Identification and characterization of repurposed small molecule inhibitors of Mycobacterium tuberculosis caseinolytic protease B (ClpB) as anti-mycobacterials.

Mycobacterium tuberculosis (Mtb) caseinolytic protease B (ClpB) is a chaperone possessing a unique ability to resolubilize the aggregated proteins in vivo. ClpB has been shown to be important for the survival of Mtb within the host. Thus, it appears to be a promising target to develop new therapeutic molecules against tuberculosis. In this study, we have screened FDA approved compounds in silico to identify inhibitors against Mtb ClpB. In our screen, several compounds interacted with ClpB. The top four compounds, namely framycetin, gentamicin, ribostamycin and tobramycin showing the highest binding energy were selected for further investigation. MD simulations and tryptophan-based quenching of ClpB-drug complexes established that the selected inhibitors stably interacted with the target protein. The inhibitor and protein complexes were found to be stabilized by hydrogen bonding, and hydrophobic interactions. Although, the compounds did not affect the ATPase activity of ClpB significantly, the protein resolubilization activity of ClpB was remarkably reduced in their presence. All four compounds potently inhibited the growth of Mtb H37Ra. The antimycobacterial activity of the compounds appears to be due the inhibition of functional ClpB oligomer formation, in turn affecting its chaperonic activity.

Inhibition of protein kinase C isozymes causes immune profile alteration and possibly decreased tumorigenesis in bladder cancer.

Protein kinase C (PRKC) isozymes activate many signaling pathways and promote tumorigenesis, which can be confirmed by masking the kinase activity. In the present study, the kinase activity of PRKC ε and ζ isozymes was masked by siRNA in bladder cancer, and the consequent gene profile was evaluated. Here, we show that the commonly dysregulated genes affected by both the isozymes were the chemokines (CXCL8 & CXCL10), adhesion molecules (ICAM1, SPP1, MMP3, VEGFA) and mutated isoform of TP53. As these same genes were upregulated in bladder cancer patients, the activity of the kinase in downregulating them is confirmed. These genes are associated with regulating the tumor microenvironment, proliferation and differentiation of cancer cells and poor prognosis. The effect of kinase masking in downregulating these genes in bladder cancer indicates the benefits PRKC inhibitors may have in managing these patients.

Role of a substrate binding pocket in the amino terminal domain of Mycobacterium tuberculosis caseinolytic protease B (ClpB) in its function.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis when infects the host encounters several stresses within the host, resulting in aggregation of its proteins. To resolve this problem Mtb uses chaperones to either repair the damage or degrade the aggregated proteins. Mtb caseinolytic protein B (ClpB) helps in the prevention of aggregation and also resolubilization of aggregated proteins in bacteria, which is important for the survival of Mtb in the host. To function optimally, ClpB associates with its co-partners DnaK, DnaJ, and GrpE. The role of N-terminal domain (NTD) of Mtb ClpB in its function is not well understood. In this context, we investigated the interaction of three substrate mimicking peptides with the NTD of Mtb ClpB in silico. A substrate binding pocket, within the NTD of ClpB comprising of residues L136, R137, E138, K142, R144, R148, V149, Y158, and Y162 forming an ɑ-helix was thus identified. The residues L136 and R137 of the ɑ-helix were found to be important for the interaction of DnaK to ClpB. Further, nine single alanine recombinant variants of the identified residues were generated. As compared to the wild-type Mtb ClpB all the Mtb ClpB variants generated in this study were found to have reduced ATPase and protein refolding activity indicating the importance of the substrate binding pocket in ClpB function. The study demonstrates that the NTD of Mtb ClpB is important for its substrate interaction activity, and the substrate binding pocket identified in this study plays a crucial role in this interaction.Communicated by Ramaswamy H. Sarma.

Immuno-informatic Prediction of B and T cell Epitopes of Cysteine Protease Allergen from Phaseolus vulgaris with Cross-reactive Potential and Population Coverage.

BACKGROUND: In-silico mapping of epitopes by immune-informatics has simplified the efforts towards understanding antigen-antibody interactions. The knowledge of allergen epitopes may help in advancing the diagnosis and therapy of allergic diseases. OBJECTIVE: This study was intended to identify B and T cell epitopes of cysteine protease allergen of Phaseolus vulgaris. METHODS: Modeller 9v20 software was used for the generation of three-dimensional model of cysteine protease and quality assessment was performed using SAVES webserver and other in silico software. Linear and conformational B and T cell epitopes were predicted via immuno-informatics based computational servers. Epitopes were synthesized and their immunoreactivity was analyzed using specific IgE ELISA with food allergy positive patient's sera. Cellular immune response of peptides was determined through basophil activation assay. Consurf and SDAP (property distance) were used to examine the evolutionary conservancy and potential cross-reactivity of predicted epitopes. MSA based positional conservancy between HDM allergen epitopes and predicted peptides was also established using IEDB epitope database. Finally, population coverage for each promiscuous T cell epitope was predicted using IEDB population coverage analysis tool. RESULTS: Cysteine protease structure was derived by homology modeling and combination of bioinformatic tools predicted three B- and three T-cell peptides by consensus method and validated computationally. ELISA with kidney bean sensitive patient's sera showed higher IgE binding of B-cell peptides as compared to T-cell or control peptides. Epitope conservancy revealed B-cell epitopes being upto 95% conserved in comparison to variable T-cell epitopes (upto 69%). B-cell peptides were crossreactive with homologous allergens based on PD values. Structural comparison of cysteine protease with Der p 1 and Der f 1 showed similar epitopic regions, validating the prediction accuracy of epitopes. Promiscuous T-cell epitopes binding to broad-spectrum class-II MHC alleles demonstrated the distribution of T-cell peptides world-wide (30-98%) and in Asian population (99%). CONCLUSION: The current approach can be applied for identification of epitopes. Analysis of crossreactive and widely-distributed specific epitopes of allergen and knowledge about their interactive surfaces will help in understanding of food allergy and related immune responses.

Role of HrcA in stress management in Mycobacterium tuberculosis.

AIM: The current study aims to understand the role of HrcA in stress response of M. tuberculosis. METHODS AND RESULTS: In this study, using an hrcA knock out mutant of M. tuberculosis it is demonstrated that the heat shock repressor, HrcA is important for countering environmental stresses pathogen faces within the host during the infection process. Also, with scanning electron microscopy, it has been shown that HrcA plays a role in maintaining the morphology and cell size of the pathogen as disruption of the hrcA gene resulted in significantly elongated bacilli. Furthermore, heat shock proteins like ClpC1, ClpB, DnaK, GroEL2, GroEL1, DnaJ2 and GroES were detected in the secretome of M. tuberculosis by mass spectrometric analysis. The study also demonstrates a strong humoral response against M. tuberculosis heat shock proteins in H37 Rv -infected mice sera. CONCLUSION: The study establishes that though hrcA is not an essential gene for M. tuberculosis, it regulates the expression of heat shock proteins during infection and disruption of hrcA gives a survival advantage to the pathogen during stress conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: HrcA plays an important role in maintaining a fine balance of heat shock proteins during infection to give adequate survival advantage and also evade immune detection.

Antimicrobial Activity of Human Eosinophil Granule Proteins.

Eosinophils secrete a number of proinflammatory mediators that include cytokines, chemokines, and granule proteins which are responsible for the initiation and maintenance of inflammatory responses. The eosinophil granule proteins, ECP, EDN, MBP, and EPO, possess antimicrobial activity against bacteria, helminths, protozoa, and viruses. In this chapter, we describe various assays used to detect and quantitate the antimicrobial activities of eosinophil granule proteins, particularly ECP and EDN. We have taken a model organism for each assay and described the method for antiviral, antihelminthic, antiprotozoan, and antibacterial activity of purified eosinophil granule proteins.

ClpB is an essential stress regulator of Mycobacterium tuberculosis and endows survival advantage to dormant bacilli.

The ability to tolerate multiple host derived stresses, resist eradication and persist within the infected individuals is central to the pathogenicity of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB). Mycobacterial survival is contingent upon sensing environmental perturbations and initiating a fitting response to counter them. Therefore, understanding of molecular mechanisms underlying stress tolerance and sensing in Mtb is critical for devising strategies for TB control. Our study aims to delineate the role of ClpB, a heat shock protein of Hsp100 family, in the general stress response and persistence mechanisms of Mtb. We demonstrate that Mtb requires ClpB to survive under stressful conditions. Additionally, we show that ClpB is necessary for the bacteria to persist in latency-like conditions such as prolonged hypoxia and nutrient-starvation. The disruption of ClpB results in aberrant cellular morphology, impaired biofilm formation and reduced infectivity of Mtb ex vivo. Our study also reports an alternative role of ClpB as a chaperokine which elicits inflammatory response in host. We conclude that ClpB is essential for Mtb to survive within macrophages, and plays a crucial part in the maintenance of dormant Mtb bacilli in latent state. The absence of ClpB in human genome makes it an attractive choice as drug target for TB.

Mechanism of HrcA function in heat shock regulation in Mycobacterium tuberculosis.

Molecular chaperones are a conserved family of proteins that are over-expressed in response to heat and other stresses. The regulation of expression of chaperone proteins plays a vital role in pathogenesis of various bacterial pathogens. In M. tuberculosis, HrcA and HspR negatively regulate heat shock protein operons by binding to their cognate DNA elements, CIRCE and HAIR respectively. In this study, we show that M. tuberculosis HrcA is able to bind to its cognate CIRCE DNA element present in the upstream regions of groES and groEL2 operons only with the help of other protein(s). It is also demonstrated that M. tuberculosis HrcA binds to a CIRCE like DNA element present in the upstream region of hrcA gene suggesting its auto-regulatory nature. In addition, we report the presence of a putative HAIR element in the upstream region of groES operon and demonstrate the binding of HspR to it. In vitro, HrcA inhibited the DNA binding activity of HspR in a dose-dependent manner. The current study demonstrates that M. tuberculosis HrcA requires other protein(s) to function, and the heat shock protein expression in M. tuberculosis is negatively regulated jointly by HrcA and HspR.

The amino-terminal domain of Mycobacterium tuberculosis ClpB protein plays a crucial role in its substrate disaggregation activity.

Mycobacterium tuberculosis (Mtb) is known to persist in extremely hostile environments within host macrophages. The ability to withstand such proteotoxic stress comes from its highly conserved molecular chaperone machinery. ClpB, a unique member of the AAA+ family of chaperones, is responsible for resolving aggregates in Mtb and many other bacterial pathogens. Mtb produces two isoforms of ClpB, a full length and an N-terminally truncated form (ClpB∆N), with the latter arising from an internal translation initiation site. It is not clear why this internal start site is conserved and what role the N-terminal domain (NTD) of Mtb ClpB plays in its function. In the current study, we functionally characterized and compared the two isoforms of Mtb ClpB. We found the NTD to be dispensable for oligomerization, ATPase activity and prevention of aggregation activity of ClpB. Both ClpB and ClpB∆N were found to be capable of resolubilizing protein aggregates. However, the efficiency of ClpB∆N at resolubilizing higher order aggregates was significantly lower than that of ClpB. Further, ClpB∆N exhibited reduced affinity for substrates as compared to ClpB. We also demonstrated that the surface of the NTD of Mtb ClpB has a hydrophobic groove that contains four hydrophobic residues: L97, L101, F140 and V141. These residues act as initial contacts for the substrate and are crucial for stable interaction between ClpB and highly aggregated substrates.

Insight into the functional role of unique determinants in RNA component of RNase P of Mycobacterium tuberculosis.

RNase P, an essential ribonucleoprotein enzyme is involved in processing 5' end of pre-tRNA molecules. All bacterial RNase P holoenzymes, including that of Mycobacterim tuberculosis, an important human pathogen contain a catalytically active RNA subunit and a protein subunit. However, the mycobacterial RNA is larger than typical bacterial RNase P RNAs. It contains the essential core structure and many unique features in the peripheral elements. In the current study, an extensive mutational analysis was performed to analyze the function of the unique features in P12, P15.A, P18 and P19 helices in the mycobacterial RNase P RNA. The study demonstrates that P12 interacts with monovalent and divalent ions and is important for the function of mycobacterial holoenzyme. The helices, P15.A and P18 appear to interact with ammonium and magnesium ions, respectively. P19 is involved in the thermostability of the RNA component as well as interaction with ammonium ions. A homology model of M. tuberculosis RNase P RNA indicates many new inter- and intra-helical interactions. The significance of the unique interactions paves way towards understanding the differential functioning of M. tuberculosis RNase P RNA, for exploring specific inhibition of the same in the pathogen to contain infection.

A ribonuclease inhibitor resistant dimer of human pancreatic ribonuclease displays specific antitumor activity.

Human pancreatic ribonuclease (HPR) and bovine seminal ribonuclease (BS-RNase) are members of the RNase A superfamily. HPR is monomeric, whereas BS-RNase is dimeric. BS-RNase has strong antitumor and cytotoxic activities. However, HPR lacks cytotoxic activity as it is inactivated by intracellular cytosolic ribonuclease inhibitor (RI). Earlier, an RI-resistant cytotoxic variant of HPR, termed HPR-KNE was generated which contained three residues Lys7, Asn71 and Glu111 of HPR, known to interact with RI, mutated to alanine. In this study, we have engineered HPR to develop two dimeric RI-resistant molecules having anti-tumor activity. By incorporating two cysteines in HPR and HPR-KNE, we generated disulfide linked dimeric HPR, and a dimer of HPR-KNE, termed as HPR-D and HPR-KNE-D respectively. HPR-KNE-D was resistant towards inhibition by RI, and was found to be highly toxic to a variety of cells. On J774A.1 cells HPR-KNE-D was >375-fold more cytotoxic than HPR, and 15-fold more toxic than HPR-D. Further, on U373 cells HPR-KNE-D was >65-fold more cytotoxic than HPR, and 9-fold more toxic than HPR-D. The study demonstrates that combining dimerization and RI-resistance results in providing potent anti-tumor activity to HPR. The cytotoxic variants of HPR will be useful in designing protein therapeutics with low immunogenicity.

Mouse eosinophil associated ribonucleases: Mechanism of cytotoxic, antibacterial and antiparasitic activities.

Ribonuclease A family is a group of proteins having similar structures and catalytic mechanism but different functions. Human eosinophil granules contain two ribonucleases belonging to the RNase A family, eosinophil cationic protein (ECP) and eosinophil derived neurotoxin (EDN). In mouse, 15 orthologs of EDN and ECP, called mouse eosinophil associated ribonucleases (mEARs) have been reported which are expressed under different pathophysiological conditions. In this study, we have characterized mEAR2, mEAR5, mEAR7 and mEAR11, and compared them with ECP for their catalytic, cytotoxic, antibacterial and antiparasitic activities. All four mEARs had cytotoxic, antibacterial and antiparasitic activities. Generally, mEAR5 and mEAR2 were more cytotoxic than mEAR7, mEAR11 and ECP. The antimicrobial activities of mEAR7 and mEAR5 were higher than those of mEAR11 and mEAR2. The cytotoxic activity appeared to be associated with the basicity and RNase activity of mEARs, whereas no such correlation was observed for antimicrobial activities. The differential selective expression of mEARs under various pathophysiological conditions may be associated with the different biological activities of various mEARs.

Denatured states of yeast cytochrome c induced by heat and guanidinium chloride are structurally and thermodynamically different.

A sequence alignment of mammalian cytochromes c with yeast iso-1-cytochrome c (y-cyt-c) shows that the yeast protein contains five extra N-terminal residues. We have been interested in understanding the question: What is the role of these five extra N-terminal residues in folding and stability of the protein? To answer this question we have prepared five deletants of y-cyt-c by sequentially removing these extra residues. During our studies on the wild type (WT) protein and its deletants, we observed that the amount of secondary structure in the guanidinium chloride (GdmCl)-induced denatured (D) state of each protein is different from that of the heat-induced denatured (H) state. This finding is confirmed by the observation of an additional cooperative transition curve of optical properties between H and D states on the addition of different concentrations of GdmCl to the already heat denatured WT y-cyt-c and its deletants at pH 6.0 and 68°C. For each protein, analysis of transition curves representing processes, native (N) state ↔ D state, N state ↔ H state, and H state ↔ D state, was done to obtain Gibbs free energy changes associated with all the three processes. This analysis showed that, for each protein, thermodynamic cycle accommodates Gibbs free energies associated with transitions between N and D states, N and H states, and H and D states, the characteristics required for a thermodynamic function. All these experimental observations have been supported by our molecular dynamics simulation studies.

Functional role of putative critical residues in Mycobacterium tuberculosis RNase P protein.

RNase P is involved in processing the 5' end of pre-tRNA molecules. Bacterial RNase P contains a catalytic RNA subunit and a protein subunit. In this study, we have analyzed the residues in RNase P protein of M. tuberculosis that differ from the residues generally conserved in other bacterial RNase Ps. The residues investigated in the current study include the unique residues, Val27, Ala70, Arg72, Ala77, and Asp124, and also Phe23 and Arg93 which have been found to be important in the function of RNase P protein components of other bacteria. The selected residues were individually mutated either to those present in other bacterial RNase P protein components at respective positions or in some cases to alanine. The wild type and mutant M. tuberculosis RNase P proteins were expressed in E. coli, purified, used to reconstitute holoenzymes with wild type RNA component in vitro, and functionally characterized. The Phe23Ala and Arg93Ala mutants showed very poor catalytic activity when reconstituted with the RNA component. The catalytic activity of holoenzyme with Val27Phe, Ala70Lys, Arg72Leu and Arg72Ala was also significantly reduced, whereas with Ala77Phe and Asp124Ser the activity of holoenzyme was similar to that with the wild type protein. Although the mutants did not suffer from any binding defects, Val27Phe, Ala70Lys, Arg72Ala and Asp124Ser were less tolerant towards higher temperatures as compared to the wild type protein. The Km of Val27Phe, Ala70Lys, Arg72Ala and Ala77Phe were >2-fold higher than that of the wild type, indicating the substituted residues to be involved in substrate interaction. The study demonstrates that residues Phe23, Val27 and Ala70 are involved in substrate interaction, while Arg72 and Arg93 interact with other residues within the protein to provide it a functional conformation.

Influence of Conformation of M. tuberculosis RNase P Protein Subunit on Its Function.

RNase P is an essential enzyme that processes 5' end leader sequence of pre-tRNA to generate mature tRNA. The bacterial RNase Ps contain a RNA subunit and one protein subunit, where the RNA subunit contains the catalytic activity. The protein subunit which lacks any catalytic activity, relaxes the ionic requirements for holoenzyme reaction and is indispensable for pre-tRNA cleavage in vivo. In the current study, we reconstituted the M. tuberculosis RNase P holoenzyme in vitro. We prepared the RNase P protein through two different strategies that differ in the conditions under which the recombinant M. tuberculosis protein, expressed in E. coli was purified. The mycobacterial RNase P protein which was purified under native conditions subsequent to isolation from inclusion bodies and in vitro renaturation, was capable of cleaving pre-tRNA specifically without the requirement of RNase P RNA. However, the preparation that was purified under denaturing conditions and refolded subsequently lacked any inherent pre-tRNA processing activity and cleaved the substrate only as a component of the holoenzyme with the RNA subunit. We found that the two RNase P protein preparations attained alternative conformations and differed with respect to their stability as well.

Insight into the role of histidine in RNR motif of protein component of RNase P of M. tuberculosis in catalysis.

RNase P, a ribonucleoprotein endoribonuclease, is involved in the 5' end processing of pre-tRNAs, with its RNA component being the catalytic subunit. It is an essential enzyme. All bacterial RNase Ps have one RNA and one protein component. A conserved RNR motif in bacterial RNase P protein components is involved in their interaction with the RNA component. In this work, we have reconstituted the RNase P of M. tuberculosis in vitro and investigated the role of a histidine in the RNR motif in its catalysis. We expressed the protein and RNA components of mycobacterial RNase P in E. coli, purified them, and reconstituted the holoenzyme in vitro. The histidine in RNR motif was mutated to alanine and asparagine by site-directed mutagenesis. The RNA component alone showed activity on pre-tRNA(ala) substrate at high magnesium concentrations. The RNA and protein components associated together to manifest catalytic activity at low magnesium concentrations. The histidine 67 in the RNR motif of M. tuberculosis RNase P protein component was found to be important for the catalytic activity and stability of the enzyme. Generally, the RNase P of M. tuberculosis functions like other bacterial enzymes. The histidine in the RNR motif of M. tuberculosis appears to be able to substitute optimally for asparagine found in the majority of the protein components of other bacterial RNase P enzymes.

Targeting c-kit receptor in neuroblastomas and colorectal cancers using stem cell factor (SCF)-based recombinant bacterial toxins.

Autocrine activation of c-kit (KIT receptor tyrosine kinase) has been postulated to be a potent oncogenic driver in small cell lung cancer, neuroblastoma (NB), and poorly differentiated colorectal carcinoma (CRC). Although targeted therapy involving tyrosine kinase inhibitors (TKIs) such as imatinib mesylate is highly effective for gastrointestinal stromal tumor carrying V560G c-kit mutation, it does not show much potential for targeting wild-type KIT (WT-KIT). Our study demonstrates the role of stem cell factor (SCF)-based toxin conjugates for targeting WT-KIT-overexpressing malignancies such as NBs and CRCs. We constructed SCF-based recombinant bacterial toxins by genetically fusing mutated form of natural ligand SCF to receptor binding deficient forms of Diphtheria toxin (DT) or Pseudomonas exotoxin A (ETA') and evaluated their efficacy in vitro. Efficient targeting was achieved in all receptor-positive neuroblastoma (IMR-32 and SHSY5Y) and colon cancer cell lines (COLO 320DM, HCT 116, and DLD-1) but not in receptor-negative breast carcinoma cell line (MCF-7) thereby proving specificity. While dose- and time-dependent cytotoxicity was observed in both neuroblastoma cell lines, COLO 320DM and HCT 116 cells, only an anti-proliferative effect was observed in DLD-1 cells. We prove that these novel targeting agents have promising potential as KIT receptor tyrosine kinase targeting system.

Role of DnaK in HspR-HAIR interaction of Mycobacterium tuberculosis.

Heat shock proteins (Hsps) are a highly conserved family of proteins. The regulation of expression of Hsps in Mycobacterium tuberculosis, is regulated both positively and negatively by alternate sigma factors and transcriptional DNA repressors, respectively. HspR is a negative regulator of expression of hsps, DnaK, ClpB, and Acr2 in M. tuberculosis. In this study, we expressed the M. tuberculosis HspR (MtHspR) in E. coli, and functionally characterized it. MtHspR independently bound to its putative cognate DNA, the HAIR element. MtHspR was found to exist in a dynamic mixture of dimeric and monomeric protein and presence of salt led to the formation of trimers which lacked the DNA binding activity. MtHspR was found to be heat stable with a Tm of 66°C. HspR-HAIR binding was stable upto 60°C suggesting that MtHspR is not the heat stress sensor. Mycobacterial DnaK was found to interact directly with MtHspR-HAIR complex in vitro in an ATP independent manner. The DnaK-HspR-HAIR binding pattern altered at high temperatures in the presence of aggregated α-casein substrate, suggesting that DnaK may indirectly be responding to heat stress in a feedback loop mechanism.

Mechanism of Anti-HIV Activity of Ribosome Inactivating Protein, Saporin.

Ribosome inactivating proteins (RIPs) are a family of proteins produced by plants, bacteria and fungi. RIPs have specific N-glycosidase activity, and they cleave a specific glycosidic bond in a universally conserved stem and loop structure within the large ribosomal RNA of all organisms. Saporin, a cytotoxic RIP from the plant Saponaria officinalis has been earlier shown to manifest its cytotoxicity by a combination of its N-glycosidase and apoptosis inducing activities. Saporin, along with many other RIPs also has strong inhibitory activity towards HIV integrase. In the current study, using two in vitro model systems, it is established that saporin inhibits propagation of HIV-1 in host cells. Saporin also showed a potent anti-HIV-1 integrase activity in vitro. Using three active site mutants of saporin, which respectively lack N-glycosidase, apoptosis inducing or both activities, it is shown that saporin's in vitro anti-HIV-1 integrase activity is independent of its N-glycosidase activity. However, for the anti-HIV activity of saporin, the apoptosis inducing activity is important.

Ribotoxin restrictocin manifests anti-HIV-1 activity through its specific ribonuclease activity.

Restrictocin, a highly specific ribonuclease produced by Aspergillus restrictus, cleaves a single phosphodiester bond in a universally conserved stem and loop structure termed sarcin/ricin loop within the large ribosomal RNA of all organisms. In the current study, we demonstrate restrictocin to manifest anti-HIV-1 activity in two model cell systems. Using two mutants of restrictocin, we further show that the anti-HIV-1 activity of restrictocin is due to its specific ribonucleolytic activity. The study suggests that restrictocin is able to recognize region(s) within HIV-1 genome as its target. Restrictocin appears to have potential as a therapeutic antiviral agent against HIV-1.