Svetlana V. Baranova

HIV-1 integrase-hydrolyzing IgM antibodies from sera of HIV-infected patients

IgG abzymes (Abzs) with different catalytic activities are a distinctive feature of various autoimmune (AI) diseases. At the same time, data concerning IgMs with catalytic activities are very limited. Electrophoretically and immunologically homogeneous IgMs were isolated from the sera of acquired immunodeficiency syndrome (AIDS) patients by chromatography on several affinity sorbents. Several rigid criteria have been applied to show that the integrase (IN)-hydrolyzing activity is an intrinsic property of IgMs from HIV-infected patients but not from healthy donors. We present evidence showing that 22 of 24 (91.7%) IgMs purified from the sera of HIV-infected patients specifically hydrolyze only HIV IN but not many other tested proteins. Usually, proteolytic antibodies of AI patients are serine protease-like or metal dependent. Only 30% of IN-hydrolyzing IgMs were inhibited by specific inhibitors of serine proteases and 60% by inhibitors of metal-dependent proteases. Unusually, a significant reduction of the activity by specific inhibitors of acidic (in 20% of IgM preparations) and thiol proteases (in 100% of IgM preparations) was observed. Although HIV infection leads to formation of antibodies to many viral and human antigens, possible biological roles for most of them are unknown. Since anti-IN IgG can efficiently hydrolyze IN, a positive role of Abzs in counteracting the infection cannot be excluded. In addition, detection of IN-hydrolyzing activity can be useful for diagnostic purposes and for assessment of the immune status in AIDS patients.

HIV-1 integrase-hydrolyzing antibodies from sera of HIV-infected patients

Autoantibodies with enzymic activities (abzymes) are a distinctive feature of autoimmune diseases. It was interesting whether Abs from patients with viral diseases can hydrolyze viral proteins. Electrophoretically and immunologically homogeneous IgGs were isolated from sera of AIDS patients by chromatography on several affinity sorbents. We present evidence showing that 89.5% IgGs purified from the sera of HIV-infected patients using several affinity resins including Sepharose with immobilized integrase specifically hydrolyze only HIV integrase (IN) but not many other tested proteins. Several rigid criteria have been applied to show that the IN-hydrolyzing activity is an intrinsic property of AIDS IgGs but not from healthy donors. Similar to autoimmune proteolytic abzymes, IN-hydrolyzing IgGs from some patients were inhibited by specific inhibitors of serine and metal-dependent proteases but a significant inhibition of the activity by specific inhibitors of acidic- and thiol-like proteases was observed for the first time. Although HIV infection leads to formation of Abs to many viral and human antigens, no possible biological role for most of them is known. Since anti-IN IgG can efficiently hydrolyze IN, a positive role of abzymes in counteracting the infection cannot be excluded. In addition, detection of IN-hydrolyzing activity can be useful for diagnostic purposes and for estimation of the immune status in AIDS patients.

In vitro initial attachment of HIV-1 integrase to viral ends: control of the DNA specific interaction by the oligomerization state

HIV-1 integrase (IN) oligomerization and DNA recognition are crucial steps for the subsequent events of the integration reaction. Recent advances described the involvement of stable intermediary complexes including dimers and tetramers in the in vitro integration processes, but the initial attachment events and IN positioning on viral ends are not clearly understood. In order to determine the role of the different IN oligomeric complexes in these early steps, we performed in vitro functional analysis comparing IN preparations having different oligomerization properties. We demonstrate that in vitro IN concerted integration activity on a long DNA substrate containing both specific viral and nonspecific DNA sequences is highly dependent on binding of preformed dimers to viral ends. In addition, we show that IN monomers bound to nonspecific DNA can also fold into functionally different oligomeric complexes displaying nonspecific double-strand DNA break activity in contrast to the well known single strand cut catalyzed by associated IN. Our results imply that the efficient formation of the active integration complex highly requires the early correct positioning of monomeric integrase or the direct binding of preformed dimers on the viral ends. Taken together the data indicates that IN oligomerization controls both the enzyme specificity and activity.

Small-angle X-ray characterization of the nucleoprotein complexes resulting from DNA-induced oligomerization of HIV-1 integrase

HIV-1 integrase (IN) catalyses integration of a DNA copy of the viral genome into the host genome. Specific interactions between retroviral IN and long terminal repeats (LTR) are required for this insertion. To characterize quantitatively the influence of the determinants of DNA substrate specificity on the oligomerization status of IN, we used the small-angle X-ray scattering (SAXS) technique. Under certain conditions in the absence of ODNs IN existed only as monomers. IN preincubation with specific ODNs led mainly to formation of dimers, the relative amount of which correlated well with the increase in the enzyme activity in the 3′-processing reaction. Under these conditions, tetramers were scarce. Non-specific ODNs stimulated formation of catalytically inactive dimers and tetramers. Complexes of monomeric, dimeric and tetrameric forms of IN with specific and non-specific ODNs had varying radii of gyration (Rg), suggesting that the specific sequence-dependent formation of IN tetramers can probably occur by dimerization of two dimers of different structure. From our data we can conclude that the DNA-induced oligomerization of HIV-1 IN is probably of importance to provide substrate specificity and to increase the enzyme activity.

Antibodies to HIV integrase catalyze site-specific degradation of their antigen

HIV-1 integrase (IN) catalyzes integration of a DNA copy of the viral genome into the host genome. In contrast to canonical proteases (trypsin, chymotrypsin and proteinase K), IgGs and IgMs isolated from HIV-infected patients by affinity chromatography on immobilized IN specifically hydrolyzed only IN but not many other tested intact globular proteins. The sites of IN cleavage determined by MALDI mass spectrometry were localized mainly within seven known immunodominant regions of IN. Thin layer chromatography analysis has shown that the abzymes (Abzs) could also cleave 17 to 22-mer oligopeptides (OPs) corresponding to the immunodominant regions of IN sequence with a much higher rate than non-specific long peptides or three- and tetrapeptides of various sequence. Therefore, a prolonged incubation of IN with AIDS IgGs and IgMs having high catalytic activity usually produces many OPs of different length. Since anti-IN IgGs and IgMs can efficiently hydrolyze IN, a positive role of the Abzs in counteracting the infection is possible.

Anti-integrase abzymes from the sera of HIV-infected patients specifically hydrolyze integrase but nonspecifically cleave short oligopeptides

In contrast to canonical proteases, total immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies (Abs) from HIV‐infected patients hydrolyze effectively only HIV integrase (IN), reverse transcriptase (RT), human casein, and serum albumin. Anti‐IN IgG and IgM isolated by chromatography on IN‐Sepharose hydrolyze specifically only IN but not many other tested proteins. Total Abs from HIV‐infected patients hydrolyze not only globular proteins but also different specific and nonspecific tri‐, tetra‐, and 20‐ to 25‐mer oligopeptides (OPs) with a higher rate than anti‐IN Abs isolated using IN‐Sepharose. A similar situation was observed for IgG from patients with multiple sclerosis and HIV‐infected patients, which after purification on myelin basic protein (MBP)–Sepharose and RT‐Sepharose specifically hydrolyze only MBP and RT, respectively. The active sites of all anti‐protein abzymes are localized on their light chains, whereas the heavy chain is responsible for the affinity of protein substrates. Interactions of intact globular proteins with both light and heavy chains of abzymes provide the specificity of protein hydrolysis. The affinity of anti‐IN and anti‐MBP abzymes for intact IN and MBP is approximately 102‐ to 105‐fold higher than for short and long specific and nonspecific OPs. The data suggest that all OPs interact mainly with the light chain of different Abs, which possesses a lower affinity for substrates, and therefore, depending on the OP sequences, their hydrolysis may be less specific or completely nonspecific. The data indicate that the relative activity of Abs not fractionated on specific protein sorbents in the hydrolysis of specific and nonspecific OPs can correspond to an average proteolytic activity of light chains of polyclonal Abs directed against many different proteins. Copyright

Antibodies from the sera of HIV-infected patients efficiently hydrolyze all human histones

Histones and their post‐translational modifications have key roles in chromatin remodeling and gene transcription. Besides intranuclear functions, histones act as damage‐associated molecular pattern molecules when they are released into the extracellular space. Administration of exogenous histones to animals leads to systemic inflammatory and toxic responses through activating Toll‐like receptors and inflammasome pathways. Here, using ELISA it was shown that sera of HIV‐infected patients and healthy donors contain autoantibodies against histones. Autoantibodies with enzymic activities (abzymes) are a distinctive feature of autoimmune diseases. It was interesting whether antibodies from sera of HIV‐infected patients can hydrolyze human histones. Electrophoretically and immunologically homogeneous IgGs were isolated from sera of HIV‐infected patients by chromatography on several affinity sorbents. We present first evidence showing that 100% of IgGs purified from the sera of 32 HIV‐infected patients efficiently hydrolyze from one to five human histones. Several rigid criteria have been applied to show that the histone‐hydrolyzing activity is an intrinsic property of IgGs of HIV‐infected patients. The relative efficiency of hydrolysis of histones (H1, H2a, H2b, H3, and H4) significantly varied for IgGs of different patients. IgGs from the sera of 40% of healthy donors also hydrolyze histones but with an average efficiency approximately 16‐fold lower than that of HIV‐infected patients. Similar to proteolytic abzymes from the sera of patients with several autoimmune diseases, histone‐hydrolyzing IgGs from HIV‐infected patients were inhibited by specific inhibitors of serine and of metal‐dependent proteases, but an unexpected significant inhibition of the activity by specific inhibitor of thiol‐like proteases was also observed. Because IgGs can efficiently hydrolyze histones, a negative role of abzymes in development of acquired immune deficiency syndrome cannot be excluded. Copyright

Catalytic antibodies from HIV-infected patients specifically hydrolyzing viral integrase suppress the enzyme catalytic activities.

Human immunodeficiency virus type 1 integrase (IN) catalyzes integration of a DNA copy of the viral genome into the host genome. It was shown previously that IN preincubation with various oligodeoxynucleotides (ODNs) induces formation of dimers and oligomers of different gyration radii; only specific ODNs stimulate the formation of catalytically active dimers. Here we have shown that preincubation of IN with specific and nonspecific ODNs leads to a significant and comparable decrease in its hydrolysis by chymotrypsin, while nonspecific ODNs protect the enzyme from the hydrolysis by trypsin worse than specific ODNs; all ODNs had little effect on the IN hydrolysis by proteinase K. In contrast to canonical proteweases, IgGs from HIV‐infected patients specifically hydrolyze only IN. While d(pT)n markedly decreased the IgG‐dependent hydrolysis of IN, d(pA)n and d(pA)n•d(pT)n demonstrated no detectable protective effect. The best protection from the hydrolysis by IgGs was observed for specific single‐ and especially double‐stranded ODNs. Although IN was considerably protected by specific ODNs, proteolytic IgGs and IgMs significantly suppressed both 3′‐processing and integration reaction catalyzed by IN. Since anti‐IN IgGs and IgMs can efficiently hydrolyze IN, a positive role of abzymes in counteracting the infection cannot be excluded. Copyright

Diversity of integrase-hydrolyzing IgGs and IgMs from sera of HIV-infected patients.

It was previously shown that small fractions of IgGs and IgMs from the sera of AIDS patients specifically hydrolyze only HIV integrase (IN) but not many other tested proteins. Here we present evidence showing that these IgGs and IgMs are extreme catalytically heterogeneous. Affinity chromatography on IN-Sepharose using elution of IgGs (or IgMs) with different concentration of NaCl and acidic buffer separated catalytic antibodies (ABs) into many AB subfractions demonstrating different values of Km for IN and kcat. Nonfractionated IgGs and IgMs possess serine-, thiol-, acidiclike, and metal-dependent proteolytic activity. Metal-dependent activity of abzymes increases in the presence of ions of different metals. In contrast to canonical proteases having one pH optimum, initial nonfractionated IgGs and IgMs demonstrate several optima at pH from 3 to 10. The data obtained show that IN-hydrolyzing polyclonal IgG and IgM of HIV-infected patients are cocktails of anti-IN ABs with different structure of the active centers possessing various affinity to IN, pH optima, and relative rates of the specific substrate hydrolysis.

Interaction of HIV-1 Reverse Transcriptase with New Minor Groove Binders and Their Conjugates with Oligonucleotides

The effect on polymerization catalyzed by reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) was studied for new nonnatural regular minor groove binders (MGBs) containing two to four imidazole, pyrrole, or thiazole residues and for MGB conjugates with oligonucleotides. Poly(A)- oligo(U), poly(A)-oligo(dT), poly(dA)-oligo(U), poly(dA)-oligo(dT), and activated DNA were used as model template-primer complexes. The half-inhibitory concentrations (I50) of the oligopeptides were shown to strongly depend on the structure of the template-primer complex and on the number and type of heterocyclic rings in the MGB. With most compounds tested, I50 varied from 7.7 × 10−3 to 1.0 × 10−5 M. Minimal affinity of MGBs was observed with the poly(A)-oligo(U) complex. However, some imidazole- and pyrrole-containing MGBs showed unusually high affinity for the complex of RT with the template-primer duplex, I50 ranging from 3 × 10−9 to 4 × 10−8 M. In most cases, conjugates of thiazole-containing MGBs with oligonucleotides completely or partly complementary to the template had an affinity one to four orders of magnitude higher than free thiazole carboxamides. Possible causes of the dependence of I50 on the structure of template-primer complexes, MGBs, and their conjugates with oligonucleotides are considered.