Constitutively dead, conditionally live HIV-1 genomes. Ex vivo implications for a live virus vaccine.

An effective vaccine against AIDS is unlikely to be available for many years. As we approach two decades since the first identification of human immunodeficiency virus, type 1 (HIV-1), currently, only one subunit vaccine candidate has reached phase 3 of clinical trials. The subunit approach has been criticized for its inability to elicit effectively cytotoxic T-lymphocyte (CTL) response, which is felt by many to be needed for protection against HIV-1 infection. In subhuman primates, a live attenuated simian immunodeficiency virus (SIV) vaccine candidate, capable of inducing CTL, has been found to confer prophylactic immunity sufficient to prevent simian AIDS. Because replication competent (live) attenuated viruses could over time revert to virulence, such a live attenuated approach has largely been dismissed for HIV-1. Here, we describe the creation of constitutively dead conditionally live (CDCL) HIV-1 genomes. These genomes are constitutively defective for the Tat/TAR axis and are conditionally dependent on tetracycline for attenuated replication with robust expression of viral antigens. Our results suggest that CDCL genomes merit consideration as safer "live" attenuated HIV-1 vaccine candidates.

Consequences of placing an intramolecular crosslink in myosin S1.

This paper describes the placement of a crosslinking agent (dibromobimane) between two thiols (Cys-522 and Cys-707) of a fragment, "S1," of the motor protein, myosin. It turns out that fastening the first anchor of the crosslinker is easy and rapid, but fastening the second anchor (Cys-522) is very temperature dependent, taking 30 min at room temperature but about a week on ice. Moreover, crystallography taken at 4 degrees C would seem to predict that the linkage is impossible, because the span of the crosslinking agent is much less than the interthiol distance. The simplest resolution of this seeming paradox is that structural fluctuations of the protein render the linkage increasingly likely as the temperature increases. Also, measurements of the affinity of MgADP for the protein, as well as the magnetic resonance of the P-atoms of the ADP once emplaced, suggest that binding the first reagent anchor to Cys-707 initiates an influence that travels to the rather distant ADP-binding site, and it is speculated what this "path of influence" might be.

Proteolytic cleavage of actin within the DNase-I-binding loop changes the conformation of F-actin and its sensitivity to myosin binding.

Effects of subtilisin cleavage of actin between residues 47 and 48 on the conformation of F-actin and on its changes occurring upon binding of myosin subfragment-1 (S1) were investigated by measuring polarized fluorescence from rhodamine-phalloidin- or 1, 5-IAEDANS-labeled actin filaments reconstructed from intact or subtilisin-cleaved actin in myosin-free muscle fibers (ghost fibers). In separate experiments, polarized fluorescence from 1, 5-IAEDANS-labeled S1 bound to non-labeled actin filaments in ghost fibers was measured. The measurements revealed differences between the filaments of cleaved and intact actin in the orientation of rhodamine probe on the rhodamine-phalloidin-labeled filaments, orientation and mobility of the C-terminus of actin, filament flexibility, and orientation and mobility of the myosin heads bound to F-actin. The changes in the filament flexibility and orientation of the actin-bound fluorophores produced by S1 binding to actin in the absence of ATP were substantially diminished by subtilisin cleavage of actin. The results suggest that loop 38-52 plays an important role, not only in maintaining the F-actin structure, but also in the conformational transitions in actin accompanying the strong binding of the myosin heads that may be essential for the generation of force and movement during actin-myosin interaction.

[Cleavage of DNA-binding loops of actin by subtilisin prevent formation of a strong type of myosin binding with actin]. / Rasshcheplenie subtilizinom DNK-sviazyvaiushchei petli aktina predotvrashchaet formirovanie sil'noi formy sviazyvaniia miozina s aktinom.

In order to elucidate the role of DNA-binding loop of actin (amino acid residues 38-52) in mechanisms of muscle contraction, polarizational fluorimetry and ghost muscle fibers, containing thin filaments reconstructed by intact and subtilisin-cleaved G-actin were used. The thin filaments were modified by fluorescent probes rhodamin-phalloidin and 1,5-IAEDANS. Changes in orientation and mobility of the probes were considered as an indication of changes in actin conformation. The stage AM of ATP hydrolysis cycle was simulated. For this purpose, thin filaments were decorated by myosin subfragment-1 (S1) in the absence of nucleotide. It has been shown that S1 binding to actin is accompanied by changes in orientation and mobility of the fluorescent probes. For intact filaments, the changes of these parameters indicate the formation of a strong binding between S1 and actin. Cleavage of DNA-binding loop by subtilisin markedly inhibits this effect. The cleavage of actin by subtilisin has also been shown to diminish the changes in fiber birefringence, which takes place at the formation of F-actin-S1 complex in the muscle fiber. The spatial organization of the actin DNA-binding loop is suggested to play an important role in determining the character of myosin interaction with actin in the ATP hydrolysis cycle.

Six putative IQ motifs of the recombinant chicken intestinal brush border myosin I are involved in calmodulin binding.

Chicken brush border myosin I has up to six IQ sequence motifs at which it may bind calmodulin. To determine the relative contributions of these motifs to calmodulin binding, fusion deletion fragments were expressed in Escherichia coli and their ability to bind calmodulin was assessed by the gel overlay technique. The first three N-terminal IQ sites showed strong binding with calmodulin. Surprisingly, the last three incomplete IQ motifs also contributed substantial calmodulin binding. The first and fourth IQ sites bound calmodulin but tended to reduce binding in combination with other sites. The data indicate that interactions among all six IQ motifs contribute to the ability of myosin I to bind calmodulin.

Comparison of the effects of calponin and a 38-kDa caldesmon fragment on formation of the "strong-binding" state in ghost muscle fibers.

We studied the conformational changes in actin filaments induced by the binding of calponin or a 38-kDa fragment of caldesmon, two actin-binding proteins known to inhibit actin-activated ATP hydrolysis by phosphorylated smooth muscle myosin. The F-actinin myosin-free muscle fibers (ghost fibers) was labeled with fluorescein-5-maleimide and the conformational change in actin was determined by polarized fluorimetry. Data show that both calponin and the 38-kDa caldesmon fragment inhibit the conformational changes in F-actin that are compatible with the "strong-binding" state between myosin heads and actin. Tropomyosin slightly reduced the effect produced by calponin, but enhances the effect produced by the 38-kDa caldesmon fragment.

[The effect of a caldesmon fragment with a mol. weight of 38 kDa and calponin on the capacity of actin to form a "strong" form of binding with myosin heads]. / Vliianie fragmenta kal'desmona s mol. massoi 38 kDa i kal'ponina na sposobnsot' aktina obrazovyvat' s golovkami miozina "sil'nuiu" formu sviazyvaniia.

Effect of calponin and 38 kD actin-binding proteolytic fragment of caldesmon on actin structure alterations, initiated by decoration of thin filaments by N-ethylmaleimide-modified skeletal myosin subfragment-1 (NEM-S1) and by phosphorylated smooth heavy meromyosin (pHMM), has been studied by polarized fluorimetry. F-actin of myosin-free ghost fiber was labeled with fluorescent probe fluoroscein-5-maleimide. Both the actin-binding regulatory proteins have been demonstrated to inhibit conformational changes of actin typical for the "strong" binding of myosin head to actin. Tropomyosin weakens the inhibitory effect of calponin and markedly increases the effect of the 38 kD fragment of caldesmon. The results indicate similarity of molecular mechanisms of the regulation of muscle contraction by calponin and the actin-binding fragment of caldesmon. It is proposed that the regulation of smooth muscle contraction by calponin and caldesmon is carried out via the inhibition of the formation of the stage AM in ATP hydrolysis cycle.

[Calponin inhibits the strong type of myosin binding with actin]. / Kal'ponin ingibiruet sil'nuiu formu sviazyvaniia miozina s aktinom.

The effects of calponin on conformational changes in actin caused by modelling of "strong" binding between actin and myosin heads have been studied using polarization fluorimetry. "Strong" binding was modelled by decoration of thin filaments by myosin subfragment I modified by N-ethylmaleimide (NEM-SI) or phosphorylated heavy meromyosin (pHMM). Changes in the actin structure were followed by orientation and mobility of the fluorescent probe--the rhodamine-phalloidin complex. It has been found that calponin cooperatively changes the actin conformation, the maximal conformational changes in actin thin filaments being observed at the calponin/actin molar ratio of about 1:7. The conformational changes in actin induced by NEM-SI and pHMM are typical of strong binding. Calponin inhibited this effect. It is suggested that the mechanism of calponin regulation of smooth muscle contractility is tightly coupled to the inhibition of formation of the stage limiting the rate of ATP hydrolysis by actomyosin.

Effect of lysine methylation and other ATPase modulators on the active site of myosin subfragment 1.

Many and diverse modifications of the myosin subfragment 1 (S-1) increase (modulate) its ATPase activity, including interaction of this particle with actin; a recent addition to these modifications is the extensive lysine modification of S-1 that seems prerequisite to crystallizing it for structure analysis. In this study we first established kinetically the ATPase modulations induced by various treatments of the myosin S-1 enzyme, and we also measured two properties of the S-1 active site--the affinity with which the site binds (a fluorescent analog of) the enzymatic nucleotide product and the access that a fluorescence quencher has to the bound ADP product--in an effort to get at the mechanism of modulation. Modulations achieved by substituting Ca2+ for the normal Mg2+ cocatalyst or by substituting Cl- for the normal carboxylate anion seem due to the product being held more loosely by the modulated enzyme. In other illustrative modulations (lysine methylation, or alkylation of Cys-707, or transition from neutral pH to pH 9.2) nucleotide product affinity and access to quencher do change, but not in a pattern explained simply by a lifting of product inhibition. Lysine methylation results in weaker binding of nucleotide product.

The effect of Ca2+ on the conformation of tropomyosin and actin in regulated actin filaments with or without bound myosin subfragment 1.

The effects of Ca2+ and myosin subfragment 1 on the conformation of tropomyosin and actin in regulated actin filaments in ghost fibers were investigated by means of the polarized fluorescence technique. Regulated thin filaments were reconstituted in skeletal muscle ghost fibers by incorporation into the fibers of either skeletal muscle troponin-tropomyosin or smooth-muscle caldesmon-calmodulin-tropomyosin complexes. Tropomyosin and actin were specifically labeled with fluorescent probes, 1,5-IAEDANS and phalloidin-rhodamine, respectively. Analysis of the fluorescence parameters indicated that the binding of Ca2+ to regulated actin filaments induces conformational changes in tropomyosin and actin that lead to the strengthening of the interaction between these two proteins and weakening of the binding of actin monomers in the filament. These changes become larger when regulated actin forms rigor links with myosin subfragment 1. No notable alterations in the position of tropomyosin relative to actin in the frontal plane of the fiber were detected either upon binding of Ca2+ or upon the additional binding of myosin subfragment 1 to regulated actin.

Polarization microfluorimetry study of interaction between myosin head and F-actin in muscle fibers.

Changes in conformation of F-actin induced by the binding of myosin molecule subfragment 1 were studied in myosin-free single ghost muscle fibers with the method of polarization microfluorimetry. The modification of the structure of subfragment 1 by proteolytic digestion with one or two cuts in subfragment 1 or degradation of 50 kDa domain did not influence the character of changes in the conformation of F-actin. The use of preparations of subfragment 1 devoid of the 20 kDa domain or both cross-linked SH1 and SH2-groups changed the character of conformational rearrangements in F-actin. The present data show that a site of interaction with actin in the 20 kDa domain plays a key role in inducing the changes in actin conformation corresponding to a "strong" form of the binding. It is supposed that transmission of changes in the conformation of the myosin head to F-actin might be important for muscle contraction.

Troponin I and caldesmon restrict alterations in actin structure occurring on binding of myosin subfragment 1.

The effect of troponin I and caldesmon on phalloidin-rhodamine- and 1,5-IAEDANS-labelled actin in skeletal muscle ghost fibers was investigated by polarized fluorescence. Both these proteins inhibited the structural alterations in the actin monomer and the increase of flexibility of actin filaments occurring on binding of myosin heads, and their effects were potentiated by tropomyosin. This immobilization of the actin filament through troponin I and caldesmon seems to originate from restriction of the relative motions of the two domains within the monomer.

[The ghost muscle fiber with thin filaments reconstructed from nonmuscle actin--a model for studying the cytoskeleton using polarization microfluorimetry]. / Tenevoe myshechnoe volokno s rekonstruirovannymi iz nemyshechnogo aktina tonkimi nitiami--model' dlia izucheniia tsitoskeleta s pomoshch'iu poliarizatsionnoi mikrofluorimetrii.

In muscle fibers which are free of myosin, tropomyosin and troponin thin filaments were reconstructed from muscle and non-muscle G-actin modified with 1,5-IAEDANS. Using polarized microfluorimetry it was shown that actin in such filaments maintained the ability to respond to conformational changes during actin interaction with subfragment of myosin (S1). The models of muscle fibers with reconstructed from non-muscle actin thin filaments are supposed to use for investigation of mechanisms of cell cytoskeleton functions with the help of polarized microfluorimetry.

[Caldesmon and myosin subfragment-1 act differently on the structural state of 1,5-IAEDANS-modified tropomyosin in ghost muscle fibers]. / Kal'desmon i subfragment-1 miozina po-raznomu vliiaiut na strukturnoe sostoianie modifitsirovannogo 1,5-IAEDANS tropomiozina v tenevykh myshechnykh voloknakh.

The effect of caldesmon (CD) and subfragment 1 of myosin (S1) on the structural state of tropomyosin (TM) modified with N-(iodoacetyl)-N-(1-naphthyl-5-sulfo)-ethylene-diamine (1.5-IAEDANS) in single myosin-free skeletal muscle fibers was studied using polarized microfluorimetry. S1 was performed from skeletal muscles of rabbits, whereas CD and TM were prepared from the smooth muscle of chicken gizzards. An analysis of experimental data revealed that CD initiates and increases the motility of 1.5-IAEDANS-TM, while S1 decreases it. In the presence of CD S1 binding to actin is accompanied by significant changes in the fluorescent label motility. It is supposed that CD and S1 induce in TM conformational changes which interfere with the protein interaction with F-actin.

[The disappearance of the dependence of actin-myosin interaction on the phosphorylation of myosin light chains in the "freezing" of the structure of heavy meromyosin by a bifunctional reagent]. / Ischeznovenie zavisimosti aktin-miozinovogo vzaimodeistviia ot fosforilirovaniia legkikh tsepei miozina pri "zamorazhivaniia" struktury tiazhelogo meromiozina bifunktsional'nym reagentom.

Using glycerinated muscle fibers, free of myosin, tropomyosin and troponin, a study was made of the structural state of F-actin modified by N-(iodoacetyl)-N'-(1-naphthyl-5-sulfo)-ethylendiamine (1.5-IAEDANS) and by rhodaminyl--phalloin at decoration of thin filaments with a proteolytic fragment of myosin--heavy meromyosin containing phosphorylated and dephosphorylated myosin light chains. The heavy meromyosin used has three SH-groups of heavy chain SH1, SH2 and SH chi modified by bifunctional reagent N,N'-n-phenylmaleimide (SH1-SH2, SH2-SH chi). At decoration of thin filaments with heavy meromyosin, some changes in polarized fluorescence of rhodaminyl--phalloin and 1.5-IAEDANS independent of phosphorylation of myosin light chains were found. Fluorescence anisotropy of the fiber was found to depend primarily on the character of heavy chain of SH-group modification. The ability of heavy chains to change their conformations is supposed to play an important role in the mechanism of myosin system modulation of muscle contraction.

[The effect of proteolysis of myosin heads on conformational changes induced by them in F-actin]. / Vliianie proteoliza miozinovykh golovok na indutsiruemye imi strukturnye perestroiki F-aktina.

The effect of limited proteolysis of myosin subfragment I (S1) on conformational changes in F-actin during the formation of a rigor F-actin-S1 complex was studied, using polarized microfluorimetry. Upon the decoration of thin filaments made up of rhodaminyl-phalloin modified F-actin with subfragment I, the anisotropy of fluorescence increased. Limited proteolysis of S1 at the junctions of 27 kD-70 kD and 27 kD-50 kD-20 kD fragments as well as the destruction of the 50 kD fragment by methanol had no effect on the nature of these changes. It was assumed that during the formation of the actomyosin complex the conformational changes in F-actin were induced by the 20 kD fragment. The 27 kD and 50 kD fragments seemed to exert a weak influence on action conformation within the complex.