Virus Hemagglutinin-neuraminidase Research Articles

Novel sialidase inhibitors suppress mumps virus replication and infection.

The prevalent human pathogen, mumps virus (MuV; orthorubulavirus parotitidis) causes various complications and serious sequelae, such as meningitis, encephalitis, deafness, and impaired fertility. Direct-acting antivirals (DAAs) targeting MuV which can prevent mumps and mumps-associated complications and sequelae are yet to be developed. Paramyxoviridae family members, such as MuV, possess viral surface hemagglutinin-neuraminidase (HN) protein with sialidase activity which facilitates efficient viral replication. Therefore, to develop DAAs targeting MuV we synthesized MuV sialidase inhibitors. It is proposed that the viral HN has a single functional site for N-acetylneuraminic acid (Neu5Ac) binding and sialidase activity. Further, the known MuV sialidase inhibitor is an analog of Neu5Ac-2,3-didehydro-2-deoxy-N-acetylneuraminic acid (DANA)-which lacks potency. DANA derivatives with higher MuV sialidase inhibitory potency are lacking. The MuV-HN-Neu5Ac binding site has a hydrophobic cavity adjacent to the C4 position of Neu5Ac. Exploiting this, here, we synthesized DANA derivatives with increasing hydrophobicity at its C4 position and created 3 novel sialidase inhibitors (Compounds 1, 2 and 3) with higher specificity for MuV-HN than DANA; they inhibited MuV replication step to greater extent than DANA. Furthermore, they also inhibited hemagglutination and the MuV infection step. The insight-that these 3 novel DANA derivatives possess linear hydrocarbon groups at the C4-hydroxyl group of DANA-could help develop highly potent sialidase inhibitors with high specificity for MuV sialidase, which may function as direct-acting MuV-specific antivirals.

The Dual-Pseudotyped Lentiviral Vector with VSV-G and Sendai Virus HN Enhances Infection Efficiency through the Synergistic Effect of the Envelope Proteins.

A gene delivery system utilizing lentiviral vectors (LVs) requires high transduction efficiency for successful application in human gene therapy. Pseudotyping allows viral tropism to be expanded, widening the usage of LVs. While vesicular stomatitis virus G (VSV-G) single-pseudotyped LVs are commonly used, dual-pseudotyping is less frequently employed because of its increased complexity. In this study, we examined the potential of phenotypically mixed heterologous dual-pseudotyped LVs with VSV-G and Sendai virus hemagglutinin-neuraminidase (SeV-HN) glycoproteins, termed V/HN-LV. Our findings demonstrated the significantly improved transduction efficiency of V/HN-LV in various cell lines of mice, cynomolgus monkeys, and humans compared with LV pseudotyped with VSV-G alone. Notably, V/HN-LV showed higher transduction efficiency in human cells, including hematopoietic stem cells. The efficient incorporation of wild-type SeV-HN into V/HN-LV depended on VSV-G. SeV-HN removed sialic acid from VSV-G, and the desialylation of VSV-G increased V/HN-LV infectivity. Furthermore, V/HN-LV acquired the ability to recognize sialic acid, particularly N-acetylneuraminic acid on the host cell, enhancing LV infectivity. Overall, VSV-G and SeV-HN synergistically improve LV transduction efficiency and broaden its tropism, indicating their potential use in gene delivery.

Evaluation of the Immunity Responses in Mice to Recombinant Bacillus subtilis Displaying Newcastle Disease Virus HN Protein Truncations.

Bacillus subtilis, a probiotic bacterium with engineering potential, is widely used for the expression of exogenous proteins. In this study, we utilized the integrative plasmid pDG364 to integrate the hemagglutinin-neuraminidase (HN) gene from Newcastle disease virus (NDV) into the genome of the B. subtilis 168 model strain. We successfully constructed a recombinant B. subtilis strain (designated B. subtilis RH) that displays a truncated HN antigen fragment on the surface of its spores and further evaluated its immunogenic effects in mice. Using ELISA, we quantified the levels of IgG in serum and secretory IgA (sIgA) in intestinal contents. The results revealed that the recombinant B. subtilis RH elicited robust specific mucosal and humoral immune responses in mice. Furthermore, B. subtilis RH demonstrated potential mucosal immune adjuvant properties by fostering the development of immune organs and augmenting the number of lymphocytes in the small intestinal villi. Additionally, the strain significantly upregulated the relative expression of inflammatory cytokines such as IL-1β, IL-6, IL-10, TNF-α, and IFN-γ in the small intestinal mucosa. In conclusion, the B. subtilis RH strain developed in this study exhibits promising mucosal immunogenic effects. It holds potential as a candidate for an anti-NDV mucosal subunit vaccine and offers a novel preventive strategy for the poultry industry against this disease.

The HN protein of Newcastle disease virus induces cell apoptosis through the induction of lysosomal membrane permeabilization.

Lysosomes are acidic organelles that mediate the degradation and recycling of cellular waste materials. Damage to lysosomes can cause lysosomal membrane permeabilization (LMP) and trigger different types of cell death, including apoptosis. Newcastle disease virus (NDV) can naturally infect most birds. Additionally, it serves as a promising oncolytic virus known for its effective infection of tumor cells and induction of intensive apoptotic responses. However, the involvement of lysosomes in NDV-induced apoptosis remains poorly understood. Here, we demonstrate that NDV infection profoundly triggers LMP, leading to the translocation of cathepsin B and D and subsequent mitochondria-dependent apoptosis in various tumor and avian cells. Notably, the released cathepsin B and D exacerbate NDV-induced LMP by inducing the generation of reactive oxygen species. Additionally, we uncover that the viral Hemagglutinin neuraminidase (HN) protein induces the deglycosylation and degradation of lysosome-associated membrane protein 1 (LAMP1) and LAMP2 dependent on its sialidase activity, which finally contributes to NDV-induced LMP and cellular apoptosis. Overall, our findings elucidate the role of LMP in NDV-induced cell apoptosis and provide novel insights into the function of HN during NDV-induced LMP, which provide innovative approaches for the development of NDV-based oncolytic agents.

Interplay of Modified Sialic Acid Inhibitors and the Human Parainfluenza Virus 1 Hemagglutinin-Neuraminidase Active Site.

In the search for effective antivirals against Paramyxoviridae, the dynamics of human parainfluenza virus type 1 hemagglutinin-neuraminidase (hPIV1-HN) inhibition offers a promising perspective. This study focuses on the potential of C5- and C4-modified 2,3-unsaturated sialic acid (DANA) inhibitors and highlights their interaction with the hPIV1-HN enzyme. We show that a strategic substitution, replacing the C5 isopropyl group in BCX 2798 with a trifluoroacetyl function, increases inhibitory potency 3- to 4-fold. At the same time, we explore the special properties of the catalytic site of hPIV1-HN, which harbors only small substituents and favors a C4 sulfonylamido function over a carbonyl function, in contrast to the C4 pocket of Newcastle disease virus hemagglutinin-neuraminidase (NDV-HN). Based on these findings, we present a newly identified potent inhibitor that has the preferred C5 trifluoroacetamido and C4 trifluorosulfonylamide groups. The results of this study pave the way for a deeper understanding of the C4 and C5 binding pockets of hPIV1-HN and promote the development of new, more selective inhibitors.

Design, Synthesis, and Antiviral Evaluation of Sialic Acid Derivatives as Inhibitors of Newcastle Disease Virus Hemagglutinin-Neuraminidase: A Translational Study on Human Parainfluenza Viruses.

Global infections with viruses belonging to the Paramyxoviridae, such as Newcastle disease virus (NDV) or human parainfluenza viruses (hPIVs), pose a serious threat to animal and human health. NDV-HN and hPIVs-HN (HN hemagglutinin-neuraminidase) share a high degree of similarity in catalytic site structures; therefore, the development of an efficient experimental NDV host model (chicken) may be informative for evaluating the efficacy of hPIVs-HN inhibitors. As part of the broad research in pursuit of this goal and as an extension of our published work on antiviral drug development, we report here the biological results obtained with some newly synthesized C4- and C5-substituted 2,3-unsaturated sialic acid derivatives against NDV. All developed compounds showed high neuraminidase inhibitory activity (IC50 0.03-13 μM). Four molecules (9, 10, 23, 24) confirmed their high in vitro inhibitory activity, which caused a significant reduction of NDV infection in Vero cells, accompanied by very low toxicity.

Execution and Design of an Anti HPIV-1 Vaccine with Multiple Epitopes Triggering Innate and Adaptive Immune Responses: An Immunoinformatic Approach.

Human Parainfluenza Virus (HPIV) Type-1, which is an anti-sense ribonucleic acid (RNA) virus belonging to the paramyxoviridae family, induces upper and lower respiratory tract infections. The infections caused by the HPIV Type-1 virus are usually confined to northwestern regions of America. HPIV-1 causes infections through the virulence of the hemagglutinin-neuraminidase (HN) protein, which plays a key role in the attachment of the viral particle with the host’s receptor cells. To the best of our knowledge, there is no effective antiviral drugs or vaccines being developed to combat the infection caused by HPIV-1. In the current study, a multiple epitope-based vaccine was designed against HPIV-1 by taking the viral HN protein as a probable vaccine candidate. The multiple epitopes were selected in accordance with their allergenicity, antigenicity and toxicity scoring. The determined epitopes of the HN protein were connected simultaneously using specific conjugates along with an adjuvant to construct the subunit vaccine, with an antigenicity score of 0.6406. The constructed vaccine model was docked with various Toll-like Receptors (TLRs) and was computationally cloned in a pET28a (+) vector to analyze the expression of vaccine sequence in the biological system. Immune stimulations carried out by the C-ImmSim Server showed an excellent result of the body’s defense system against the constructed vaccine model. The AllerTop tool predicted that the construct was non-allergen with and without the adjuvant sequence, and the VaxiJen 2.0 with 0.4 threshold predicted that the construct was antigenic, while the Toxinpred predicted that the construct was non-toxic. Protparam results showed that the selected protein was stable with 36.48 instability index (II) scores. The Grand average of Hydropathicity or GRAVY score indicated that the constructed protein was hydrophilic in nature. Aliphatic index values (93.53) confirmed that the construct was thermostable. This integrated computational approach shows that the constructed vaccine model has a potential to combat laryngotracheobronchitis infections caused by HPIV-I.

Molecular Mechanisms of Anti-Neoplastic and Immune Stimulatory Properties of Oncolytic Newcastle Disease Virus.

Oncolytic viruses represent interesting anti-cancer agents with high tumor selectivity and immune stimulatory potential. The present review provides an update of the molecular mechanisms of the anti-neoplastic and immune stimulatory properties of the avian paramyxovirus, Newcastle Disease Virus (NDV). The anti-neoplastic activities of NDV include (i) the endocytic targeting of the GTPase Rac1 in Ras-transformed human tumorigenic cells; (ii) the switch from cellular protein to viral protein synthesis and the induction of autophagy mediated by viral nucleoprotein NP; (iii) the virus replication mediated by viral RNA polymerase (large protein (L), associated with phosphoprotein (P)); (iv) the facilitation of NDV spread in tumors via the membrane budding of the virus progeny with the help of matrix protein (M) and fusion protein (F); and (v) the oncolysis via apoptosis, necroptosis, pyroptosis, or ferroptosis associated with immunogenic cell death. A special property of this oncolytic virus consists of its potential for breaking therapy resistance in human cancer cells. Eight examples of this important property are presented and explained. In healthy human cells, NDV infection activates the RIG-MAVs immune signaling pathway and establishes an anti-viral state based on a strong and uninhibited interferon α,ß response. The review also describes the molecular determinants and mechanisms of the NDV-mediated immune stimulatory effects, in which the viral hemagglutinin-neuraminidase (HN) protein plays a prominent role. The six viral proteins provide oncolytic NDV with a special profile in the treatment of cancer.

Characterization of Natural and Synthetic Sialoglycans Targeting the Hemagglutinin-Neuraminidase of Mumps Virus.

The inhibition of surface viral glycoproteins offers great potential to hamper the attachment of viruses to the host cells surface and the spreading of viral infection. Mumps virus (MuV) is the etiological agent of the mumps infectious disease and causes a wide spectrum of mild to severe symptoms due to the inflammation of the salivary glands. Here we focus our attention on the hemagglutinin-neuraminidase (HN) isolated from MuV SBL-1 strain. We describe the molecular features of host sialoglycans recognition by HN protein by means of NMR, fluorescence assays and computational studies. Furthermore, we also describe the synthesis of a N-acetylneuraminic acid-derived thiotrisaccharide targeting the viral protein, and the corresponding 3D-complex. Our results provide the basis to improve the design and synthesis of potent viral hemagglutinin-neuraminidase inhibitors.

Bi/tri-specific antibodies (HN-Fc-CD16 and HN-Fc-IL-15-CD16) cross-linking natural killer (NK)-CD16 and Newcastle Disease Virus (NDV)-HN, enhanced NK activation for cancer immunotherapy

Cancer/tumor cells infected with the “avian paramyxovirus Newcastle Disease Virus (TC-NDV)” express the viral hemagglutinin-neuraminidase (HN) on the cell surface that is used as both the danger signal and anchor for bi/tri-specific antibodies (bs/tsAbs).We constructed a bs-Ab (HN-Fc-CD16) that bindsto HN and natural killer (NK)-CD16 receptor (FcgRIII)and a ts-Ab (HN-Fc-IL15-CD16) harbouring NK-activating cytokine “IL-15” within the bs-Ab.In silicoand computational predictions indicated proper exposure of both Abs in bs/tsAbs.Properbinding of thebi/tsAbstoHN on surface of TC-NDVandCD16+-cells was demonstrated by flow cytometry.The bi/tsAbstriggeredspecificcytotoxicity of NK cells againstTC-NDVand elicited substantial IFN-γproduction by activated NK cells(higher for ts-Ab) that sound promising for cancer immunotherapy purposes.

Cellular CARD11 Inhibits the Fusogenic Activity of Newcastle Disease Virus via CBM Signalosome-Mediated Furin Reduction in Chicken Fibroblasts.

Newcastle disease virus (NDV) causes an infectious disease that poses a major threat to poultry health. Our previous study identified a chicken brain-specific caspase recruitment domain-containing protein 11 (CARD11) that was upregulated in chicken neurons and inhibited NDV replication. This raises the question of whether CARD11 plays a role in inhibiting viruses in non-neural cells. Here, chicken fibroblasts were used as a non-neural cell model to investigate the role. CARD11 expression was not significantly upregulated by either velogenic or lentogenic NDV infection in chicken fibroblasts. Viral replication was decreased in DF-1 cells stably overexpressing CARD11, while viral growth was significantly increased in the CARD11-knockdown DF-1 cell line. Moreover, CARD11 colocalized with the viral P protein and aggregated around the fibroblast nucleus, suggesting that an interaction existed between CARD11 and the viral P protein; this interaction was further examined by suppressing viral RNA polymerase activity by using a minigenome assay. Viral replication was inhibited by CARD11 in fibroblasts, and this result was consistent with our previous report in chicken neurons. Importantly, CARD11 was observed to reduce the syncytia induced by either velogenic virus infection or viral haemagglutinin-neuraminidase (HN) and F cotransfection in fibroblasts. We found that CARD11 inhibited the expression of the host protease furin, which is essential for cleavage of the viral F protein to trigger fusogenic activity. Furthermore, the CARD11-Bcl10-MALT1 (CBM) signalosome was found to suppress furin expression, which resulted in a reduction in the cleavage efficiency of the viral F protein to further inhibit viral syncytia. Taken together, our findings mainly demonstrated a novel CARD11 inhibitory mechanism for viral fusogenic activity in chicken fibroblasts, and this mechanism explains the antiviral roles of this molecule in NDV pathogenesis.

Residues 315 and 369 in HN Protein Contribute to the Thermostability of Newcastle Disease Virus.

Thermostable Newcastle disease virus (NDV) vaccines have been widely used in areas where a “cold-chain” is not reliable. However, the molecular mechanism of NDV thermostability remains poorly understood. In this work, we constructed chimeric viruses by exchanging viral fusion (F) and/or hemagglutinin-neuraminidase (HN) genes between the heat-resistant strain HR09 and thermolabile strain La Sota utilizing a reverse genetic system. The results showed that only chimeras with HN derived from the thermostable virus exhibited a thermostable phenotype at 56°C. The hemagglutinin (HA) and neuraminidase (NA) activities of chimeras with HN derived from the HR09 strain were more thermostable than those containing HN from the La Sota strain. Then, we used molecular dynamics simulation at different temperatures (310 K and 330 K) to measure the HN protein of the La Sota strain. The conformation of an amino acid region (residues 315–375) was observed to fluctuate. Sequence alignment of the HN protein revealed that residues 315, 329, and 369 in the La Sota strain and thermostable strains differed. Whether the three amino acid substitutions affected viral thermostability was investigated. Three mutant viruses based on the thermolabile strain were generated by substituting one, two or three amino acids at positions 315, 369, and 329 in the HN protein. In comparison with the parental virus, the mutant viruses containing mutations S315P and I369V possessed higher thermostablity and HA titers, NA and fusion activities. Taken together, these data indicate that the HN gene of NDV is a major determinant of thermostability, and residues 315 and 369 have important effects on viral thermostability.

High-Yield Expression and Purification of Recombinant Influenza Virus Proteins from Stably-Transfected Mammalian Cell Lines.

Influenza viruses infect millions of people each year, resulting in significant morbidity and mortality in the human population. Therefore, generation of a universal influenza virus vaccine is an urgent need and would greatly benefit public health. Recombinant protein technology is an established vaccine platform and has resulted in several commercially available vaccines. Herein, we describe the approach for developing stable transfected human cell lines for the expression of recombinant influenza virus hemagglutinin (HA) and recombinant influenza virus neuraminidase (NA) proteins for the purpose of in vitro and in vivo vaccine development. HA and NA are the main surface glycoproteins on influenza virions and the major antibody targets. The benefits for using recombinant proteins for in vitro and in vivo assays include the ease of use, high level of purity and the ability to scale-up production. This work provides guidelines on how to produce and purify recombinant proteins produced in mammalian cell lines through either transient transfection or generation of stable cell lines from plasmid creation through the isolation step via Immobilized Metal Affinity Chromatography (IMAC). Collectively, the establishment of this pipeline has facilitated large-scale production of recombinant HA and NA proteins to high purity and with consistent yields, including glycosylation patterns that are very similar to proteins produced in a human host.

Structural basis for Glycan-receptor binding by mumps virus hemagglutinin-neuraminidase

Mumps virus is one of the main cause of respiratory illnesses in humans, especially children. Among the viral surface glycoproteins, the hemagglutinin – neuraminidase, MuV-HN, plays key roles in virus entry into host cells and infectivity, thus representing an ideal target for the design of novel inhibitors. Here we report the detailed analysis of the molecular recognition of host cell surface sialylated glycans by the viral glycoprotein MuV-HN. By a combined use of NMR, docking, molecular modelling and CORCEMA-ST, the structural features of sialoglycans/MuV-HN complexes were revealed. Evidence for a different enzyme activity toward longer and complex substrates compared to unbranched ligands was also examined by an accurate NMR kinetic analysis. Our results provide the basis for the structure-based design of effective drugs against mumps-induced diseases.

Large-Scale Expression and Purification of Mumps Virus Hemagglutinin-Neuraminidase for Structural Analyses and Glycan-Binding Assays.

Many viruses utilize cell-surface glycans as receptors for host cell entry. Viral surface glycoproteins specifically interact with glycan motifs, which strongly contributes to viral tropism. Recently, the interactions between host cell glycan receptors and the mumps virus (MuV) hemagglutinin-neuraminidase (MuV-HN) protein were characterized by determining the co-crystal structure of MuV-HN in complex with glycan receptors. Here, we describe protocols for large-scale expression, purification and crystallization of MuV-HN proteins for structural analyses and glycan-binding assays with the overarching goal of investigating glycan-protein interactions.

Role of the head-stalk linker region (aa115-122) of Newcastle disease virus haemagglutinin-neuraminidase in regulating fusion triggering.

To gain insights into the role of the head-stalk linker region in the fusion triggering, we constructed mutants by deleting or substituting the linker region (115-NGAANNSG-122) of Newcastle disease virus (NDV) haemagglutinin-neuraminidase (HN) with the corresponding sequences of other paramyxoviruses. The results showed that these HN mutants exhibited different levels of fusion-triggering activity, but most of them maintained comparable levels with wide-type HN in both receptor recognition and neuraminidase activity. We tried to figure out reasons for fusion alteration through assessing the expression and the oligomeric state of HN mutants. Moreover, four mutants with significant fusion changes were introduced into the headless HN stem (HN1-123) to intensively investigate the role of the linker region in fusion triggering. Consequently, the stability of HN oligomers and the structural integrity of the 4 helical-bundle of stalk have complicated influences on the alteration of fusion-triggering activities for different mutants. These data suggested that the head-stalk linker could regulate the fusion triggering at both full-length and headless HN levels.

Roles of the highly conserved amino acids in the second receptor binding site of the Newcastle disease virus HN protein

BackgroundThe paramyxovirus haemagglutinin-neuraminidase (HN) is a multifunctional protein that is responsible for attachment to receptors, removal of receptors from infected cells to prevent viral self-aggregation (neuraminidase, NA) and fusion promotion. It is commonly accepted that there are two receptor binding sites in the globular head of HN, and the second receptor binding site is only involved in the function of receptor binding and fusion promotion.Methods10 conserved residues in the second receptor binding site of Newcastle disease virus (NDV) HN were chosen and substituted to alanine (A). The desired mutants were examined to detect the functional change in hemadsorption (HAD) ability, NA activity and fusion promotion ability.ResultsThe HAD and fusion promotion ability of mutants C172A, R174A, C196A, D198A, Y526A and E547A were abolished. Compared with wild-type (wt) HN, the HAD of mutants T167A, S202A and R516A decreased to 55.81, 44.53, 69.02%, respectively, and the fusion promotion ability of these three mutants decreased to 54.74, 49.46, 65.26%, respectively; however, mutant G171A still maintained fusion promotion ability comparable with wt HN but had impaired HAD ability. All the site-directed mutations altered the NA activity of NDV HN without affecting protein cell surface expression.ConclusionsThe data suggest that mutants C172A, R174A, C196A, D198A, Y526A and E547A do not allow the conformational change that is required for fusion promotion ability and HAD activity, while the other mutants only affect the conformational change to a limited extent, except mutant G171A with intact fusion promotion ability. Overall, the conserved amino acids in the second receptor binding site, especially residues C172, R174, C196, D198, Y526 and E547, are crucial to normal NDV HN protein function.

Role of Structural Fluctuations in Allosteric Stimulation of Paramyxovirus Hemagglutinin-Neuraminidase

Complexity in understanding allosteric stimulation of the hemagglutinin-neuraminidase (HN) protein of paramyxoviruses by host sialic acids (SIAs) stems from (1) unavailability of structure in its SIA-bound state and (2) the observation that this process is temperature sensitive. To consider simultaneously SIA's effect on structure and thermal fluctuations, we use molecular dynamics and simulate the dimeric form of the Newcastle disease virus HN. We find that SIA induces only minor structural changes in individual monomers, yet it reorients dimer interface by 10°. Interface reorientation is accompanied by constriction of SIA binding groove and enhanced fluctuations of interfacial residues that disrupt hydrophobic interactions and favor creation of new salt bridges. Supervised machine learning analysis of non-equilibrium data reveals that the allosteric signal is not formed from a directed sequence of these events. Altogether, we propose a detailed model of the initial events of allosteric stimulation, consistent with experiments on engineered mutations.

Hemagglutinin–Neuraminidase and fusion genes are determinants of NDV thermostability

Newcastle disease (ND) caused by infections with virulent strains of Newcastle disease virus (NDV) continues to be a threat for poultry industry worldwide. The prospect of developing a thermostable and effective NDV vaccine is still highly desirable. To investigate the determinants of thermostability in NDV, we generated recombinant NDV strains by exchanging viral hemagglutinin–neuraminidase (HN) gene or by mutating the fusion (F) gene. The results showed that the HN and F protein were both determinants of NDV thermostability. With increased thermostability, the HN protein-chimeric virus showed significantly reduced neuraminidase and hemadsorption activities, but its hemolytic activity was retained. We also found that changing the amino acid in the F protein cleavage sites, affected the thermostability as well as the pathogenicity and fusogenic capacity of the virus. Taken together, our results suggest that HN and F proteins both contribute to the thermostability of NDV, and other viral biological activities change as the thermostability of the virus changes. These findings should be of benefit to the development of a thermostable and efficacious NDV vaccine.

Antitumor effect of the Newcastle disease viral hemagglutinin-neuraminidase gene is expressed through an oncolytic adenovirus effect in osteosarcoma cells.

Newcastle disease virus (NDV) can specifically kill cancer cells and has less toxicity to normal cells. The hemagglutinin-neuraminidase (HN) protein is an important structural protein in NDV pathogenesis and has been postulated as a promising candidate for antitumor therapy. The aim of this study was to investigate the anticancer potential of recombinant adenovirus Ad-HN-PEG3p-E1a. An MTS assay was performed to determine viral proliferation after viral infection, the data showed that the proliferation ability of osteosarcoma cells decreased, whereas there was no significant change in normal hepatic cells. DAPI and Annexin V experiments showed that osteosarcoma cells were killed because of apoptosis, active oxygen content, and augmented mitochondrial membrane potential loss. Caspase Activity Assay Kits were used to detect the caspase-3 activities of the treated OS-732 for increased expression. Western blot analysis showed that cytochrome C increased significantly and apoptosis of the virus was confirmed in tumor cells. In-vivo experiments show that NDV has an inhibitory effect on tumor growth. The recombinant adenovirus, which is composed of a HN protein and progressive increment promoter PEG3p, could inhibit the growth of OS-732 and promote the apoptosis of tumor cells. However, there was no clear relationship with normal cell (L02) apoptosis.