Biomedical subjects

P J Fay

Publications and source records attributed to P J Fay.

At least 19 recordsLinked to original sources

PubMed1998

The A2 subunit of factor VIIIa modulates the active site of factor IXa.

Factor VIIIa, the protein cofactor for factor IXa, is comprised of A1, A2, and A3-C1-C2 subunits. Isolated subunits of factor VIIIa were examined for their ability to accelerate the factor IXa-catalyzed activation of factor X. The A2 subunit enhanced the kcat for this conversion by 100-fold whereas the Km for factor X was unaffected. The apparent Kd for the interaction of A2 subunit with factor IXa was approximately 300 nM. Similar results were obtained using purified A2 expressed as the isolated domain in Chinese hamster ovary cells, although this material was less stable than the factor VIIIa-derived material. Isolated A1 and A3-C1-C2 subunits showed no effect on the rate of factor X conversion. A2 subunit increased the fluorescence anisotropy of fluorescein-Phe-Phe-Arg-factor IXa (Deltar = 0.015) and markedly increased anisotropy in the presence of factor X (Deltar = 0.057), suggesting that it contributes to the orientation of the factor IXa active site and its relation to substrate. A synthetic peptide to A2 residues 558-565 inhibited the A2-dependent enhancement of factor X activation with an IC50 = 40 microM, a value similar to its Ki for inhibition of the intrinsic factor Xase (105 microM). These results indicate that the isolated A2 subunit modulates the active site of factor IXa and identifies a functional role for this subunit in factor VIIIa.

Animals↗

PubMed1998

Effects of copper on the structure and function of factor VIII subunits: evidence for an auxiliary role for copper ions in cofactor activity.

Factor VIII, a divalent metal ion-dependent heterodimer, contains a single copper atom, but the role of this metal in the structure and function of the cofactor is unclear. Earlier results showed that the dissociated heavy and light chains of factor VIII could be recombined in the presence of Ca(II) or Mn(II) but not Cu(II) to yield functional protein [Fay, P. J. (1988) Arch. Biochem. Biophys. 262, 525-531]. Inclusion of Cu(I) or Cu(II) inhibited the Mn(II)- or Ca(II)-dependent reconstitution of factor VIII with an IC50 approximately 10 micro M. The heavy chain was the susceptible subunit with inhibition by copper ion resulting from its reduced affinity for light chain. On the other hand, Mn(II)-dependent factor VIII reconstitutions performed with Cu(II) light chain and native heavy chain occurred at an accelerated rate (approximately 10-fold) and yielded an enhanced activity ( approximately 50%), likely reflecting an increased specific activity of the heterodimer. Cu ions enhanced the activity of EDTA-treated factor VIII in the presence of Ca(II) but not in its absence, suggesting that EDTA-treated factor VIII is not equivalent to separated subunits and that copper ions are auxiliary to ions that mediate reconstitution. Conformational analyses showed that the ellipticities and extrinsic fluorescence of both subunits were differentially affected by Cu(II) and Mn(II). These structural effects were fully reversed by EDTA. The metal ions had little if any effect on the conformation of intact factor VIII or the A1/A3-C1-C2 dimer. Mn(II) and Cu(II) stabilized the factor VIII light chain, and the latter stabilized the A1 subunit derived from the heavy chain, yielding similar thermal denaturation profiles that were distinct from that observed for the Ca(II)-stabilized subunits. Thus both subunits of factor VIII bind copper ions, and the effects of this binding differ from the interactions observed with Ca(II) or Mn(II). These data support a model where copper in factor VIII likely functions to increase specific activity of the heterodimer rather than directly mediating the intersubunit interaction.

Calcium↗

PubMed1998

Binding of tissue-plasminogen activator to fibrin: effect of ultrasound.

Ultrasound reversibly alters the structure of polymerized fibrin, an effect that could influence tissue-plasminogen activator (t-PA) binding. We have, therefore, characterized the effects of ultrasound on binding of t-PA to fibrin using a novel system in which radiolabeled, active-site blocked, single chain tissue-plasminogen activator flowed through a fibrin gel at constant rate, and specific binding was determined by monitoring incorporation of radiolabel. Results using polymerized fibrin were compared with those using a surface of fibrin immobilized on Sepharose beads in a similar system. Interaction of t-PA with surface-immobilized fibrin involved two classes of binding sites (Kd = 31 nmol/L and 244 nmol/L) and a maximum binding ratio of 3.8 mol t-PA/mol fibrin. Ultrasound increased Kd for the high affinity site to 46 nmol/L (P < .0001), but it had no significant effects on the Kd 244 nmol/L site nor on Bmax. Tissue-plasminogen activator binding to noncrosslinked fibrin involved two sites with Kds of 267 nmol/L and 952 nmol/L, while a single Kd 405 nmol/L site was identified for crosslinked fibrin. Ultrasound had no significant effect on the binding affinity for noncrosslinked fibrin, but Bmax was increased in the presence of ultrasound, from 31 mumol/L to 43 mumol/L (P < .0001). Ultrasound decreased the Kd for crosslinked fibrin to 343 nmol/L (P = .026) and also increased Bmax from 22 mumol/L to 25 mumol/L (P = .015). Ultrasound also affected the kinetics of t-PA binding to fibrin, significantly accelerating the rate of dissociation by 77% +/- 5% for noncrosslinked fibrin and by 69% +/- 3% for crosslinked fibrin (P < .001 for each). These results indicate that ultrasound exposure accelerates t-PA binding, alters binding affinity, and increases maximum binding to polymerized fibrin, effects that may result from ultrasound-induced changes in fibrin structure.

Binding Sites↗

PubMed1998

Control of initiation of viral plus strand DNA synthesis by HIV reverse transcriptase.

Human immunodeficiency virus reverse transcribes its single-stranded RNA genome making a DNA copy. As synthesis proceeds, the RNA is simultaneously degraded to oligomers; one of these, the polypurine tract, primes synthesis of a plus strand DNA. The viral reverse transcriptase (RT) degrades all of the non-polypurine tract oligomers. We show that unlike other DNA polymerases the retroviral RT can bind either end of an annealed RNA primer, the 5'-end for degradation and the 3'-end for synthesis. The competition between the two binding modes at any primer determines whether it will be extended or degraded. The 5'-end binding can be suppressed in at least two ways. The sequence of the primer can be such that a region at the 5'-end is unannealed or a DNA primer can be annealed just adjacent to the 5'-end of the RNA primer. This promotes binding of RT to the RNA 3'-end, allowing a primer that would normally be degraded to be extended. Implications for human immunodeficiency virus replication and antiviral therapy are discussed.

Binding Sites↗

PubMed1998

Interaction of the A1 subunit of factor VIIIa and the serine protease domain of factor X identified by zero-length cross-linking.

We have previously used a solid phase binding assay to localize a Factor X (FX) interactive site to the acidic C-terminus of the A1 subunit of FVIIIa (Lapan KA, Fay PJ. J Biol Chem 1997; 272: 2082-2088). The complex of FVIII-FX was made covalent following reaction with the zero-length cross-linking reagent 1-ethyl-3-(3dimethylaminopropyl-)carbodiimide hydrochloride (EDC). Western blotting of the thrombin-cleaved complex showed that the Al subunit of FVIIIa associated with FX heavy chain. The FX-A1 product was also detected following cross-linking to the A1/A3-C1-C2 dimer, but not the activated protein C-cleaved A1(336)/A3-C1-C2 form, indicating that a residue(s) in the region spanning Met337-Arg372 contributed to the intermolecular ion pair(s). A synthetic peptide to this acidic region (FVIII337-372) cross-linked to FX and the product was alkaline resistant indicating that amide linkage(s) were formed. Sequence analysis of the FX-FVIII337-372 adduct suggested that the first 12 NH2-terminal residues of the FX and peptide do not participate in cross-link formation. Conversion of the cross-linked product to FXa by RVV-X showed that the peptide was associated with the serine protease-forming domain of the heavy chain. These results indicate that the association of FVIIIa and FX occurs from a salt linkage(s) formed between residues of the A1 acidic C-terminus of the cofactor (within residues 349-372) and the serine protease-forming domain of the substrate.

Cross-Linking Reagents↗

PubMed1997

Interacting regions in the A1 and A2 subunits of factor VIIIa identified by zero-length cross-linking.

Factor VIIIa is a heterotrimer of A1, A2, and A3-C1-C2 subunits, the activity of which is labile due to a weak affinity interaction of the A2 subunit with the A1/A3-C1-C2 dimer. We have used the zero-length cross-linking reagent, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), to localize regions of interaction within the A1 and A2 subunits. Reaction of factor VIIIa with EDC resulted in the formation of a cross-linked product of approximately 90 kD consisting of the A1 and A2 subunits as judged by Western blotting. Alkaline resistance of this product indicated an amide rather than ester linkage. Factor VIIIa activity decreased as the concentration of cross-linked product increased, suggesting that flexibility in the inter-subunit interaction may be required for proper cofactor function. This product was not formed in the contiguous A1-A2 domains of factor VIII, suggesting that, upon cofactor activation, a conformational change occurs that leads to the formation of a new interdomainal salt bridge(s). Reaction of the EDC-treated factor VIIIa with activated protein C (APC), which cleaves the A1 subunit at Arg336 and bisects the A2 subunit at Arg562, resulted in the formation of an approximately 30 kD product that contains the C-terminus region of A1 covalently linked to the N-terminal half of the A2. The approximately 90 kD cross-linked product was generated after reaction of A2 subunit with A1/A3-C1-C2 dimer but not with A1(336)/A3-C1-C2, a form of the dimer produced by APC cleavage and lacking the C-terminal acidic region of A1. A synthetic peptide corresponding to this acidic region (Met337-Arg372) was found to covalently cross-link to the isolated A2 subunit in 1:1 stoichiometry, suggesting that this region is both necessary and sufficient for the interaction of the A1 and A2 subunits. Sequence analysis of this product suggested that Glu344 in the A1 peptide may contribute to the cross-linkage. These results indicate that activation of factor VIII results in formation of a new ionic linkage(s) localized to the acidic C-terminal region of A1 and the N-terminal half of A2.

Cross-Linking Reagents↗

PubMed1997

Evidence for a unique mechanism of strand transfer from the transactivation response region of HIV-1.

We previously found that strand transfer by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is promoted at sites where RT pauses during synthesis. In this report, strand transfer is measured within the 5' transactivation response region (TAR) of HIV-1 RNA. We hypothesized that the stable hairpin structure of TAR would induce RT pausing, promoting RNase H-directed cleavage of the template and subsequent transfer at that site. We further predicted that HIV-1 nucleocapsid protein (NC), known to melt secondary structures, would decrease transfer. We show that TAR created a strong pause site for RT, but NC significantly promoted strand transfer. The effect of NC is specific, since other single strand binding proteins failed to stimulate transfer. In another unexpected outcome, preferred positions of internal transfer were not at the pause site but were in the upper stem and loop of TAR. Thus, we propose a new mechanism for transfer within TAR described by an interactive hairpin model, in which association between the donor and the acceptor templates within the TAR stem promotes transfer. The model is consistent with the observed stimulation of strand transfer by NC. The model is applicable to internal and replicative end transfer.

Base Sequence↗

PubMed1997

Mutations within the primer grip region of HIV-1 reverse transcriptase result in loss of RNase H function.

Human immunodeficiency virus (HIV) DNA synthesis is accompanied by degradation of genomic RNA by the RNase H of reverse transcriptase (RT). Two different modes of RNase H activity appear necessary for complete RNA removal. In one, occurring during minus strand synthesis, positioning of the RNase H is determined by binding of the polymerase active site to the DNA 3'-end. In the other, used for removal of remaining RNA fragments, positioning of RT for RNase H-directed cleavage is determined by the RNA 5'-ends. We attempted to identify RT amino acids responsible for these modes of positioning. Twelve RT mutants, each with one alanine replacement in residues 224 to 235, known as the primer grip region, were examined for catalytic abilities. Six of the examined primer grip mutants, although distant from the RNase H active site were altered in their ability to cleave RNA. The mutants P226A, F227A, G231A, Y232A, E233A, and H235A failed to perform RNA 5'-end-directed RNase H cleavage in heparin-challenged reactions. The last four mutants also lacked DNA synthesis and DNA 3'-end-directed RNase H cleavage activities in challenged reactions. Since mutants P226A and F227A carried out these latter reactions normally, these two residues specifically influence 5'-RNA-directed RNase H catalysis.

Binding Sites↗

PubMed1997

Localization of a factor X interactive site in the A1 subunit of factor VIIIa.

The protein cofactor, factor (F) VIIIa, is required for the efficient conversion of the substrate FX to FXa by the serine protease FIXa. The interaction between human FVIII (and its constituent subunits) and FX was characterized using a solid phase binding assay performed in the absence of phospholipid and FIXa. Saturable binding of FX to heterodimeric FVIII, the FVIII heavy chain (contiguous A1-A2 domains), the FVIIIa-derived A1/A3-C1-C2 dimer, and the isolated A1 subunit was observed with estimated Kd values ranging from approximately 1 to 3 microM. The interaction of FX with FVIII was inhibited by moderate ionic strength and was Ca2+-dependent, consistent with the salt sensitivity observed in a phospholipid-independent FXa generation assay. Negligible binding to FX was observed for the isolated A2 and A3-C1-C2 subunits of FVIIIa, suggesting that the A1 subunit of FVIII contains a primary binding site for FX. A synthetic peptide to the COOH-terminal acidic region of the A1 subunit, designated FVIII337-372, bound FX and effectively competed with A1 for FX binding (Ki = approximately 16 microM). Cross-linking between the FVIII337-372 peptide and the FX heavy chain was observed following reaction with 1-ethyl-3-[(diethylamino)propyl]carbodiimide. The presence of FX reduced the rate of activated protein C-catalyzed cleavage at Arg336 by approximately 5-fold. These results identify a primary FX interactive site on the cofactor of the intrinsic FXase.

Electrophoresis, Polyacrylamide Gel↗

PubMed1996

Strand displacement synthesis in the central polypurine tract region of HIV-1 promotes DNA to DNA strand transfer recombination.

Two distinct plus strand initiation sites have been identified in human immunodeficiency virus (HIV), the central polypurine tract (cPPT) and the polypurine tract located just upstream of the U3 region (U3-PPT). When synthesis from the U3-PPT reaches the cPPT, the elongating primer causes limited strand displacement of the product created from the cPPT. We examined whether reverse transcriptase (RT) catalyzed strand transfer recombination is promoted by this process. Using a substrate having the viral sequence of the displaced region, we measured transfer of an elongating DNA primer from a donor DNA to an acceptor DNA. Strand transfer synthesis was only efficient when RT was performing strand displacement synthesis. Transfer efficiency was directly related to acceptor concentration but independent of the reaction time. Transfer could occur to acceptors containing 80, 40, or 20 nucleotides of homology with the template DNA. Using different acceptors, we found that DNA to DNA transfer occurred at positions throughout the donor template, except near the 5' end. This shows that a number of the sequences downstream of the cPPT region can promote transfer, but once synthesis has progressed to the point where the downstream segment is completely displaced transfer is not allowed. When the DNA to DNA transfer reactions were performed using a template containing nonviral sequences, the transfer efficiency dropped significantly. This indicates that transfer efficiency is determined by the sequences of the templates used. HIV-RT RNase H-dependent strand transfer between RNA templates is well documented. We propose a quite different mechanism for DNA to DNA transfer, consistent with the ability of RNase H minus RT to perform this reaction. If these DNA to DNA transfer events occur in vivo, they will result in plus strand recombination.

DNA Primers↗

PubMed1996

Exposed hydrophobic sites in factor VIII and isolated subunits.

Hydrophobic sites on the surface of factor VIII, factor VIIIa, and their derived subunits were evaluated using the fluorescent, apolar probe, bisanilinonapthalsulfonic acid (bis-ANS). Two hydrophobic sites, with indicated affinities for the probe, were identified on factor VIII (Kd = 0.2 and 1.22 microM), the isolated heavy chain (HC; Kd = 0.21 and 1.44 microM), and light chain (LC; Kd = 0.04 and 0.22 microM). Comparison of these values and fluorescence emission maxima parameters suggested that the higher affinity site on each isolated subunit contributes to the divalent metal ion-dependent, intersubunit interaction while the two lower affinity sites are retained on the surface of the factor VIII heterodimer. A single bis-ANS site was identified on both the A1/A3-C1-C2 dimer (Kd = 0.19 microM) and A2 subunit (Kd = 0.11 microM), whereas two sites, equivalent to the sites of factor VIII, were observed on factor VIIIa. These results suggested the absence of interactive hydrophobic sites between A2 subunit and dimer, a major conformational change around the hydrophobic site in A2 upon dissociation, and the lack of accessible hydrophobic regions on the B domain of HC. Ca2+ reduced the emission intensity of bis-ANS bound to the isolated LC, HC, and A1 subunit, but not the A2 subunit. Reconstitution of factor VIII activity from isolated HC and LC was inhibited by >90% in the presence of 20 microM bis-ANS, whereas this concentration of probe had no effect on the reconstitution of FVIIIa from the A1/A3-C1-C2 dimer and A2 subunit. These results indicate that intersubunit hydrophobic interactions are important for the metal ion-dependent association between A1 and A3 domains, but are not required for the metal ion-independent interaction between A2 subunit and the A1/A3-C1-C2 dimer in factor VIIIa.

Anilino Naphthalenesulfonates↗

PubMed1996

Misincorporation by HIV-1 reverse transcriptase promotes recombination via strand transfer synthesis.

Genome heterogeneity in retroviruses derives from poor fidelity of the reverse transcriptase (RT) and recombination via RT-catalyzed strand transfer synthesis. RTs lack proofreading ability, and they proficiently extend primers with mismatched termini. Recombination reactions carried out in vitro are accompanied by a high frequency of base substitution errors, suggesting a relationship. Here we provide evidence that misincorporation during RNA-directed DNA synthesis promotes strand transfer recombination. Experiments involved measurement of DNA synthesis, RNase H-directed cleavage, and strand transfer synthesis from preformed mismatched primers on RNA templates by human immunodeficiency virus (HIV) RT in vitro. A significant pause in synthesis occurred from a G(primer). rA(template) mismatch compared to the synthesis from a correctly paired (T.A) primer. The misincorporation-induced pause allowed an unusually large area of RT-RNase H-directed cleavage of the template RNA beneath the primer. Strand transfer to an acceptor molecule with sequence identical to the template RNA was about 50% more efficient than if the primer had had a correctly paired terminus. Overall transfer was measured over a large region of homology. Assuming that enhanced transfer occurs primarily at the site of the mismatch, the actual increase in transfer at that site must have been 1-2 orders of magnitude. Inclusion of a different acceptor molecule with complete complementarity to the originally mismatched 3' primer terminus resulted in an additional 2-fold increase in strand transfer efficiency. Overall, these results suggest the mechanism by which misincorporation during minus strand DNA synthesis in retroviral replication would promote high frequency recombination.

DNA Primers↗

PubMed1996

Differences in mutagenesis during minus strand, plus strand and strand transfer (recombination) synthesis of the HIV-1 gene in vitro.

We have developed an HIV nef-Escherichia coli lacZ fusion system in vitro that allows the detection of low frequency mutations, including frameshifts, deletions and insertions. A portion of the nef gene that encompasses a hypervariable region was fused in-frame with a downstream lacZalpha peptide coding region. The resulting lacZalpha peptide fusion protein remained functional. Any frameshift mutations in the nef insert would put the downstream lacZ alpha peptide gene out of frame, eliminating alpha complementation. With this system we compared the error rates of frameshift mutations that arise during DNA-directed and RNA-directed DNA synthesis. Results showed that DNA-directed and RNA-directed DNA synthesis did not contribute equally to the generation of mutations. DNA-directed DNA synthesis generated frameshift mutations at a frequency approximately 10-fold higher than those arising from RNA-directed DNA synthesis. RNA-directed DNA synthesis in the presence of acceptor templates showed an increase in mutation rate and differences in the mutation spectrum. The enhancement of mutation rate was caused by the appearance of mutations at three new locations that correlated with likely recombination sites. Results indicate that recombination is another source of mutations during viral replication.

Base Sequence↗

PubMed1996

Relationship between plus strand DNA synthesis removal of downstream segments of RNA by human immunodeficiency virus, murine leukemia virus and avian myeloblastoma virus reverse transcriptases.

During retroviral reverse transcription the genomic RNA is degraded by the RNase H activity of reverse transcriptase (RT). Previous results suggest that after RNA-directed DNA synthesis, fragments of RNA remain annealed to the newly synthesized DNA [DeStefano et al.(1991) J. Biol.Chem. 266, 7423-7431]. These must be removed to allow synthesis of the second DNA strand. We measured the ability of HIV-, AMV- and MuLV-RT to coordinate DNA-dependent DNA synthesis and removal of downstream segments of RNA. The substrates employed were DNA templates having upstream DNA and downstream RNA primers. We found that none of the wild type RTs elongated the upstream DNA without simultaneous degradation of the RNA. Consistent with these results, HIV-, AMV- and MuLV-RT showed relatively higher affinity for RNA than for DNA oligonucleotides bound to a DNA template. Differences were observed in the RNA degradation and DNA extension patterns generated by the different RTs. AMV-RT degraded the RNA to segments 11-12 nt long, and readily elongated the upstream DNA to the end of the template. MuLV- and HIV-RT degraded the RNA primarily to segments 15-16 nt long. At low concentrations of the latter two RTs, the DNA primer stalled when it encountered the 5'-end of the RNA. In sufficient excess, all of the RTs elongated the upstream primer without stalling. Even though we were unable to detect displacement of the downstream RNA by the wild type RTs, MuLV- and HIV-RT lacking RNase H, were able to elongate the upstream DNA to the end of the template without degradation of the RNA. This suggests that degradation of downstream pieces of RNA is not absolutely required before synthesis of the plus strand DNA. The implications of these findings for viral replication are discussed.

Animals↗

PubMed1996

Model for the factor VIIIa-dependent decay of the intrinsic factor Xase. Role of subunit dissociation and factor IXa-catalyzed proteolysis.

The intrinsic factor Xase complex (FXase) is comprised of a serine protease, FIXa, and a protein cofactor, FVIIIa, assembled on a phospholipid surface. Activity of FXase decays with time and reflects the lability of FVIIIa. Two mechanisms potentially contribute to this decay: (i) a weak affinity interaction between the FVIIIa A2 subunit and Al/A3-Cl-C2 dimer and (ii) FVIIIa inactivation resulting from FIXa-catalyzed proteolysis of the Al subunit. At low reactant concentrations (0.5 nm FVIIIa; 5 nm FIXa), FXase decay is governed by the inter-FVIIIa subunit affinity and residual activity approaches a value consistent with this equilibrium, as judged by reactions containing exogenous A2 subunit. Analysis using a mutant form of FVIII (FVIIIR336I) possessing an altered FIXa cleavage site, showed similar rates of FXase decay (0.12 min(-1)) and confirmed the lack of contribution of proteolysis under these conditions. When the concentration of FIXa was increased 10-fold, the initial rate of decay of FXase containing native FVIIIa increased (0.82 min(-1)) and paralleled the rate of proteolysis of Al subunit. However, the rate of decay of FXase containing the FVIIIaR336I was reduced (0.048 min(-1)) consistent with the elevated concentration of FIXa stabilizing the labile subunit structure of the cofactor. Reconstitution of FVIII with FIXa-cleaved light chain showed that cleavage at the alternate FIXa site (A3 domain) was not inhibitory to FXase. The presence of substrate FX resulted in a 10-fold reduction in the rate of FIXa-catalyzed proteolysis of FVIIIa. These results suggest a model whereby decay of FXase results from both FVIIIa subunit dissociation and FIXa-catalyzed cleavage, dependent upon the relative concentration of reactants, with greater contribution of the former at low values and, in the absence of substrate, greater contribution of the latter at high values.

Amino Acid Sequence↗

PubMed1996

Activated protein C-catalyzed proteolysis of factor VIIIa alters its interactions within factor Xase.

Factor VIIIa, the cofactor for the factor IXa-dependent conversion of factor X to factor Xa, is proteolytically inactivated by activated protein C (APC). APC cleaves at two sites in factor VIIIa, Arg336, near the C terminus of the A1 subunit; and Arg562, bisecting the A2 subunit (Fay, P., Smudzin, T., and Walker, F. (1991) J. Biol. Chem. 266, 20139-20145). Factor VIIIa increased the fluorescence anisotropy of fluorescein-Phe-Phe-Arg factor IXa (Fl-FFR-FIXa; Kd = 42.4 nM), whereas cleavage of factor VIIIa by APC eliminated this property. Isolation of the APC-cleaved A1/A3-C1-C2 dimer (A1336/A3-C1-C2), and the fragments derived from cleaved A2 subunit (A2N/A2C), permitted dissection of the roles of individual cleavages in cofactor inactivation. Intact A1/A3-C1-C2 dimer increased Fl-FFR-FIXa anisotropy and bound factor X in a solid phase assay, while these activities were absent in the A1336/A3-C1-C2. However, the residues removed by this cleavage, Met337 Arg372, did not directly participate in these functions since neither a synthetic peptide to this sequence nor an anti-peptide polyclonal antibody blocked these activities using intact dimer. CD spectral analysis of the intact and truncated dimers indicated reduced alpha and/or beta content in the latter. The A1/A3-C1-C2 dimer plus A2 subunit reconstitutes cofactor activity and produced a factor VIIIa-like effect on the anisotropy of Fl-FFR-FIXa. However, when A2 was replaced by the A2N/A2C fragments, the resulting fluorescence signal was equivalent to that observed with the dimer alone. These results indicate that APC inactivates the cofactor at two levels within the intrinsic factor Xase complex. Cleavage of either subunit modulates the factor IXa active site, suggesting an essential synergy of interactive sites in factor VIIIa. Furthermore, cleavage of the A1 site alters the conformation of a factor X binding site within that subunit, thereby reducing the affinity of cofactor for substrate.

Amino Acid Sequence↗

PubMed1996

Strand displacement synthesis of the long terminal repeats by HIV reverse transcriptase.

According to the current model for retroviral replication, strand displacement of the long terminal repeat (LTR) is a necessary step during plus strand DNA synthesis in vivo. We have investigated the ability of human immunodeficiency virus reverse transcriptase (HIV-RT) to synthesize in vitro over a 634-nucleotide HIV LTR DNA template, having or lacking a single full-length DNA downstream primer. The presence of the downstream primer resulted in an approximately 12-fold reduction in the rate of upstream primer elongation. Addition of Escherichia coli single-stranded binding protein (SSB) or human replication protein A (RP-A) enhanced strand displacement synthesis; however, addition of HIV nucleocapsid protein (NC) did not. The presence of excess single-stranded DNA complementary to the downstream primer did not stimulate displacement synthesis. Interestingly, we observed that the elongating upstream primer could readily transfer to this DNA. This observation suggests that recombination is favored during strand displacement synthesis in vivo.

Base Sequence↗

PubMed1996

Helix structure and ends of RNA/DNA hybrids direct the cleavage specificity of HIV-1 reverse transcriptase RNase H.

RNA/DNA hybrids in human immunodeficiency virus (HIV) replication are cleaved by HIV-1 reverse transcriptase (RT) H in locations determined by hybrid structure. Minus strand DNA synthesis is accompanied by cleavage of template viral RNA directed by RT positioned at the growing 3' DNA end. Some RNA remains as oligomers annealed to the new DNA strand and is cut by RTs positioned at the 5' RNA ends. We constructed substrates to the test the hypothesis that internal helix structure, rather than strand end structure, drives the RT to position at 3' DNA and 5' RNA ends. On substrates with an RNA primer recessed on a DNA template, the 5' end of the RNA had a dominant role in the determination of RNase H cleavage positions. If the 5' end region of the RNA could not anneal, cleavage would not occur. Nevertheless, we obtained evidence that helix structure promotes the binding of RT to the end of the helical region closest to the 5' RNA/3' DNA end. When a DNA primer recessed on an RNA template had a 3' unannealed region, cleavage occurred, with RT positioned solely by helical structure at the 5' RNA/3' DNA end of the annealed region of the hybrid. Using substrates having RNA primers annealed to circular DNA templates, we showed that cleavage can be independent of the presence of a DNA 3'end and is directed by the 5' RNA end. Overall, the results suggest that the RT initially binds an internal region of the hybrid and then is driven in the direction to encounter a 3' DNA or 5' RNA end, where it is positioned for catalysts by the strand end. The requirement for two modes of RNA cleavage in viral replication and the unexpected requirement for the 5' RNA end structure are discussed.

Base Sequence↗