Role of the Cytoplasmic Tail Amino Acid Sequences of Newcastle Disease Virus Hemagglutinin-Neuraminidase Protein in Virion Incorporation,Cell Fusion,and Pathogenicity

To determine the role of amino acid sequences of the hemagglutinin-neuraminidase (HN) cytoplasmic tail in Newcastle disease virus (NDV) replication and pathogenicity, we generated recombinant NDVs with a deletion or point mutation in the N-terminal cytoplasmic tail. The first 2-amino-acid deletion in the cytoplasmic tail did not affect the biological characteristics of NDV. However, a 4-amino-acid deletion and the substitution of alanine for serine at position 6 affected cell fusion, pathogenicity, and colocalization of the HN and M proteins of NDV, indicating that these residues of the HN cytoplasmic tail are critical for its specific incorporation into virions.Newcastle disease virus (NDV) causes a highly contagious respiratory and neurologic disease in chickens, leading to severe economic losses in the poultry industry worldwide (1). NDV is a member of the family Paramyxoviridae and has a nonsegmented, negative-sense RNA genome consisting of six genes (3′-NP-P-M-F-HN-L-5′) (7). Infection of host cells by NDV is accomplished through the interaction of two surface glycoproteins, the fusion (F) and hemagglutinin-neuraminidase (HN) proteins. The F protein directs the membrane fusion between the viral and cellular membranes, while the HN protein mediates attachment to sialic acid, has neuraminidase activity, and plays a role in fusion promotion (4).The HN protein of NDV is a type II transmembrane glycoprotein and possesses three spatially distinct domains: the ectodomain, transmembrane domain, and cytoplasmic tail. The globular ectodomain contains the sites for receptor binding and neuraminidase activity, and the transmembrane domain anchors to viral envelopes (8). The cytoplasmic tail domain contains 26 highly conserved amino acids whose functions are not well-known. In a plasmid-based expression system, truncation (23 amino acids) of the cytoplasmic tail caused improper orientation of the HN protein in the membrane insertion (13). In other paramyxoviruses, cytoplasmic tails of the HN proteins are known to play crucial roles in virus budding and assembly (10, 12). Our unsuccessful attempt to recover a recombinant NDV (rNDV) with complete deletion of the HN cytoplasmic tail also suggested that the cytoplasmic tail is required for assembly and budding of NDV. Therefore, in this study, we determined the role of amino acid sequences of the cytoplasmic tail in the NDV replication cycle. Since essential regions of the HN cytoplasmic tail for virus replication are unknown, we consecutively deleted the first 6 nucleotides (nt), 12 nt, or 18 nt of the HN cytoplasmic tail in a full-length antigenomic cDNA of NDV intermediate virulent (mesogenic) strain Beaudette C (BC) (6), thus maintaining the “rule of six” for the NDV genome (Fig. ​(Fig.1A).1A). rNDVs were recovered using our standard protocol (6). We recovered rNDVs containing 2-amino-acid deletion and 4-amino-acid deletion of the HN cytoplasmic tail (rBC/HNΔ2 and rBC/HNΔ4, respectively), indicating that only these 4 amino acids are dispensable in generating infectious virions. Since rNDV containing 6-amino-acid deletion of the HN cytoplasmic tail could not be recovered, we wanted to know the role of amino acids at positions 5 and 6 in NDV replications. The serine residue at position 6 is a potential phosphorylation site. Therefore, to determine whether phosphorylation at this site is crucial for recovery of NDV, we additionally generated rNDVs with substitution of alanine and glutamic acid for serine (rBC/HNS6A and rBC/HNS6E, respectively) to confirm its crucial role in the recovery of rNDV.Open in a separate windowFIG. 1.Constructs of recombinant NDVs containing a deletion or point mutation in the N-terminal cytoplasmic tail of the HN protein and replication and fusion index of recovered viruses in infected cells. (A) Consecutively, 6 nt, 12 nt, or 18 nt of mRNA of the HN cytoplasmic tail in a full-length antigenomic cDNA of NDV was deleted. Deletions in the HN cytoplasmic tails are indicated by the large boldface dashes. In addition, serine at position 6 was substituted with alanine and glutamic acid was substituted by changing guanine to cytidine and adenosine, respectively. (B) In vitro replication of the mutant viruses was determined in virus-infected DF-1 cells at an MOI of 0.01. The viral titers were determined by plaque assay. (C) The fusion index was determined in virus-infected Vero cells at an MOI of 0.1. Cells were stained with hematoxylin-eosin, and the fusion index was calculated as a mean number of nuclei per cell. The assay was performed three times.In vitro replication of recovered viruses was determined by plaque assay in virus-infected DF-1 cells at a multiplicity of infection (MOI) of 0.01 (5). All mutant viruses and the parental virus, rBC, grew to similar titers, indicating that alteration of the HN cytoplasmic tails did not affect their in vitro replication (Fig. ​(Fig.1B).1B). Although the rBC/HNΔ4 mutant had grown well up to 24 h postinfection, a reduction of the viral titer was detected thereafter with rapid and extensive induction of syncytia. Therefore, we determined fusion promotion activity of the mutant viruses by quantitating syncytia in virus-infected Vero cells at an MOI of 0.1 at 30 h postinfection (8) and confirmed increased fusion promotion activity of rBC/HNΔ4 followed by rBC/HNS6A compared to that of rBC (Fig. ​(Fig.1C).1C). Similarly, enhanced fusion activity was observed in other cytoplasmic tail-truncated paramyxoviruses, such as simian virus 5 and measles virus (2, 9). It has been postulated that interaction of matrix (M) protein with the cytoplasmic tails of the glycoproteins involves in a fusion-refractory conformation at the early stage of viral maturation (2). Therefore, these altered HN cytoplasmic tails could assist NDV in gaining its cell fusion competence by modulating this fusion-refractory conformation.In general, the levels of the HN protein contents on the surfaces of virus-infected cells and in the virus particles were more affected by point mutation of serine than by truncation of the cytoplasmic tail. We analyzed surface expression of the HN protein on virus-infected DF-1 cells at an MOI of 0.1. At 24 h postinfection, the cells were labeled with a monoclonal antibody against the NDV HN protein followed by anti-Alexa Fluor 488 conjugate, fixed with 4% paraformaldehyde, and analyzed by a fluorescence-activated cell sorter (AriaII; BD Bioscience) with Flowjo program (Tree Star, Inc.) (Fig. ​(Fig.2A).2A). The percentages of cells expressing the HN proteins were 89 (rBC), 78 (rBC/HNΔ2), 71 (rBC/HNΔ4), 64 (rBC/HNS6A), and 53 (rBC/HNS6E). To analyze incorporation of the HN proteins into the viral particles, the parental and mutant viruses harvested from allantoic fluid samples were purified through a 30% sucrose cushion. The viral proteins were separated on an 8% sodium dodecyl sulfate-polyacrylamide gel (Fig. ​(Fig.2B).2B). We first examined whether the mutant viruses incorporated the same levels of other viral proteins. This assay was performed by determining the ratios of the P protein to M protein. We found that similar levels of the P and M proteins were present among the different mutant viruses (Fig. ​(Fig.2B).2B). We then measured the levels of the HN proteins incorporated into the virus particles by determining the ratios of the HN protein to M protein (Fig. ​(Fig.2C).2C). The pattern of incorporation of the HN proteins into the virus particles was similar with their cell surface expression. The HN protein contents of rBC/HNΔ2 and rBC/HNΔ4 were not significantly different from that of the parental virus (P > 0.05), indicating that truncation of the cytoplasmic tail did not impair its incorporation into the viral particles. In contrast, substitution of glutamic acid for serine decreased incorporation of the HN protein into the viral particles, indicating that serine plays an important role in both cell surface expression of the HN protein and its incorporation into the viral particles.Open in a separate windowFIG. 2.Effect of alteration of the HN cytoplasmic tail on incorporation of the HN proteins into viral particles and their surface expression in DF-1 cells. (A) Surface expression of the NDV HN protein in DF-1 cells was analyzed by a fluorescence-activated cell sorter. At 24 h postinfection, DF-1 cells infected with each virus were stained with monoclonal antibody against the HN protein followed by anti-Alexa Fluor 488 conjugate. (B) Ultracentrifuge-purified viruses from infected allantoic fluid samples were separated by electrophoresis, and the gel was then stained with Coomassie brilliant blue. (C) Ratios of HN protein to M-protein levels from the parental virus and the HN cytoplasmic tail mutant viruses were quantified.We further determined the effect of cytoplasmic tail alteration on the pathogenicity of NDV in embryonated eggs and chicks (Table ​(Table1).1). The mean death time (MDT) was determined as the mean time (h) for the minimum lethal dose of virus to kill all the embryos after inoculation of 9-day-old specific-pathogen-free (SPF) embryonated chicken eggs with virus (1). The criteria for classifying the virulence of NDV strains are as follows: virulent strains take <60 h to kill embryos, intermediate virulent strains take 60 to 90 h to kill embryos, and avirulent strains take >90 h to kill embryos. Two mutant viruses (rBC/HNΔ2 and rBC/HNS6E) showed similar values of MDT compared to rBC (59 h). In contrast, the MDTs of rBC/HNΔ4 and rBC/HNS6A were 50 h and 51 h, respectively. Increased pathogenicity of these two mutants was also confirmed by an intracerebral pathogenicity index (ICPI) test in 1-day-old SPF chicks (1). The scale of the ICPI value in evaluating the virulence of NDV strains is from 0.00 (avirulent strains) to 2.00 (highly virulent NDV strains). The rBC/HNΔ4 virus had the highest ICPI value (1.61), followed by rBC/HNS6A (ICPI value of 1.58), among the parental and mutant viruses, probably due to their enhanced fusion promotion activity. In contrast, rBC/HNS6E had the lowest ICPI value (1.41), which would be associated with decreased HN protein contents detected in the viral particles and virus-infected cells. In our previous study, decreased HN protein contents in virus particles due to complete deletion of 5′ untranslated regions of the HN gene also resulted in attenuation of the virus in chickens (14). Consistently, rBC/HNΔ2 showed biological characteristics and pathogenicity similar to those of the parental virus, suggesting that aspartic acid and arginine are indispensable for the HN cytoplasmic tail of NDV.

TABLE 1.

Pathogenicity of the HN cytoplasmic tail mutant viruses in embryonated eggs and chicks

新城疫病毒F蛋白细胞融合活性位点中保守氨基酸基因突变分析

为了确定新城疫病毒融合蛋白(F)分子上活性位点中保守氨基酸在F蛋白的细胞融合作用,弄清F细胞融合的分子机理,采用基因定点突变法,创造一个酶切位点,用酶切反应初步筛选突变株,然后用DNA序列分析进一步确定,并于真核细胞内进行表达,Giemsa染色定性和指示基因法定量检测细胞融合功能,荧光强度分析(FACS)检测表达效率情况。结果表明,NDV F第117位苯丙氨酸(F)突变成亮氨酸(L)时对细胞融合作用没有显著影响。R112和K115同为保守序列,分别突变为G时,细胞融合活性只有原来的44%,下降了56%。细胞表面表达效率没有明显的改变。N147突变为K时,细胞融合活性明显下降,只有原来的15%,而细胞表面表达效率没有明显的改变。L154为保守序列,突变为K时,细胞融合活性消失,说明L154是一个非常关键的氨基酸,对维持F蛋白的细胞融合活性非常重要。细胞表面表达效率也有所下降(为原来的94%)。D462属于高度保守氨基酸,当突变为N时,细胞融合活性消失,但经细胞表面表达效率分析证明,此突变蛋白未表达于细胞表面,证明在细胞浆转运至细胞表面的过程中发生了问题。当突变为R和E时,细胞融合活性未发生改变,但细胞表面表达效率有所下降,分别为野毒株的63%和44%。说明NDV F分子上与HN相互作用的特异性区域中的某些保守氨基酸在细胞融合中发挥着重要作用,对F蛋白的折叠、加工、转运等,发挥着不同作用,从而影响F蛋白的细胞融合作用和/或在细胞表面的表达量。     

Cell Fusion by Newcastle Disease Virus in the Absence of RNA Synthesis

OUR studies on the cytopathic effects of Newcastle disease virus (NDV) grown in chick embryo fibroblast cell cultures have shown that the principal cytopathic effect involves the formation of polykaryocytes by cell fusion (our unpublished work). This ability is related directly to the virulence of the infecting strain; those pathogenic for chick embryos readily induce cell fusion whereas avirulent strains induce little or no fusion¹. We now report that, although protein synthesis is required for NDV-induced cell fusion, RNA synthesis is not. Furthermore, blocking of RNA synthesis significantly increases cell fusion by avirulent strains.     

Cell Fusion by Various Strains of Newcastle Disease Virus and Their Virulence

Some properties of eight strains of Newcastle disease virus (cell-fusing ability, hemolysin, heat stability of hemagglutinin or of hemolysin) do not correlate with virulence of these strains.     

Full Conversion of the Hemagglutinin-Neuraminidase Specificity of the Parainfluenza Virus 5 Fusion Protein by Replacement of 21 Amino Acids in Its Head Region with Those of the Simian Virus 41 Fusion Protein

For most parainfluenza viruses, a virus type-specific interaction between the hemagglutinin-neuraminidase (HN) and fusion (F) proteins is a prerequisite for mediating virus-cell fusion and cell-cell fusion. The molecular basis of this functional interaction is still obscure partly because it is unknown which region of the F protein is responsible for the physical interaction with the HN protein. Our previous cell-cell fusion assay using the chimeric F proteins of parainfluenza virus 5 (PIV5) and simian virus 41 (SV41) indicated that replacement of two domains in the head region of the PIV5 F protein with the SV41 F counterparts bestowed on the PIV5 F protein the ability to induce cell-cell fusion on coexpression with the SV41 HN protein while retaining its ability to induce fusion with the PIV5 HN protein. In the study presented here, we furthered the chimeric analysis of the F proteins of PIV5 and SV41, finding that the PIV5 F protein could be converted to an SV41 HN-specific chimeric F protein by replacing five domains in the head region with the SV41 F counterparts. The five SV41 F-protein-derived domains of this chimera were then divided into 16 segments; 9 out of 16 proved to be not involved in determining its specificity for the SV41 HN protein. Finally, mutational analyses of a chimeric F protein, which harbored seven SV41 F-protein-derived segments, revealed that replacement of at most 21 amino acids of the PIV5 F protein with the SV41 F-protein counterparts was enough to convert its HN protein specificity.     

Conditional Dependence of Fusion from Within and Other Cell Membrane Alterations by Newcastle Disease Virus

Fusion from within (FFWI) by Newcastle disease virus occurs optimally in medium maintained at pH 8.2, whereas fusion from without is relatively insensitive to the pH of the medium in the range of 7.0 to 8.3. The pH-sensitive events in FFWI take place in the synthesis of the hypothetical fusion factor rather than in the response to it. pH pulse and pH shift experiments have localized the pH-sensitive events between 4 and 6.5 h postinfection (a period of synthesis of proteins required for FFWI), but before the fusion process. The pH sensitivity is not due to a pH-sensitive interference phenomenon. Virus production and the appearance of hemadsorbing cell surfaces are also pH sensitive, but for these functions the pH optima depend upon the virus strains tested. The independence of FFWI, hemadsorption, and virus production is discussed. Also discussed are the possible roles of virus-specific proteins in the fusion process.     

Structure of the Ulster Strain Newcastle Disease Virus Hemagglutinin-Neuraminidase Reveals Auto-Inhibitory Interactions Associated with Low Virulence

Paramyxovirus hemagglutinin-neuraminidase (HN) plays roles in viral entry and maturation, including binding to sialic acid receptors, activation of the F protein to drive membrane fusion, and enabling virion release during virus budding. HN can thereby directly influence virulence and in a subset of avirulent Newcastle disease virus (NDV) strains, such as NDV Ulster, HN must be proteolytically activated to remove a C-terminal extension not found in other NDV HN proteins. Ulster HN is 616 amino acids long and the 45 amino acid C-terminal extension present in its precursor (HN₀) form has to be cleaved to render HN biologically active. Here we show that Ulster HN contains an inter-subunit disulfide bond within the C-terminal extension at residue 596, which regulates HN activities and neuraminidase (NA) domain dimerization. We determined the crystal structure of the dimerized NA domain containing the C-terminal extension, which extends along the outside of the sialidase β-propeller domain and inserts C-terminal residues into the NA domain active site. The C-terminal extension also engages a secondary sialic acid binding site present in NDV HN proteins, which is located at the NA domain dimer interface, that most likely blocks its attachment function. These results clarify how the Ulster HN C-terminal residues lead to an auto-inhibited state of HN, the requirement for proteolytic activation of HN₀ and associated reduced virulence.     

Structures of Merkel Cell Polyomavirus VP1 Complexes Define a Sialic Acid Binding Site Required for Infection

The recently discovered human Merkel cell polyomavirus (MCPyV or MCV) causes the aggressive Merkel cell carcinoma (MCC) in the skin of immunocompromised individuals. Conflicting reports suggest that cellular glycans containing sialic acid (Neu5Ac) may play a role in MCPyV infectious entry. To address this question, we solved X-ray structures of the MCPyV major capsid protein VP1 both alone and in complex with several sialylated oligosaccharides. A shallow binding site on the apical surface of the VP1 capsomer recognizes the disaccharide Neu5Ac-α2,3-Gal through a complex network of interactions. MCPyV engages Neu5Ac in an orientation and with contacts that differ markedly from those observed in other polyomavirus complexes with sialylated receptors. Mutations in the Neu5Ac binding site abolish MCPyV infection, highlighting the relevance of the Neu5Ac interaction for MCPyV entry. Our study thus provides a powerful platform for the development of MCPyV-specific vaccines and antivirals. Interestingly, engagement of sialic acid does not interfere with initial attachment of MCPyV to cells, consistent with a previous proposal that attachment is mediated by a class of non-sialylated carbohydrates called glycosaminoglycans. Our results therefore suggest a model in which sialylated glycans serve as secondary, post-attachment co-receptors during MCPyV infectious entry. Since cell-surface glycans typically serve as primary attachment receptors for many viruses, we identify here a new role for glycans in mediating, and perhaps even modulating, post-attachment entry processes.     

新城疫病毒F和HN蛋白的氨基酸序列对其毒力的影响

新城疫病毒(Newcastle disease virus,NDV)属副粘病毒科副粘病毒属,为不分节的负极性单股RNA病毒,有囊膜.它虽只有一个血清型,但不同毒株的致病力差异较大,有强毒株、中毒株和弱毒株之分.业已证明,NDV不存在先天性控制病毒致病性基因,不同的毒株都含有相同的基因组和结构蛋白质组分.     

Investigations of Amino Acids in the ATP Binding Site of 5,10-Methenyltetrahydrofolate Synthetase

5-Formyltetrahydrofolate is a compound that is administered as a rescue agent in methotrexate chemotherapy and in 5-fluorouracil chemotherapy for synergistic effects. It has also recently been suggested to play a role in bacterial resistance to antifolate therapy. 5,10-methenyltetrahydrofolate synthetase (MTHFS) is the only enzyme known to catalyze the conversion of this compound to 5,10-methenyltetrahydrofolate along with the hydrolysis of ATP to ADP. To better understand the roles of specific amino acids in the ATP binding pocket of this enzyme, we used site-directed mutagenesis to create 10 modified forms of the Mycoplasma pneumoniae ortholog. The Michaelis constant (K_m) for each substrate and the turnover number (k_cat) was determined for each mutant to help elucidate the role of individual amino acids. Data were compared to crystal structures of human and M. pneumoniae orthologs of MTHFS. Results were largely consistent with a simple coulombic and proximity model; the larger the predicted charges of an interaction and the closer those interactions were to the phosphate transferred between the substrates, the greater the reduction in ATP binding and catalytic activity of the enzyme.     

The Receptor Binding Site of Feline Leukemia Virus Surface Glycoprotein Is Distinct from the Site Involved in Virus Neutralization

The external surface glycoprotein (SU) of feline leukemia virus (FeLV) contains sites which define the viral subgroup and induce virus-neutralizing antibodies. The subgroup phenotypic determinants have been located to a small variable region, VR1, towards the amino terminus of SU. The sites which function as neutralizing epitopes in vivo are unknown. Recombinant SU proteins were produced by using baculoviruses that contained sequences encoding the SUs of FeLV subgroup A (FeLV-A), FeLV-C, and two chimeric FeLVs (FeLV-215 and FeLV-VC) in which the VR1 domain of FeLV-A had been replaced by the corresponding regions of FeLV-C isolates. The recombinant glycoproteins, designated Bgp70-A, -C, -215, and -VC, respectively, were similar to their wild-type counterparts in several immunoblots and inhibited infection of susceptible cell lines in a subgroup-specific manner. Thus, Bgp70-A interfered with infection by FeLV-A, whereas Bgp70-C, -VC, and -215 did not. Conversely, Bgp70-C, -VC, and -215 blocked infection with FeLV-C, while Bgp70-A had no effect. These results indicate that the site on SU which binds to the FeLV cell surface receptor was preserved in the recombinant glycoproteins. It was also found that the recombinant proteins were able to bind naturally occurring neutralizing antibodies. Bgp70-A, -VC, and -215 interfered with the action of anti-FeLV-A neutralizing antibodies, whereas Bgp70-C did not. Furthermore, Bgp70-C interfered with the action of anti-FeLV-C neutralizing antibodies, while the other proteins did not. These results indicate that the neutralizing epitope(s) of FeLV SU lies outside the subgroup-determining VR1 domain.     

Different Regions of the Newcastle Disease Virus Fusion Protein Modulate Pathogenicity

Newcastle disease virus (NDV), also designated as Avian paramyxovirus type 1 (APMV-1), is the causative agent of a notifiable disease of poultry but it exhibits different pathogenicity dependent on the virus strain. The molecular basis for this variability is not fully understood. The efficiency of activation of the fusion protein (F) is determined by presence or absence of a polybasic amino acid sequence at an internal proteolytic cleavage site which is a major determinant of NDV virulence. However, other determinants of pathogenicity must exist since APMV-1 of high (velogenic), intermediate (mesogenic) and low (lentogenic) virulence specify a polybasic F cleavage site. We aimed at elucidation of additional virulence determinants by constructing a recombinant virus that consists of a lentogenic NDV Clone 30 backbone and the F protein gene from a mesogenic pigeon paramyxovirus-1 (PPMV-1) isolate with an intracerebral pathogenicity index (ICPI) of 1.1 specifying the polybasic sequence R-R-K-K-R*F motif at the cleavage site. The resulting virus was characterized by an ICPI of 0.6, indicating a lentogenic pathotype. In contrast, alteration of the cleavage site G-R-Q-G-R*L of the lentogenic Clone 30 to R-R-K-K-R*F resulted in a recombinant virus with an ICPI of 1.36 which was higher than that of parental PPMV-1. Substitution of different regions of the F protein of Clone 30 by those of PPMV-1, while maintaining the polybasic amino acid sequence at the F cleavage site, resulted in recombinant viruses with ICPIs ranging from 0.59 to 1.36 suggesting that virulence is modulated by regions of the F protein other than the polybasic cleavage site.     

Identification of Key Amino Acid Residues Involved in the Localization of Sorting Nexin 10 and Induction of Vacuole Formation

Biochemistry (Moscow) - Sorting nexin 10 (SNX10) induces formation of vacuoles participating in the endosome morphogenesis in mammalian cells, but the key amino acids involved in this function have...     

Dioxygenases Without Requirement for Cofactors: Identification of Amino Acid Residues Involved in Substrate Binding and Catalysis, and Testing for Rate-Limiting Steps in the Reaction of 1H-3-Hydroxy-4-oxoquinaldine 2,4-dioxygenase

1H-3-Hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod), catalyzing cleavage of its heteroaromatic substrate to form carbon monoxide and N-acetylanthranilate, belongs to the α/β hydrolase fold family of enzymes. Analysis of protein variants suggested that Hod has adapted active-site residues of the α/β hydrolase fold for the dioxygenolytic reaction. H251 was recently shown to act as a general base to abstract a proton from the organic substrate. Residue S101, which corresponds to the nucleophile of the catalytic triad of α/β-hydrolases, presumably participates in binding the heteroaromatic substrate. H102 and residues located in the topological region of the triad’s acidic residue appear to influence O₂ binding and reactivity. A tyrosine residue might be involved in the turnover of the ternary complex [HodH⁺–3,4-dioxyquinaldine dianion–O₂]. Absence of viscosity effects and kinetic solvent isotope effects suggests that turnover of the ternary complex, rather than substrate binding, product release, or proton movements, involves the rate-determining step in the reaction catalyzed by Hod.     

A Single Amino Acid Alteration in the Human Parainfluenza Virus Type 3 Hemagglutinin-Neuraminidase Glycoprotein Confers Resistance to the Inhibitory Effects of Zanamivir on Receptor Binding and Neuraminidase Activity

Entry and fusion of human parainfluenza virus type 3 (HPF3) requires interaction of the viral hemagglutinin-neuraminidase (HN) glycoprotein with its sialic acid receptor. 4-Guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid (4-GU-DANA; zanamivir), a sialic acid transition-state analog designed to fit the influenza virus neuraminidase catalytic site, possesses antiviral activity at nanomolar concentrations in vitro. We have shown previously that 4-GU-DANA also inhibits both HN-mediated binding of HPF3 to host cell receptors and HN's neuraminidase activity. In the present study, a 4-GU-DANA-resistant HPF3 virus variant (ZM1) was generated by serial passage in the presence of 4-GU-DANA. ZM1 exhibited a markedly fusogenic plaque morphology and harbored two HN gene mutations resulting in two amino acid alterations, T193I and I567V. Another HPF3 variant studied in parallel, C-0, shared an alteration at T193 and exhibited similar plaque morphology but was not resistant to 4-GU-DANA. Neuraminidase assays revealed a 15-fold reduction in 4-GU-DANA sensitivity for ZM1 relative to the wild type (WT) and C-0. The ability of ZM1 to bind sialic acid receptors was inhibited 10-fold less than for both WT and C-0 in the presence of 1 mM 4-GU-DANA. ZM1 also retained infectivity at 15-fold-higher concentrations of 4-GU-DANA than WT and C-0. A single amino acid alteration at HN residue 567 confers these 4-GU-DANA-resistant properties. An understanding of ZM1 and other escape variants provides insight into the effects of this small molecule on HN function as well as the role of the HN glycoprotein in HPF3 pathogenesis.