properties of HIV-1 RT and (iii) the structure-function relationship of the HIV-1 RT in view of the known three dimensional structure.
DNA, Viral↗
Recognition by viral and cellular DNA polymerases of nucleosides bearing bases with nonstandard hydrogen bonding patterns.
The ability of DNA polymerases (pols) to catalyze the template-directed synthesis of duplex oligonucleotides containing a nonstandard Watson-Crick base pair between a nucleotide bearing a 5-(2,4-diaminopyrimidine) heterocycle (d kappa) and a nucleotide bearing either deoxyxanthosine (dX) or N1-methyloxoformycin B (pi) has been investigated. The kappa-X and kappa-pi base pairs are jointed by a hydrogen bonding pattern different from and exclusive of those joining the AT and GC base pairs. Reverse transcriptase from human immunodeficiency virus type 1 (HIV-1) incorporates dXTP into an oligonucleotide opposite d kappa in a template with good fidelity. With lower efficiency and fidelity, HIV-1 reverse transcriptase also incorporates d kappa TP opposite dX in the template. With d pi in the template, no incorporation of d kappa TP was observed with HIV reverse transcriptase. The Klenow fragment of DNA pol I from Escherichia coli does not incorporate d kappa TP opposite dX in a template but does incorporate dXTP opposite d kappa. Bovine DNA pols alpha, beta, and epsilon accept neither dXTP opposite d kappa nor d kappa TP opposite d pi. DNA pols alpha and epsilon (but not beta) incorporate d kappa TP opposite dX in a template but discontinue elongation after incorporating a single additional base. These results are discussed in light of the crystal structure for pol beta and general considerations of how polymerases must interact with an incoming base pair to faithfully copy genetic information.
Base Composition↗
The large subunit of HIV-1 reverse transcriptase interacts with beta-actin.
HIV-1 reverse transcriptase is a dimeric enzyme mainly involved in the replication of the viral genome. A filamentous phage cDNA expression library from human lymphocytes was used to select cellular proteins interacting with HIV-1 reverse transcriptase Affinity selections using the bacterially expressed monomeric large subunit of reverse transcriptase (p66) yielded host beta-actin. This clone was expressed as glutathione-S-transferase fusion protein which was identified by using a specific antibody against beta-actin. Furthermore we show that also the eukaryotic beta-actin binds to either the large subunit of reverse transcriptase or to the Pol precursor polyprotein in vitro. The reverse transcriptase/beta-actin interaction might be important for the secretion of HIV-1 virions.
Actins↗
Feline immunodeficiency virus reverse transcriptase: expression, functional characterization, and reconstitution of the 66- and 51-kilodalton subunits.
The two subunits of the feline immunodeficiency virus (FIV) reverse transcriptase (RT) were cloned and functionally expressed in Escherichia coli. The recombinant proteins are enzymatically active as homodimers (p66 and p51) as well as a heterodimer p66/p51. The biochemical properties of the FIV RT are very similar to those of the counterpart of the human immunodeficiency virus type 1 in being an RNA-dependent and DNA-dependent DNA polymerase. When a double-stranded DNA containing a small gap of 26 nucleotides was tested, we found a new activity of the FIV RT p66/p51 heterodimer--the cat viral enzyme could perform strand displacement DNA synthesis of approximately 300 bases. The FIV RT homodimer p66 alone could carry out limited strand displacement DNA synthesis, but this activity was stimulated by the p51 subunit at a molar ratio of one molecule of p66 to five molecules of p51. On the other hand, the homodimeric p51 itself was unable to fill a small gap of 26 nucleotides in a double-stranded DNA substrate and was not active by itself in strand displacement DNA synthesis. These data are in agreement with an earlier finding of strand displacement DNA synthesis by human immunodeficiency virus type 1 RT (M. Hottiger, V.N. Podust, R.L. Thimmig, C.S. McHenry, and U. Hübscher. J. Biol. Chem. 269:986-991, 1994). Our data therefore suggest a general and important function of lentiviral p51 subunits in strand displacement DNA synthesis which appears to be required in later stages of the lentiviral replication cycle, when DNA-dependent DNA synthesis occurs on double-stranded DNA.
Amino Acid Sequence↗
Strand displacement activity of the human immunodeficiency virus type 1 reverse transcriptase heterodimer and its individual subunits.
By using a DNA substrate with defined gap size, we found that human immunodeficiency virus type 1 reverse transcriptase (HIV-RT) was able to perform strand displacement DNA synthesis. This activity was not affected first by calf thymus proliferating cell nuclear antigen and replication factor C and second by Escherichia coli single-stranded DNA-binding protein, which together allow DNA polymerase delta to perform strand displacement DNA synthesis (Podust, V., and Hübscher, U. (1993) Nucleic Acids Res. 21, 841-846). 3'-Azido-2',3'-dideoxythymidine triphosphate inhibited displacement completely, indicating that DNA synthesis is required for this reaction. The HIV-RT p66 polypeptide alone could perform limited strand displacement DNA synthesis, whereas the HIV-RT p51 polypeptide was completely inactive, likely due to its inability to replicate extensively on a M13 DNA template. On the other hand the HIV-RT p51 polypeptide enhanced the strand displacement activity of the HIV-RT p66 subunit at a molar ratio of 4:1, mainly by chasing short products into longer ones. Furthermore, kinetic experiments after complementation of HIV-RT p66 with HIV-RT p51 indicated that HIV-RT p51 can restore rate and extent of strand displacement activity by HIV-RT p66 compared with the HIV-RT heterodimer p66/p51, suggesting a function of the 51-kDa polypeptide.
DNA↗
Calf thymus DNA polymerase delta: purification, biochemical and functional properties of the enzyme after its separation from DNA polymerase alpha, a DNA dependent ATPase and proliferating cell nuclear antigen.
We have established a novel procedure to purify calf thymus DNA polymerase delta from cytoplasmic extracts. The enzyme has typical properties of DNA polymerase delta including a 3' - greater than 5' exonuclease activity and efficiently replicates natural occurring genomes such as primed single-stranded M13 DNA and single-stranded porcine circovirus DNA, this last one thanks to an associated or contaminating primase activity. A processivity of at least a thousand bases was evident and this in the apparent absence of proliferating cell nuclear antigen. The enzyme was purified through a procedure that allows the simultaneous isolation of DNA polymerase delta, DNA polymerase alpha-primase and a DNA dependent ATPase. All these enzymes coeluted from a phosphocellulose column. After chromatography on hydroxylapatite DNA polymerase delta separated from the coeluting DNA polymerase alpha and DNA dependent ATPase. Separation of the latter two was achieved on heparin-Sepharose. DNA polymerase delta was further purified by heparin-Sepharose and fast protein liquid chromatography. Purified DNA polymerase delta was resistant to the DNA polymerase alpha inhibitors BuPdGTP and BuAdATP and did not react with DNA polymerase alpha monoclonal and polyclonal antibodies. Based on this isolation protocol we can start to test biochemically the hypothesis whether DNA polymerase delta and DNA polymerase alpha might act coordinately at the replication fork as leading and lagging strand replicases, respectively.
Adenosine Triphosphatases↗