Covalently attached oligodeoxyribonucleotides induce RNase activity of a short peptide and modulate its base specificity

New artificial ribonucleases, conjugates of short oligodeoxyribonucleotides with peptides containing alternating arginine and leucine, were synthesized and characterized in terms of their catalytic activity and specificity of RNA cleavage. The conjugates efficiently cleave different RNAs within single-stranded regions. Depending on the sequence and length of the oligonucleotide, the conjugates display either G–X>>Pyr–A or Pyr–A>>G–X cleavage specificity. Preferential RNA cleavage at G–X phosphodiester bonds was observed for conjugate NH₂-Gly-[ArgLeu]₄-CCAAACA. The conjugates function as true catalysts, exhibiting reaction turnover up to 175 for 24 h. Our data show that in the conjugate the oligonucleotide plays the role of a factor which provides an ‘active‘ conformation of the peptide via intramolecular interactions, and that it is the peptide residue itself which is responsible for substrate affinity and catalysis.     

Highly efficient catalytic RNA cleavage by the cooperative action of two Cu(II) complexes embodied within an antisense oligonucleotide

Based on our recent studies of RNA cleavage by oligonucleotide–terpyridine·Cu(II) complex 5′- and/or 3′-conjugates, we designed 2′-O-methyloligonucleotides with two terpyridine-attached nucleosides at contiguous internal sites. To connect the 2′-terpyridine-modified uridine residue at the 5′-side to the 5′-O-terpyridyl nucleoside residue at the 3′-side, a dimethoxytrityl derivative of 5-hydroxypropyl-5′-O-terpyridyl-2′-deoxyuridine-3′-phosphoramidite was newly synthesized. Using this unit, we constructed two terpyridine conjugates, with either an unusual phophodiester bond or the bond extended by a propanediol(s)-containing linker. Cleavage reactions of the target RNA oligomer, under the conditions of conjugate excess in the presence of Cu(II), indicated that the conjugates precisely cleaved the RNA at the predetermined site and that one propanediol-containing linker was the most appropriate for inducing high cleavage activity. Furthermore, a comparison of the activity of the propanediol agent with those of the control conjugates with one complex confirmed that the two complexes are required for efficient RNA cleavage. The reaction of the novel cleaver revealed a bell-shaped pH–rate profile with a maximum at pH ~7.5, which is a result of the cooperative action of the complexes. In addition, we demonstrated that the agent catalytically cleaves an excess of the RNA, with the kinetic parameter k_cat/K_m = 0.118 nM^–1 h^–1.     

Modular construction for function of a ribonucleoprotein enzyme: the catalytic domain of Bacillus subtilis RNase P complexed with B. subtilis RNase P protein

The bacterial RNase P holoenzyme catalyzes the formation of the mature 5′-end of tRNAs and is composed of an RNA and a protein subunit. Among the two folding domains of the RNase P RNA, the catalytic domain (C-domain) contains the active site of this ribozyme. We investigated specific binding of the Bacillus subtilis C-domain with the B.subtilis RNase P protein and examined the catalytic activity of this C-domain–P protein complex. The C-domain forms a specific complex with the P protein with a binding constant of ~0.1 µM. The C-domain–P protein complex and the holoenzyme are equally efficient in cleaving single-stranded RNA (~0.9 min^–1 at pH 7.8) and substrates with a hairpin–loop 3′ to the cleavage site (~40 min^–1). The holoenzyme reaction is much more efficient with a pre-tRNA substrate, binding at least 100-fold better and cleaving 10–500 times more efficiently. These results demonstrate that the RNase P holoenzyme is functionally constructed in three parts. The catalytic domain alone contains the active site, but has little specificity and affinity for most substrates. The specificity and affinity for the substrate is generated by either the specificity domain of RNase P RNA binding to a T stem–loop-like hairpin or RNase P protein binding to a single-stranded RNA. This modular construction may be exploited to obtain RNase P-based ribonucleoprotein complexes with altered substrate specificity.     

Protein cofactors and substrate influence Mg2+-dependent structural changes in the catalytic RNA of archaeal RNase P

The ribonucleoprotein (RNP) form of archaeal RNase P comprises one catalytic RNA and five protein cofactors. To catalyze Mg²⁺-dependent cleavage of the 5′ leader from pre-tRNAs, the catalytic (C) and specificity (S) domains of the RNase P RNA (RPR) cooperate to recognize different parts of the pre-tRNA. While ∼250–500 mM Mg²⁺ renders the archaeal RPR active without RNase P proteins (RPPs), addition of all RPPs lowers the Mg²⁺ requirement to ∼10–20 mM and improves the rate and fidelity of cleavage. To understand the Mg²⁺- and RPP-dependent structural changes that increase activity, we used pre-tRNA cleavage and ensemble FRET assays to characterize inter-domain interactions in Pyrococcus furiosus (Pfu) RPR, either alone or with RPPs ± pre-tRNA. Following splint ligation to doubly label the RPR (Cy3-RPR_{C domain} and Cy5-RPR_{S domain}), we used native mass spectrometry to verify the final product. We found that FRET correlates closely with activity, the Pfu RPR and RNase P holoenzyme (RPR + 5 RPPs) traverse different Mg²⁺-dependent paths to converge on similar functional states, and binding of the pre-tRNA by the holoenzyme influences Mg²⁺ cooperativity. Our findings highlight how Mg²⁺ and proteins in multi-subunit RNPs together favor RNA conformations in a dynamic ensemble for functional gains.     

G-specific RNA-cleaving Conjugates of Short Peptides and Oligodeoxyribonucleotides

Abstract

Artificial ribonucleases, conjugates of short oligodeoxyribonucleotides and peptides built of arginine, leucine, proline, and serine, were synthesized and assessed in terms of ribonuclease activity and specificity of RNA cleavage. A specific group of the conjugates was identified that display T1-ribonuclease-like activity and cleave RNA predominantly at G-X sequences. Circular dichroism study of the structures of the most active conjugates, free peptide (LR)4G, and oligonucleotides revealed that conjugation of oligonucleotide to the peptide results in a specific peptide folding that possibly provides ribonuclease activity to the conjugate.     

Synthesis, cellular uptake and HIV-1 Tat-dependent trans-activation inhibition activity of oligonucleotide analogues disulphide-conjugated to cell-penetrating peptides

Oligonucleotides composed of 2′-O-methyl and locked nucleic acid residues complementary to HIV-1 trans-activation responsive element TAR block Tat-dependent trans-activation in a HeLa cell assay when delivered by cationic lipids. We describe an improved procedure for synthesis and purification under highly denaturing conditions of 5′-disulphide-linked conjugates of 3′-fluorescein labelled oligonucleotides with a range of cell-penetrating peptides and investigate their abilities to enter HeLa cells and block trans-activation. Free uptake of 12mer OMe/LNA oligonucleotide conjugates to Tat (48–58), Penetratin and R₉F₂ was observed in cytosolic compartments of HeLa cells. Uptake of the Tat conjugate was enhanced by N-terminal addition of four Lys or Arg residues or a second Tat peptide. None of the conjugates entered the nucleus or inhibited trans-activation when freely delivered, but inhibition was obtained in the presence of cationic lipids. Nuclear exclusion was seen for free delivery of Tat (48–58), Penetratin and R₉ conjugates of 16mer phosphorothioate OMe oligonucleotide. Uptake into human fibroblast cytosolic compartments was seen for Tat, Penetratin, R₉F₂ and Transportan conjugates. Large enhancements of HeLa cell uptake into cytosolic compartments were seen when free Tat peptide was added to Tat conjugate of 12mer OMe/LNA oligonucleotide or Penetratin peptide to Penetratin conjugate of the same oligonucleotide.     

Catalytic mechanism of Escherichia coli ribonuclease III: kinetic and inhibitor evidence for the involvement of two magnesium ions in RNA phosphodiester hydrolysis

Escherichia coli ribonuclease III (RNase III; EC 3.1.24) is a double-stranded(ds)-RNA-specific endonuclease with key roles in diverse RNA maturation and decay pathways. E.coli RNase III is a member of a structurally distinct superfamily that includes Dicer, a central enzyme in the mechanism of RNA interference. E.coli RNase III requires a divalent metal ion for activity, with Mg²⁺ as the preferred species. However, neither the function(s) nor the number of metal ions involved in catalysis is known. To gain information on metal ion involvement in catalysis, the rate of cleavage of the model substrate R1.1 RNA was determined as a function of Mg²⁺ concentration. Single-turnover conditions were applied, wherein phosphodiester cleavage was the rate-limiting event. The measured Hill coefficient (n_H) is 2.0 ± 0.1, indicative of the involvement of two Mg²⁺ ions in phosphodiester hydrolysis. It is also shown that 2-hydroxy-4H-isoquinoline-1,3-dione—an inhibitor of ribonucleases that employ two divalent metal ions in their catalytic sites—inhibits E.coli RNase III cleavage of R1.1 RNA. The IC₅₀ for the compound is 14 μM for the Mg²⁺-supported reaction, and 8 μM for the Mn²⁺-supported reaction. The compound exhibits noncompetitive inhibitory kinetics, indicating that it does not perturb substrate binding. Neither the O-methylated version of the compound nor the unsubstituted imide inhibit substrate cleavage, which is consistent with a specific interaction of the N-hydroxyimide with two closely positioned divalent metal ions. A preliminary model is presented for functional roles of two divalent metal ions in the RNase III catalytic mechanism.     

G-specific RNA-cleaving conjugates of short peptides and oligodeoxyribonucleotides

Artificial ribonucleases, conjugates of short oligodeoxyribonucleotides and peptides built of arginine, leucine, proline, and serine, were synthesized and assessed in terms of ribonuclease activity and specificity of RNA cleavage. A specific group of the conjugates was identified that display T1-ribonuclease-like activity and cleave RNA predominantly at G-X sequences. Circular dichroism study of the structures of the most active conjugates, free peptide (LR)4G, and oligonucleotides revealed that conjugation of oligonucleotide to the peptide results in a specific peptide folding that possibly provides ribonuclease activity to the conjugate.     

N(omega)-arginine dimethylation modulates the interaction between a Gly/Arg-rich peptide from human nucleolin and nucleic acids

We studied the interaction between a synthetic peptide (sequence Ac-GXGGFGGXGGFXGGXGG-NH₂, where X = arginine, N^ω,N^ω-dimethylarginine, DMA, or lysine) corresponding to residues 676–692 of human nucleolin and several DNA and RNA substrates using double filter binding, melting curve analysis and circular dichroism spectroscopy. We found that despite the reduced capability of DMA in forming hydrogen bonds, N^ω,N^ω-dimethylation does not affect the strength of the binding to nucleic acids nor does it have any effect on stabilization of a double-stranded DNA substrate. However, circular dichroism studies show that unmethylated peptide can perturb the helical structure, especially in RNA, to a much larger extent than the DMA peptide.     

The naturally trans-acting ribozyme RNase P RNA has leadzyme properties

Divalent metal ions promote hydrolysis of RNA backbones generating 5′OH and 2′;3′P as cleavage products. In these reactions, the neighboring 2′OH act as the nucleophile. RNA catalyzed reactions also require divalent metal ions and a number of different metal ions function in RNA mediated cleavage of RNA. In one case, the LZV leadzyme, it was shown that this catalytic RNA requires lead for catalysis. So far, none of the naturally isolated ribozymes have been demonstrated to use lead to activate the nucleophile. Here we provide evidence that RNase P RNA, a naturally trans-acting ribozyme, has leadzyme properties. But, in contrast to LZV RNA, RNase P RNA mediated cleavage promoted by Pb²⁺ results in 5′ phosphate and 3′OH as cleavage products. Based on our findings, we infer that Pb²⁺ activates H₂O to act as the nucleophile and we identified residues both in the substrate and RNase P RNA that most likely influenced the positioning of Pb²⁺ at the cleavage site. Our data suggest that Pb²⁺ can promote cleavage of RNA by activating either an inner sphere H₂O or a neighboring 2′OH to act as nucleophile.     

Sequence-specific artificial ribonucleases. I. Bis-imidazole-containing oligonucleotide conjugates prepared using precursor-based strategy

Antisense oligonucleotide conjugates, bearing constructs with two imidazole residues, were synthesized using a precursor-based technique employing post-synthetic histamine functionalization of oligonucleotides bearing methoxyoxalamido precursors at the 5′-termini. The conjugates were assessed in terms of their cleavage activities using both biochemical assays and conformational analysis by molecular modelling. The oligonucleotide part of the conjugates was complementary to the T-arm of yeast tRNA^Phe (44–60 nt) and was expected to deliver imidazole groups near the fragile sequence C₆₁-ACA-G₆₅ of the tRNA. The conjugates showed ribonuclease activity at neutral pH and physiological temperature resulting in complete cleavage of the target RNA, mainly at the C₆₃–A₆₄ phosphodiester bond. For some constructs, cleavage was completed within 1–2 h under optimal conditions. Molecular modelling was used to determine the preferred orientation(s) of the cleaving group(s) in the complexes of the conjugates with RNA target. Cleaving constructs bearing two imidazole residues were found to be conformationally highly flexible, adopting no preferred specific conformation. No interactions other than complementary base pairing between the conjugates and the target were found to be the factors stabilizing the ‘active’ cleaving conformation(s).     

RNase III cleavage demonstrates a long range RNA: RNA duplex element flanking the hepatitis C virus internal ribosome entry site

Here, we show that Escherichia coli Ribonuclease III cleaves specifically the RNA genome of hepatitis C virus (HCV) within the first 570 nt with similar efficiency within two sequences which are ~400 bases apart in the linear HCV map. Demonstrations include determination of the specificity of the cleavage sites at positions C²⁷ and U³³ in the first (5′) motif and G⁴³⁹ in the second (3′) motif, complete competition inhibition of 5′ and 3′ HCV RNA cleavages by added double-stranded RNA in a 1:6 to 1:8 weight ratio, respectively, 50% reverse competition inhibition of the RNase III T7 R1.1 mRNA substrate cleavage by HCV RNA at 1:1 molar ratio, and determination of the 5′ phosphate and 3′ hydroxyl end groups of the newly generated termini after cleavage. By comparing the activity and specificity of the commercial RNase III enzyme, used in this study, with the natural E.coli RNase III enzyme, on the natural bacteriophage T7 R1.1 mRNA substrate, we demonstrated that the HCV cuts fall into the category of specific, secondary RNase III cleavages. This reaction identifies regions of unusual RNA structure, and we further showed that blocking or deletion of one of the two RNase III-sensitive sequence motifs impeded cleavage at the other, providing direct evidence that both sequence motifs, besides being far apart in the linear RNA sequence, occur in a single RNA structural motif, which encloses the HCV internal ribosome entry site in a large RNA loop.     

Sequence-specific cleavage of the RNA strand in DNA–RNA hybrids by the fusion of ribonuclease H with a zinc finger

Ribonucleases (RNases) are valuable tools applied in the analysis of RNA sequence, structure and function. Their substrate specificity is limited to recognition of single bases or distinct secondary structures in the substrate. Currently, there are no RNases available for purely sequence-dependent fragmentation of RNA. Here, we report the development of a new enzyme that cleaves the RNA strand in DNA–RNA hybrids 5 nt from a nonanucleotide recognition sequence. The enzyme was constructed by fusing two functionally independent domains, a RNase HI, that hydrolyzes RNA in DNA–RNA hybrids in processive and sequence-independent manner, and a zinc finger that recognizes a sequence in DNA–RNA hybrids. The optimization of the fusion enzyme’s specificity was guided by a structural model of the protein-substrate complex and involved a number of steps, including site-directed mutagenesis of the RNase moiety and optimization of the interdomain linker length. Methods for engineering zinc finger domains with new sequence specificities are readily available, making it feasible to acquire a library of RNases that recognize and cleave a variety of sequences, much like the commercially available assortment of restriction enzymes. Potentially, zinc finger-RNase HI fusions may, in addition to in vitro applications, be used in vivo for targeted RNA degradation.     

RNA-ligand interactions: affinity and specificity of aminoglycoside dimers and acridine conjugates to the HIV-1 Rev response element

Semisynthetic aminoglycoside derivatives may provide a means to selectively target viral RNA sites, including the HIV-1 Rev response element (RRE). The design, synthesis, and evaluation of derivatives based upon neomycin B, kanamycin A, and tobramycin conjugates of 9-aminoacridine are presented. To evaluate the importance of the acridine moiety, a series of dimeric aminoglycosides as well as unmodified "monomeric" aminoglycosides have also been evaluated for their nucleic acid affinity and specificity. Fluorescence-based binding assays that use ethidium bromide or Rev peptide displacement are used to quantify the affinities of these compounds to various nucleic acids, including the RRE, tRNA, and duplex DNA. All the modified aminoglycosides exhibit a high affinity for the Rev binding site on the RRE (K(d) 相似文献   

Conformational change in the Bacillus subtilis RNase P holoenzyme–pre-tRNA complex enhances substrate affinity and limits cleavage rate

Ribonuclease P (RNase P) is a ribonucleoprotein complex that catalyzes the 5′ maturation of precursor tRNAs. To investigate the mechanism of substrate recognition in this enzyme, we characterize the thermodynamics and kinetics of Bacillus subtilis pre-tRNA^Asp binding to B. subtilis RNase P holoenzyme using fluorescence techniques. Time courses for fluorescein-labeled pre-tRNA binding to RNase P are biphasic in the presence of both Ca(II) and Mg(II), requiring a minimal two-step association mechanism. In the first step, the apparent bimolecular rate constant for pre-tRNA associating with RNase P has a value that is near the diffusion limit and is independent of the length of the pre-tRNA leader. Following formation of the initial enzyme–substrate complex, a unimolecular step enhances the overall affinity of pre-tRNA by eight- to 300-fold as the length of the leader sequence increases from 2 to 5 nucleotides. This increase in affinity is due to a decrease in the reverse rate constant for the conformational change that correlates with the formation of an optimal leader–protein interaction in the RNase P holoenzyme–pre-tRNA complex. Furthermore, the forward rate constant for the conformational change becomes rate limiting for cleavage under single-turnover conditions at high pH, explaining the origin of the observed apparent pK_a in the RNase P-catalyzed cleavage reaction. These data suggest that a conformational change in the RNase P•pre-tRNA complex is coupled to the interactions between the 5′ leader and P protein and aligns essential functional groups at the cleavage active site to enhance efficient cleavage of pre-tRNA.     

Rearrangement of a 1,3-trans-[Pt(NH3)2[(GXG)-N7G,N7G]] intrastrand cross-link into interstrand cross-links within RNA duplexes

The cross-linking reaction described previously in the DNA and 2′-O-methyl RNA series is extended to RNA duplexes. A 17mer single-stranded RNA containing the 1,3-trans-{Pt(NH₃)₂[(GAG)-N7G,N7G]} intrastrand chelate, named G*AG* (* indicating a platinated base) gives, upon pairing with the complementary RNA strand, the G*AG/CUC* interstrand cross-link. The rate of the reaction in 200 mM NaClO₄ is similar to that observed for DNA–RNA duplexes. It depends on the added Na⁺ or Mg²⁺ cation and on its concentration. RNA duplexes containing GA/GA or AG/AG tandem mismatches in the rearrangement triplet core were also studied. The major interstrand cross-links, G*AG/CGA* and G*AG/AGC*, are accompanied by a minor one involving the central G of the CGA or AGC complementary sequence G*AG/CG*A and G*AG/AG*C. In 200 mM NaClO₄, the G*A/GA tandem mismatch does not modify the rate of the cross-linking rearrangement whereas the AG*/AG mismatch slows it down by a factor of four. Our results reflect the predominance of the local structure of the rearrangement core over the nucleophility of the cross-linking base. They also show that the reaction could be used to trap tertiary structures of naturally occurring RNAs, including those with the commonly encountered GA/GA mismatch.     

RNase H mediated cleavage of RNA by cyclohexene nucleic acid (CeNA)

Cyclohexene nucleic acid (CeNA) forms a duplex with RNA that is more stable than a DNA–RNA duplex (ΔT_m per modification: +2°C). A cyclohexenyl A nucleotide adopts a 3′-endo conformation when introduced in dsDNA. The neighbouring deoxynucleotide adopts an O4′-endo conformation. The CeNA:RNA duplex is cleaved by RNase H. The V_max and K_m of the cleavage reaction for CeNA:RNA and DNA:RNA is in the same range, although the k_cat value is about 600 times lower in the case of CeNA:RNA.     

The N-terminal half-domain of the long form of tRNase Z is required for the RNase 65 activity

Transfer RNA (tRNA) 3′ processing endoribonuclease (tRNase Z) is an enzyme responsible for the removal of a 3′ trailer from pre-tRNA. There exists two types of tRNase Z: one is a short form (tRNase ZS) that consists of 300–400 amino acids, and the other is a long form (tRNase ZL) that contains 800–900 amino acids. Here we investigated whether the short and long forms have different preferences for various RNA substrates. We examined three recombinant tRNase ZSs from human, Escherichia coli and Thermotoga maritima, two recombinant tRNase ZLs from human and Saccharomyces cerevisiae, one tRNase ZL from pig liver, and the N- and C-terminal half regions of human tRNase ZL for cleavage of human micro-pre-tRNA^Arg and the RNase 65 activity. All tRNase ZLs cleaved the micro-pre-tRNA and showed the RNase 65 activity, while all tRNase ZSs and both half regions of human tRNase ZL failed to do so with the exception of the C-terminal half, which barely cleaved the micro-pre-tRNA. We also show that only the long forms of tRNase Z can specifically cleave a target RNA under the direction of a new type of small guide RNA, hook RNA. These results indicate that indeed tRNase ZL and tRNase ZS have different substrate specificities and that the differences are attributed to the N-terminal half-domain of tRNase ZL. Furthermore, the optimal concentrations of NaCl, MgCl₂ and MnCl₂ differed between tRNase ZSs and tRNase ZLs, and the K_m values implied that tRNase ZLs interact with pre-tRNA substrates more strongly than tRNase ZSs.     

RNA structure-dependent uncoupling of substrate recognition and cleavage by Escherichia coli ribonuclease III

Members of the ribonuclease III superfamily of double-strand-specific endoribonucleases participate in diverse RNA maturation and decay pathways. Ribonuclease III of the gram-negative bacterium Escherichia coli processes rRNA and mRNA precursors, and its catalytic action can regulate gene expression by controlling mRNA translation and stability. It has been proposed that E.coli RNase III can function in a non-catalytic manner, by binding RNA without cleaving phosphodiesters. However, there has been no direct evidence for this mode of action. We describe here an RNA, derived from the T7 phage R1.1 RNase III substrate, that is resistant to cleavage in vitro by E.coli RNase III but retains comparable binding affinity. R1.1[CL3B] RNA is recognized by RNase III in the same manner as R1.1 RNA, as revealed by the similar inhibitory effects of a specific mutation in both substrates. Structure-probing assays and Mfold analysis indicate that R1.1[CL3B] RNA possesses a bulge– helix–bulge motif in place of the R1.1 asymmetric internal loop. The presence of both bulges is required for uncoupling. The bulge–helix–bulge motif acts as a ‘catalytic’ antideterminant, which is distinct from recognition antideterminants, which inhibit RNase III binding.     

Anti-HIV-1 activity of anti-TAR polyamide nucleic acid conjugated with various membrane transducing peptides

The transactivator responsive region (TAR) present in the 5′-NTR of the HIV-1 genome represents a potential target for antiretroviral intervention and a model system for the development of specific inhibitors of RNA–protein interaction. Earlier, we have shown that an anti-TAR polyamide nucleotide analog (PNA_TAR) conjugated to a membrane transducing (MTD) peptide, transportan, is efficiently taken up by the cells and displays potent antiviral and virucidal activity [B. Chaubey, S. Tripathi, S. Ganguly, D. Harris, R. A. Casale and V. N. Pandey (2005) Virology, 331, 418–428]. In the present communication, we have conjugated five different MTD peptides, penetratin, tat peptide, transportan-27, and two of its truncated derivatives, transportan-21 and transportan-22, to a 16mer PNA targeted to the TAR region of the HIV-1 genome. The individual conjugates were examined for their uptake efficiency as judged by FACScan analysis, uptake kinetics using radiolabeled conjugate, virucidal activity and antiviral efficacy assessed by inhibition of HIV-1 infection/replication. While FACScan analysis revealed concentration-dependent cellular uptake of all the PNA_TAR–peptide conjugates where uptake of the PNA_TAR–penetratin conjugate was most efficient as >90% MTD was observed within 1 min at a concentration of 200 nM. The conjugates with penetratin, transportan-21 and tat-peptides were most effective as an anti-HIV virucidal agents with IC₅₀ values in the range of 28–37 nM while IC₅₀ for inhibition of HIV-1 replication was lowest with PNA_TAR–transportan-27 (0.4 μM) followed by PNA_TAR–tat (0.72 μM) and PNA_TAR–penetratin (0.8 μM). These results indicate that anti-HIV-1 PNA conjugated with MTD peptides are not only inhibitory to HIV-1 replication in vitro but are also potent virucidal agents which render HIV-1 virions non-infectious upon brief exposure.