Characterization of the mechanical unfolding of RNA pseudoknots

The pseudoknot is an important RNA structural element that provides an excellent model system for studying the contributions of tertiary interactions to RNA stability and to folding kinetics. RNA pseudoknots are also of interest because of their key role in the control of ribosomal frameshifting by viral RNAs. Their mechanical properties are directly relevant to their unfolding by ribosomes during translation. We have used optical tweezers to study the kinetics and thermodynamics of mechanical unfolding and refolding of single RNA molecules. Here we describe the unfolding of the frameshifting pseudoknot from infectious bronchitis virus (IBV), three constituent hairpins, and three mutants of the IBV pseudoknot. All four pseudoknots cause −1 programmed ribosomal frameshifting. We have measured the free energies and rates of mechanical unfolding and refolding of the four frameshifting pseudoknots. Our results show that the IBV pseudoknot requires a higher force than its corresponding hairpins to unfold. Furthermore, its rate of unfolding changes little with increasing force, in contrast with the rate of hairpin unfolding. The presence of Mg²⁺ significantly increases the kinetic barriers to unfolding the IBV pseudoknot, but has only a minor effect on the hairpin unfolding. The greater mechanical stability of pseudoknots compared to hairpins, and their kinetic insensitivity to force supports the hypothesis that −1 frameshifting depends on the difficulty of unfolding the mRNA.     

Potent inhibition of human influenza H5N1 virus by oligonucleotides derived by SELEX

New therapeutics are urgently needed for the treatment of pandemic influenza caused by H5N1 influenza virus mutants. Aptamer was a promising candidate for treatment and prophylaxis of influenza virus infections. In this study, systemic evolution of ligands through exponential enrichment (SELEX) was used to screen DNA aptamers targeted to recombinant HA1 proteins of the H5N1 influenza virus. After 11 rounds of selection, DNA aptamers that bind to the HA1 protein were isolated and shown to have different binding capacities. Among them, aptamer 10 had the strongest binding to the HA1 protein, and had an inhibitory effect on H5N1 influenza virus, as shown by the hemagglutinin and MTT assays. These results should aid the development of new drugs for the prevention and control of influenza virus infections.     

Possible involvement of coaxially stacked double pseudoknots in the regulation of −1 programmed ribosomal frameshifting in RNA viruses

?1 programmed ribosomal frameshifting (PRF) in viruses is often stimulated by a pseudoknot downstream from the slippery sequence. At the PRF junction of HIV-1, transmissible gastroenteritis virus (TGEV), Barmah Forest virus (BFV), Fort Morgan virus (FMV), and Equine arteritis virus (EAV), we identified potential double pseudoknots in either a tandem mode or embedded mode. In viruses with tandem pseudoknots (5′PK & 3′PK), the slippery sequence is encompassed in the 5′PK. The ribosome needs to unwind the 5′PK to get to the slippery sequence. In HIV-1, the 3′PK and several alternative structures are mutually exclusive. Disruption of the tandem pseudoknots may enable one of the alternative structures to form as the effective frameshift stimulator. In TGEV/BFV/FMV, the 3′PK is a conventional frameshift stimulator. In all cases, the tandem pseudoknots may slow down the ribosome before it reaches the conventional PRF signals. In EAV, a compact pseudoknot is embedded within loop2 of the otherwise conventional frameshift-stimulating pseudoknot. All double pseudoknots have the potential to stack their stems coaxially. We built structural models of the HIV-1 and EAV double pseudoknots to show that both the tandem and embedded modes are feasible and reasonable. We hypothesize that the fundamental reason for the viruses to utilize coaxially stacked double pseudoknots is to increase the overall stability of the frameshift regulating structure, and avoid an ultra-stable single pseudoknot which may become a ribosomal roadblock. Our results significantly expand the repertoire of RNA structures and dynamics that may potentially involve in ?1 PRF regulation.     

Inhibition of Influenza A Virus Infection In Vitro by Peptides Designed In Silico

Influenza A viruses are enveloped, segmented negative single-stranded RNA viruses, capable of causing severe human respiratory infections. Currently, only two types of drugs are used to treat influenza A infections, the M2 H⁺ ion channel blockers (amantadine and rimantadine) and the neuraminidase inhibitors (NAI) (oseltamivir and zanamivir). Moreover, the emergence of drug-resistant influenza A virus strains has emphasized the need to develop new antiviral agents to complement or replace the existing drugs. Influenza A virus has on the surface a glycoprotein named hemagglutinin (HA) which due to its important role in the initial stage of infection: receptor binding and fusion activities of viral and endosomal membranes, is a potential target for new antiviral drugs. In this work we designed nine peptides using several bioinformatics tools. These peptides were derived from the HA1 and HA2 subunits of influenza A HA with the aim to inhibit influenza A virus infection. The peptides were synthetized and their antiviral activity was tested in vitro against several influenza A viral strains: Puerto Rico/916/34 (H1N1), (H1N1)pdm09, swine (H1N1) and avian (H5N2). We found these peptides were able to inhibit the influenza A viral strains tested, without showing any cytotoxic effect. By docking studies we found evidence that all the peptides were capable to bind to the viral HA, principally to important regions on the viral HA stalk, thus could prevent the HA conformational changes required to carry out its membranes fusion activity.     

Recombinant Parainfluenza Virus 5 Expressing Hemagglutinin of Influenza A Virus H5N1 Protected Mice against Lethal Highly Pathogenic Avian Influenza Virus H5N1 Challenge

A safe and effective vaccine is the best way to prevent large-scale highly pathogenic avian influenza virus (HPAI) H5N1 outbreaks in the human population. The current FDA-approved H5N1 vaccine has serious limitations. A more efficacious H5N1 vaccine is urgently needed. Parainfluenza virus 5 (PIV5), a paramyxovirus, is not known to cause any illness in humans. PIV5 is an attractive vaccine vector. In our studies, a single dose of a live recombinant PIV5 expressing a hemagglutinin (HA) gene of H5N1 (rPIV5-H5) from the H5N1 subtype provided sterilizing immunity against lethal doses of HPAI H5N1 infection in mice. Furthermore, we have examined the effect of insertion of H5N1 HA at different locations within the PIV5 genome on the efficacy of a PIV5-based vaccine. Interestingly, insertion of H5N1 HA between the leader sequence, the de facto promoter of PIV5, and the first viral gene, nucleoprotein (NP), did not lead to a viable virus. Insertion of H5N1 HA between NP and the next gene, V/phosphorprotein (V/P), led to a virus that was defective in growth. We have found that insertion of H5N1 HA at the junction between the small hydrophobic (SH) gene and the hemagglutinin-neuraminidase (HN) gene gave the best immunity against HPAI H5N1 challenge: a dose as low as 1,000 PFU was sufficient to protect against lethal HPAI H5N1 challenge in mice. The work suggests that recombinant PIV5 expressing H5N1 HA has great potential as an HPAI H5N1 vaccine.     

Thermodynamic analysis of conserved loop-stem interactions in P1-P2 frameshifting RNA pseudoknots from plant Luteoviridae

The RNA genomes of plant luteovirids beet western yellows virus (BWYV), potato leaf roll virus (PLRV), and pea enation mosaic virus (PEMV RNA1; PEMV-1) contain a short mRNA pseudoknotted motif overlapping the P1 and P2 open reading frames required for programmed -1 mRNA ribosomal frameshifting. The relationship between structure, stability, and function is poorly understood in these RNA systems. A m(5)-C(8)-substituted BWYV RNA is employed to establish that the BWYV P1-P2 pseudoknot is protonated at cytidine 8 in loop L1 (delta(N(3)H)+ = 12.98 ppm), which stabilizes a C(+.)(G-C) major groove base triple by Delta(DeltaG(37))(protonation) = 3.1 (+/-0.4) kcal mol(-1). The stabilities of both the PLRV and PEMV-1 P1-P2 pseudoknots are also strongly pH-dependent, with Delta(DeltaG(37))(protonation) = 2.1 (+/-0.2) kcal mol(-1) for the PEMV-1 pseudoknot despite a distinct structural context. As previously found for the BWYV pseudoknot [Nixon and Giedroc (2000) J. Mol. Biol. 296, 659], both the PLRV and PEMV-1 RNAs are stabilized by DeltaH > or = 30 kcal mol(-)(1) in excess of secondary structure predictions, attributed to loop L2-stem S1 minor groove triplex interactions. BWYV RNAs containing single 2'-deoxy or A --> G substitutions that disrupt L2-S1 hydrogen bonding are strongly destabilized with Delta(DeltaG(37))(folding) (pH = 7.0) ranging from approximately 1.8 (+/-0.3) to > or =4.0 kcal mol(-1), relative to the wild-type BWYV RNA. These findings suggest that each member of this family of pseudoknots adopts a tightly folded structure that maximizes the cooperativity and complementarity of L1-S2 and L2-S1 loop-stem interactions required in part to offset the low intrinsic stability of the short three base pair pseudoknot stem S2.     

Isolation of Recombinant Phage Antibodies Targeting the Hemagglutinin Cleavage Site of Highly Pathogenic Avian Influenza Virus

Highly pathogenic avian influenza (HPAI) H5N1 viruses, which have emerged in poultry and other wildlife worldwide, contain a characteristic multi-basic cleavage site (CS) in the hemagglutinin protein (HA). Because this arginine-rich CS is unique among influenza virus subtypes, antibodies against this site have the potential to specifically diagnose pathogenic H5N1. By immunizing mice with the CS peptide and screening a phage display library, we isolated four antibody Fab fragment clones that specifically bind the antigen peptide and several HPAI H5N1 HA proteins in different clades. The soluble Fab fragments expressed in Escherichia coli bound the CS peptide and the H5N1 HA protein with nanomolar affinity. In an immunofluorescence assay, these Fab fragments stained cells infected with HPAI H5N1 but not those infected with a less virulent strain. Lastly, all the Fab clones could detect the CS peptide and H5N1 HA protein by open sandwich ELISA. Thus, these recombinant Fab fragments will be useful novel reagents for the rapid and specific detection of HPAI H5N1 virus.     

RNA aptamers that bind the nucleocapsid protein contain pseudoknots

We screened two independent RNA libraries consisting of molecules of 50 nucleotides of random sequence, one of which had additional viral psi-sequences to isolate RNA aptamers that bound to the mature form of the nucleocapsid (NC) protein of Human Immunodeficiency Virus Type-1 (HIV-1). Surface Plasmon Resonance measurements and gel shift assays showed that the RNA aptamers bound with high affinity and specificity. We employed RNase footprinting to characterize the RNA structures and to map their protein binding sites. Most of the selected RNA aptamers contained a plausible pseudoknot in addition to the characteristic stem-loop structure. Moreover, the pseudoknots were part of the NC binding sites. We propose that higher order structures such as pseudoknots may constitute binding motifs for nucleic acid binding proteins, especially for NC protein, which is a nucleic acid chaperone.     

Crystal structure of a luteoviral RNA pseudoknot and model for a minimal ribosomal frameshifting motif

To understand the role of structural elements of RNA pseudoknots in controlling the extent of -1-type ribosomal frameshifting, we determined the crystal structure of a high-efficiency frameshifting mutant of the pseudoknot from potato leaf roll virus (PLRV). Correlations of the structure with available in vitro frameshifting data for PLRV pseudoknot mutants implicate sequence and length of a stem-loop linker as modulators of frameshifting efficiency. Although the sequences and overall structures of the RNA pseudoknots from PLRV and beet western yellow virus (BWYV) are similar, nucleotide deletions in the linker and adjacent minor groove loop abolish frameshifting only with the latter. Conversely, mutant PLRV pseudoknots with up to four nucleotides deleted in this region exhibit nearly wild-type frameshifting efficiencies. The crystal structure helps rationalize the different tolerances for deletions in the PLRV and BWYV RNAs, and we have used it to build a three-dimensional model of the PRLV pseudoknot with a four-nucleotide deletion. The resulting structure defines a minimal RNA pseudoknot motif composed of 22 nucleotides capable of stimulating -1-type ribosomal frameshifts.     

Isolation of Single-Stranded DNA Aptamers That Distinguish Influenza Virus Hemagglutinin Subtype H1 from H5

Surface protein hemagglutinin (HA) mediates the binding of influenza virus to host cell receptors containing sialic acid, facilitating the entry of the virus into host cells. Therefore, the HA protein is regarded as a suitable target for the development of influenza virus detection devices. In this study, we isolated single-stranded DNA (ssDNA) aptamers binding to the HA1 subunit of subtype H1 (H1-HA1), but not to the HA1 subunit of subtype H5 (H5-HA1), using a counter-systematic evolution of ligands by exponential enrichment (counter-SELEX) procedure. Enzyme-linked immunosorbent assay and surface plasmon resonance studies showed that the selected aptamers bind tightly to H1-HA1 with dissociation constants in the nanomolar range. Western blot analysis demonstrated that the aptamers were binding to H1-HA1 in a concentration-dependent manner, yet were not binding to H5-HA1. Interestingly, the selected aptamers contained G-rich sequences in the central random nucleotides region. Further biophysical analysis showed that the G-rich sequences formed a G-quadruplex structure, which is a distinctive structure compared to the starting ssDNA library. Using flow cytometry analysis, we found that the aptamers did not bind to the receptor-binding site of H1-HA1. These results indicate that the selected aptamers that distinguish H1-HA1 from H5-HA1 can be developed as unique probes for the detection of the H1 subtype of influenza virus.     

Evidence for an RNA pseudoknot loop-helix interaction essential for efficient -1 ribosomal frameshifting.

RNA pseudoknots are structural elements that participate in a variety of biological processes. At -1 ribosomal frameshifting sites, several types of pseudoknot have been identified which differ in their organisation and functionality. The pseudoknot found in infectious bronchitis virus (IBV) is typical of those that possess a long stem 1 of 11-12 bp and a long loop 2 (30-164 nt). A second group of pseudoknots are distinguishable that contain stems of only 5 to 7 bp and shorter loops. The NMR structure of one such pseudoknot, that of mouse mammary tumor virus (MMTV), has revealed that it is kinked at the stem 1-stem 2 junction, and that this kinked conformation is essential for efficient frameshifting. We recently investigated the effect on frameshifting of modulating stem 1 length and stability in IBV-based pseudoknots, and found that a stem 1 with at least 11 bp was needed for efficient frameshifting. Here, we describe the sequence manipulations that are necessary to bypass the requirement for an 11 bp stem 1 and to convert a short non-functional IBV-derived pseudoknot into a highly efficient, kinked frameshifter pseudoknot. Simple insertion of an adenine residue at the stem 1-stem 2 junction (an essential feature of a kinked pseudoknot) was not sufficient to create a functional pseudoknot. An additional change was needed: efficient frameshifting was recovered only when the last nucleotide of loop 2 was changed from a G to an A. The requirement for an A at the end of loop 2 is consistent with a loop-helix contact similar to those described in other RNA tertiary structures. A mutational analysis of both partners of the proposed interaction, the loop 2 terminal adenine residue and two G.C pairs near the top of stem 1, revealed that the interaction was essential for efficient frameshifting. The specific requirement for a 3'-terminal A residue was lost when loop 2 was increased from 8 to 14 nt, suggesting that the loop-helix contact may be required only in those pseudoknots with a short loop 2.     

A Characteristic Bent Conformation of RNA Pseudoknots Promotes –1 Frameshifting during Translation of Retroviral RNA

The structures of four different RNA pseudoknots that provide one of the signals required for ribosomal frameshifting in mouse mammary tumor virus have been determined by NMR. The RNA pseudoknots have similar sequences and assume similar secondary structures, but show significantly different frameshifting efficiencies. The three-dimensional structures of one frameshifting and one non-frameshifting RNA pseudoknot had been determined previously by our group. Here we determine the structures of two new RNA pseudoknots, and relate the structures of all four pseudoknots to their frameshifting abilities. The two efficient frameshifting pseudoknots adopt characteristic bent conformations with stem 1 bending towards the major groove of stem 2. In contrast, the two poor frameshifting pseudoknots have structures very different from each other and from the efficient frameshifters. One has linear, coaxially stacked stems, the other has stems twisted and bent, but in the opposite direction to the efficient frameshifters. Changes in loop size that favor bending (shorter loops) increase frameshifting efficiency; longer loops that allow linear arrangement of the stems decrease frameshifting. Frameshifting pseudoknots in feline immunodeficiency virus and simian retrovirus have different loop sequences, but the sequences at their stem junctions imply the same bent conformation as in the mouse mammary tumor viral RNA. The requirement for a precise pseudoknot conformation for efficient frameshifting strongly implies that a specific interaction occurs between the viral RNA pseudoknot and the host protein-synthesizing machinery.     

An NMR and mutational study of the pseudoknot within the gene 32 mRNA of bacteriophage T2: insights into a family of structurally related RNA pseudoknots.

NMR methods were used to investigate a series of mutants of the pseudoknot within the gene 32 messenger RNA of bacteriophage T2, for the purpose of investigating the range of sequences, stem and loop lengths that can form a similar pseudoknot structure. This information is of particular relevance since the T2 pseudoknot has been considered a representative of a large family of RNA pseudoknots related by a common structural motif, previously referred to as 'common pseudoknot motif 1' or CPK1. In the work presented here, a mutated sequence with the potential to form a pseudoknot with a 6 bp stem2 was shown to adopt a pseudoknot structure similar to that of the wild-type sequence. This result is significant in that it demonstrates that pseudoknots with 6 bp in stem2 and a single nucleotide in loop1 are indeed feasible. Mutated sequences with the potential to form pseudoknots with either 5 or 8 bp in stem2 yielded NMR spectra that could not confirm the formation of a pseudoknot structure. Replacing the adenosine nucleotide in loop1 of the wild-type pseudoknot with any one of G, C or U did not significantly alter the pseudoknot structure. Taken together, the results of this study provide support for the existence of a family of similarly structured pseudoknots with two coaxially stacked stems, either 6 or 7 bp in stem2, and a single nucleotide in loop1. This family includes many of the pseudoknots predicted to occur downstream of the frameshift or readthrough sites in a significant number of viral RNAs.     

High sensitivity RNA pseudoknot prediction

Most ab initio pseudoknot predicting methods provide very few folding scenarios for a given RNA sequence and have low sensitivities. RNA researchers, in many cases, would rather sacrifice the specificity for a much higher sensitivity for pseudoknot detection. In this study, we introduce the Pseudoknot Local Motif Model and Dynamic Partner Sequence Stacking (PLMM_DPSS) algorithm which predicts all PLM model pseudoknots within an RNA sequence in a neighboring-region-interference-free fashion. The PLM model is derived from the existing Pseudobase entries. The innovative DPSS approach calculates the optimally lowest stacking energy between two partner sequences. Combined with the Mfold, PLMM_DPSS can also be used in predicting complicated pseudoknots. The test results of PLMM_DPSS, PKNOTS, iterated loop matching, pknotsRG and HotKnots with Pseudobase sequences have shown that PLMM_DPSS is the most sensitive among the five methods. PLMM_DPSS also provides manageable pseudoknot folding scenarios for further structure determination.     

A mutant RNA pseudoknot that promotes ribosomal frameshifting in mouse mammary tumor virus.

A single A-->G mutation that changes a potential A.U base pair to a G.U pair at the junction of the stems and loops of a non-frameshifting pseudoknot dramatically increases its frameshifting efficiency in mouse mammary tumor virus. The structure of the non-frameshifting pseudoknot APK has been found to be very different from that of pseudoknots that cause efficient frameshifting [Kang,H., Hines,J.V. and Tinoco,I. (1995) J. Mol. Biol. , 259, 135-147]. The 3-dimensional structure of the mutant pseudoknot was determined by restrained molecular dynamics based on NMR-derived interproton distance and torsion angle constraints. One striking feature of the mutant pseudoknot compared with the parent pseudoknot is that a G.U base pair forms at the top of stem 2, thus leaving only 1 nt at the junction of the two stems. The conformation is very different from that of the previously determined non-frameshifting parent pseudoknot, which lacks the A.U base pair at the top of the stem and has 2 nt between the stems. However, the conformation is quite similar to that of efficient frameshifting pseudoknots whose structures were previously determined by NMR. A single adenylate residue intervenes between the two stems and interrupts their coaxial stacking. This unpaired nucleotide produces a bent structure. The structural similarity among the efficient frameshifting pseudoknots indicates that a specific conformation is required for ribosomal frameshifting, further implying a specific interaction of the pseudoknot with the ribosome.     

Prediction of RNA pseudoknots using heuristic modeling with mapping and sequential folding

Predicting RNA secondary structure is often the first step to determining the structure of RNA. Prediction approaches have historically avoided searching for pseudoknots because of the extreme combinatorial and time complexity of the problem. Yet neglecting pseudoknots limits the utility of such approaches. Here, an algorithm utilizing structure mapping and thermodynamics is introduced for RNA pseudoknot prediction that finds the minimum free energy and identifies information about the flexibility of the RNA. The heuristic approach takes advantage of the 5' to 3' folding direction of many biological RNA molecules and is consistent with the hierarchical folding hypothesis and the contact order model. Mapping methods are used to build and analyze the folded structure for pseudoknots and to add important 3D structural considerations. The program can predict some well known pseudoknot structures correctly. The results of this study suggest that many functional RNA sequences are optimized for proper folding. They also suggest directions we can proceed in the future to achieve even better results.     

A phylogenetically conserved sequence within viral 3'' untranslated RNA pseudoknots regulates translation.

Both the 68-base 5' leader (omega) and the 205-base 3' untranslated region (UTR) of tobacco mosaic virus (TMV) promote efficient translation. A 35-base region within omega is necessary and sufficient for the regulation. Within the 3' UTR, a 52-base region, composed of two RNA pseudoknots, is required for regulation. These pseudoknots are phylogenetically conserved among seven viruses from two different viral groups and one satellite virus. The pseudoknots contained significant conservation at the secondary and tertiary levels and at several positions at the primary sequence level. Mutational analysis of the sequences determined that the primary sequence in several conserved positions, particularly within the third pseudoknot, was essential for function. The higher-order structure of the pseudoknots was also required. Both the leader and the pseudoknot region were specifically recognized by, and competed for, the same proteins in extracts made from carrot cell suspension cells and wheat germ. Binding of the proteins is much stronger to omega than the pseudoknot region. Synergism was observed between the TMV 3' UTR and the cap and to a lesser extent between omega and the 3' UTR. The functional synergism and the protein binding data suggest that the cap, TMV 5' leader, and 3' UTR interact to establish an efficient level of translation.     

Predicting the Antigenic Structure of the Pandemic (H1N1) 2009 Influenza Virus Hemagglutinin

The pandemic influenza virus (2009 H1N1) was recently introduced into the human population. The hemagglutinin (HA) gene of 2009 H1N1 is derived from “classical swine H1N1” virus, which likely shares a common ancestor with the human H1N1 virus that caused the pandemic in 1918, whose descendant viruses are still circulating in the human population with highly altered antigenicity of HA. However, information on the structural basis to compare the HA antigenicity among 2009 H1N1, the 1918 pandemic, and seasonal human H1N1 viruses has been lacking. By homology modeling of the HA structure, here we show that HAs of 2009 H1N1 and the 1918 pandemic virus share a significant number of amino acid residues in known antigenic sites, suggesting the existence of common epitopes for neutralizing antibodies cross-reactive to both HAs. It was noted that the early human H1N1 viruses isolated in the 1930s–1940s still harbored some of the original epitopes that are also found in 2009 H1N1. Interestingly, while 2009 H1N1 HA lacks the multiple N-glycosylations that have been found to be associated with an antigenic change of the human H1N1 virus during the early epidemic of this virus, 2009 H1N1 HA still retains unique three-codon motifs, some of which became N-glycosylation sites via a single nucleotide mutation in the human H1N1 virus. We thus hypothesize that the 2009 H1N1 HA antigenic sites involving the conserved amino acids will soon be targeted by antibody-mediated selection pressure in humans. Indeed, amino acid substitutions predicted here are occurring in the recent 2009 H1N1 variants. The present study suggests that antibodies elicited by natural infection with the 1918 pandemic or its early descendant viruses play a role in specific immunity against 2009 H1N1, and provides an insight into future likely antigenic changes in the evolutionary process of 2009 H1N1 in the human population.     

Selection of an antiviral RNA aptamer against hemagglutinin of the subtype H5 avian influenza virus

Avian influenza is an acute viral respiratory disease caused by RNA viruses of the family Orthomyxoviridae. The influenza A virus subtype H5 can cause severe illness and results in almost 100% mortality rate among livestock. Hemagglutinin (HA) present in the virus envelope plays an essential role in the initiation of viral infection. In this study, we investigated the efficacy of using HA as a target for antiviral therapy through nucleic acid aptamers. After purification of the receptor binding domain (HA1) of HA protein, activity of recombinant HA1 was confirmed by using hemagglutination assay. We selected RNA aptamer candidates after 15 rounds of iterative Systematic Evolution of Ligands by EXponential enrichment (SELEX) targeting the biologically active HA protein. The selected RNA aptamer HAS15-5, which specifically binds to HA1, exhibited significant antiviral efficacy according to the results of a hemagglutination inhibition assay using egg allantoic fluids harboring the virus. Thus, the RNA aptamer HAS15-5, which acts by blocking and inhibiting the receptor-binding domain of viral HA, can be developed as a novel antiviral agent against type H5 avian influenza virus.