Neuraminidase activity of blue eye disease porcine rubulavirus: Specificity, affinity and inhibition studies

Research in Veterinary Science 114 (2017) 218–224

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Neuraminidase activity of blue eye disease porcine rubulavirus: Specificity, affinity and inhibition studies

MARK

Gerardo Santos-Lópeza,⁎, María T. Borraz-Argüellob, Luis Márquez-Domíngueza, Juan Carlos Flores-Alonsoa, Humberto Ramírez-Mendozac, Bernard Priemd,e, Sébastien Fortd,e, Verónica Vallejo-Ruiza, Julio Reyes-Leyvaa, Irma Herrera-Camachof,⁎⁎ a

Laboratorio de Biología Molecular y Virología, Centro de Investigación Biomédica de Oriente, Instituto Mexicano del Seguro Social, Metepec, Puebla, Mexico Departamento de Ingeniería en Biotecnología Universidad Politécnica de Puebla, Calpan, Puebla, Mexico c Departamento de producción Animal Cerdos, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, México, D.F., Mexico d Université Grenoble Alpes, CERMAV, F-38000 Grenoble, France e CNRS, CERMAV, F-38000 Grenoble, France f Laboratorio de Bioquímica, Centro de Química, Instituto de Ciencias, Universidad Autónoma de Puebla, Puebla, Mexico b

A R T I C L E I N F O

A B S T R A C T

Keywords: Blue eye disease LPMV Hemagglutinin-neuraminidase HN Rubulavirus

Porcine rubulavirus (PorPV), also known as La Piedad Michoacan Virus (LPMV) causes encephalitis and reproductive failure in newborn and adult pigs, respectively. The hemagglutinin–neuraminidase (HN) glycoprotein is the most exposed and antigenic of the virus proteins. HN plays central roles in PorPV infection; i.e., it recognizes sialic acid-containing cell receptors that mediate virus attachment and penetration; in addition, its neuraminidase (sialic acid releasing) activity has been proposed as a virulence factor. This work describes the purification and characterization of PorPV HN protein (isolate PAC1). The specificity of neuraminidase is restricted to sialyl(α2,3)lactose (3SL). HN showed typical Michaelis-Menten kinetics with fetuin as substrate (km = 0.029 μM, Vmax = 522.8 nmol min− 1 mg− 1). When 3SL was used as substrate, typical cooperative kinetics were found (S50 = 0.15 μM, Vmax = 154.3 nmol min− 1 mg− 1). The influenza inhibitor zanamivir inhibited the PorPV neuraminidase with IC50 of 0.24 μM. PorPV neuraminidase was activated by Ca2 + and inhibited by nucleoside triphosphates with the level of inhibition depending on phosphorylation level. The present results open possibilities to study the role of neuraminidase in the pathogenicity of PorPV infection and its potential inhibitors.

1. Introduction Porcine rubulavirus, also known as La Piedad Michoacan Virus (LPMV), is a virus of veterinary importance reported in Mexico in 1980. Formerly abbreviated as PoRV and currently as PorPV (King et al., 2012), this virus causes the blue eye disease (BED) in pigs, characterized by neurological, respiratory and reproductive manifestations accompanied by corneal opacity in 1–10% of cases (Moreno-Lopez et al., 1986; Stephan et al., 1988). PorPV belongs to the Rubulavirus genus of the Paramyxoviridae family. It has a single-stranded negative-sense RNA genome of 15,180 nucleotides that contains six genes encoding for the following proteins:

nucleoprotein (NP), large protein (L) and phosphoprotein (P), associated with the nucleocapsid; and matrix (M), fusion (F) and hemagglutinin-neuraminidase (HN) proteins associated with the viral envelope. NP, L and P proteins play key roles during viral transcription and replication, while M protein is involved in virion assembly. HN and F proteins are necessary for viral entry into the host cell and also for the spread of infection to other cells (Linne et al., 1992; Santos-López et al., 2004; Wang et al., 2007). HN is a multifunctional protein. It recognizes the receptor on the cell surface and possesses an enzymatic activity (neuraminidase, E.C. 3.2.1.18), which is proposed as a virulence factor and promotes membrane fusion by activation of the F protein (Lamb and Parks,

Abbreviations: PorPV, Porcine rubulavirus; HN, hemagglutinin-neuraminidase; BED, blue eye disease ⁎ Correspondence to: G. Santos-López, Laboratorio de Biología Molecular y Virología, Centro de Investigación Biomédica de Oriente, Instituto Mexicano del Seguro Social, HGZ #5, Km. 4.5 Carretera Federal Atlixco-Metepec, 74360 Metepec, Puebla, Mexico. ⁎⁎ Correspondence to: I. Herrera-Camacho, Laboratorio de Bioquímica y Biología Molecular, Centro de Química del Instituto de Ciencias (ICUAP), Edificio 103F, Ciudad Universitaria, Benemérita Universidad Autónoma de Puebla, 72592 Puebla, Puebla, Mexico. E-mail addresses: [email protected] (G. Santos-López), [email protected] (I. Herrera-Camacho). http://dx.doi.org/10.1016/j.rvsc.2017.05.008 Received 24 December 2016; Received in revised form 4 May 2017; Accepted 6 May 2017 0034-5288/ © 2017 Elsevier Ltd. All rights reserved.

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PK-15 cells for 120 h at 37 °C. Infected supernatants were harvested and clarified by centrifugation at 3200 rpm/20 min/4 °C.

2013). PorPV HN protein (strain LPMV; La Piedad Michoacán, Mexico, 1984) specifically interacts with alpha 2,3-linked sialic acids on host cell receptors (Reyes-Leyva et al., 1993). These molecules are highly expressed in the central nervous system (CNS) of newborn pigs and in the reproductive tissues of adult pigs (Vallejo et al., 2000). These sialic acids play a central role in LPMV infection in cell culture (Reyes-Leyva et al., 1997) and CNS (Mendoza-Magana et al., 2007). In addition, the HN glycoprotein is highly antigenic and the humoral response is predominantly directed against this protein in pigs infected with PorPV (Hernandez et al., 1998). This has led to propose the HN protein as a vaccine target to prevent infection. There are two main reports of recombinant HN protein capable of inducing antibodies (CerritenoSanchez et al., 2016; Cuevas-Romero et al., 2016a). One of them induced the neutralization of PorPV in cell culture (Cerriteno-Sanchez et al., 2016), demonstrating the relevance of HN as a potential target for vaccine or antiviral drug design against BED. In a previous study we published the genetic changes observed at the HN gene in several field PorPV isolates collected during a 14-year period. Most of the mutations were predicted to locate at surface level, putatively caused by selective pressure of the host immune response (Sanchez-Betancourt et al., 2008; Sanchez-Betancourt et al., 2012). The presence of mutations has been directly associated with antigenic variation and the probable escape of the current vaccines against PorPV (Escobar-Lopez et al., 2012). It is interesting to note that the HN activities of some of these isolates differ. Analyzing three of those PorPV isolates: LPMV (1984), PAC1 (1990) and PAC3 (1992), we observed differences in hemagglutinating and neuraminidase properties. Other differences in cell culture were also determined: i.e. fusogenic and lytic properties. The cytolysis level correlated with neuraminidase activity of the distinct viruses. Accordingly, we hypothesized that neuraminidase may be related with the cell lysis in vitro and possibly has a role in the pathogenic properties of PorPV in vivo (Borraz-Arguello et al., 2008). These findings are similar to those of other paramyxoviral infections such as parainfluenza virus 3, in which evidence indicates that the neuraminidase activity influences the outcome of viral infection (Huberman et al., 1995). Due to the relevance of this protein, we decided to study additional features of the neuraminidase activity of PorPV. In order to carry out these objectives we purified the HN protein of the PAC1 isolate, and a series of kinetic studies were carried out using several sialylated substrates.

2.4. Cloning and sequencing of the HN gene The HN gene of the PorPV-PAC1 was amplified by RT-PCR using the primers HNRVP1 (5′-ATG TCT CAA TTA GGG ACT GAT C-3′) and HNRVP2 (5′-TTT CTT AAA GTA AGG GAT TTT G-3′), which produce a 1786 bp fragment that contains the complete open reading frame. The RT-PCR product was cloned into the pJET1.2/blunt Cloning Vector (CloneJET PCR Cloning Kit, Fermentas) according to the manufacturer's instructions. The cloned product was sequenced with pJET1.2 forward and reverse sequencing primers (included in the CloneJET PCR Cloning Kit) and HN-L1 (5′-GTCTATTCTCACCGCCATTC-3′), HN-L2 (5′GACGCACTCTGTATTGTTCC-3′), HNRVPSs (5′-CCCCCACAACAA CTCTTGACTCTT-3′), HNRVPSa (5′-TAACGCATGTTGCTGGACAGTGA3′) primers, using the GenomeLab Dye Terminator Cycle Sequencing Kit and the automatic sequencer GenomeLab GeXP Genetic Analysis System (Beckman-Coulter, Pasadena, CA, USA). Phylogenetic analyses were conducted in MEGA5 using the Neighbor-joining method (Saitou and Nei, 1987; Tamura et al., 2011). 2.5. Virus purification Virions in cell culture supernatants were concentrated by centrifugation at 31,000 × g for 1 h at 4 °C in a SS-34 rotor (Sorvall, Newtown, CT, USA). Pellets were re-suspended in TEN (0.1 M NaCl, 10 mM TrisHCl pH 8.0, 1 mM EDTA pH 8.0) buffer and submitted to sucrose density gradient (30–60% in TEN buffer) by ultracentrifugation at 100,000 × g for 4 h at 4 °C in a SW-28 rotor (Beckman Instruments, Fullerton, CA) (Reyes-Leyva et al., 2007). The bands that contained virus were collected, resuspended in TEN and pelleted through a 30% sucrose cushion at the same centrifugation conditions as above, then dialysed for 24 h against TEN. 2.6. HN extraction and purification HN purification was performed according to Thompson et al., 1988 (Thompson et al., 1988), with some modifications. Purified viruses were incubated with 1% Triton X-114 and 1 M KCl for 12 h at 4 °C with soft mixing to extract the HN protein from the viral membranes. The mixture was centrifuged at 190,000 × g for 1 h at 4 °C (50Ti rotor, Beckman). The supernatant was recovered and proteins were precipitated with 75% ammonium sulfate. Pellets were re-suspended and dialyzed for 12 h in TEN buffer. Protein concentration was calculated by the Bradford method (Bradford, 1976) through the purification process; the purity of samples was examined by 10% denaturing polyacrylamide gel electrophoresis (Laemmli, 1970).

2. Material and methods 2.1. Reagents and media The culture medium, supplements, and antibiotics were purchased from Invitrogen-Gibco (Carlsbad, CA, USA). Reagents used in analytical methods (NaCl, Tris-HCl, EDTA, periodic acid, thiobarbituric acid, HCl, sodium (meta)arsenite, colominic acid, nucleotides and zanamivir) were purchased from Sigma Chemical Co. (San Louis, MO, USA) except where otherwise indicated. Sialyl(α2,3)lactose and sialyl(α2,6)lactose were produced and purified as previously was published (Drouillard et al., 2010; Priem et al., 2002).

2.7. Neuraminidase assays 200 μl of the reaction mixture (enzyme, substrate, and sodium acetate buffer, pH 4) were incubated for 30 min at 37 °C. The reaction was stopped by boiling for 2 min. Neuraminic acid released by the HN protein was measured using the modified thiobarbituric acid method previously reported (Santos-Lopez et al., 2004). The free neuraminic acid concentration was calculated in correlation to a standard curve of pure 5-N-acetyl neuraminic acid.

2.2. Cells Porcine kidney PK-15 cells were grown in Eagle's minimal essential medium (MEM) supplemented with 5% neonate bovine serum (NBS), Lglutamine (100 mg/ml), penicillin (100 U/ml), and streptomycin (100 mg/ml).

2.8. Kinetic assays Purified HN protein or virus extracts were incubated with the following sialylated molecules as possible substrates: sialyl(α2,3)lactose (3SL), sialyl(α2,6)lactose (6SL), colominic (polysialic) acid and bovine serum fetuin. The neuraminic acid released from each substrate was determined as indicated above. Specific activity (SA) was reported

2.3. Virus culture Porcine rubulavirus isolate PAC1, from the 1990 BED outbreak in Michoacan, Mexico (Borraz-Arguello et al., 2008) was propagated in 219

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as nmol of released sialic acid per min per mg of protein. The kinetic parameters Km or S50 and Vmax were calculated using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, U.S.A.).

2.9. Neuraminidase inhibition To study the neuraminidase inhibition, purified HN protein was preincubated with increasing concentrations of inhibitor molecules (monodi- and tri-phosphate nucleotides and zanamivir) for 30 min at 25 °C and neuraminidase activity was determined as mentioned above. Enzyme inhibition analyses were performed using GraphPad Prism 5.

2.10. Hemagglutination tests HA activity was assayed in 96-wells U bottomed microassay plates using two-fold serial dilutions. One percent suspensions of goat erythrocytes in PBS buffer (10 mM sodium phosphate, 150 mM sodium chloride, pH 7.2) were incubated with HN protein or viral extracts at 4 °C for 1 h. Hemagglutinating titers were expressed as the reciprocal of the highest dilution with agglutinating activity. Comparative titers through the purification process were expressed in HAU per mg of extract or virus protein (Borraz-Arguello et al., 2008; Reyes-Leyva et al., 1993).

Fig. 1. Phylogenetic tree based on the amino acid sequences of PAC1 HN protein and other PorPV strains. The evolutionary history was inferred using the Neighbor-Joining method. The optimal tree with the sum of the branch length = 0.06300547 is shown. The tree is drawn to scale with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Evolutionary distances are in the units of the number of amino acid substitutions per site. There was a total of 576 positions in the final dataset. Evolutionary analyses were conducted in MEGA5.

3. Results 3.1. HN gene sequence of PorPV-PAC1

Table 1 Functional amino acids of PorPV-PAC1 hemagglutinin-neuraminidase compared to the crystallized SV5 and NDV HN.

The HN gene from the PAC1 virus was amplified by RT-PCR and cloned into the pJet1.2-blunt vector originating the pJHNP1 plasmid. The sequence of the HN gene from PAC1 was determined, deposited in GenBank (KP229773) and compared with other complete HN sequences available in GenBank. PAC1 HN showed between 97.5 and 99.6% identity with the other PorPV HN sequences. The PAC1 HN sequence clustered into group A of the filogenetic tree, along with the first sequence reported for PorPV, LPMV/1984, (99.4% identity). The closest PorPV isolates were PAC4/1993, Mx/Jalisco/2007 and Mx/2/ Michoacan/2009, which presented 99.6% identity with the PAC1 HN sequence (Fig. 1). The predicted PAC1 HN amino acid sequence possesses all the residues related to neuraminidase activity that have been previously reported for paramyxoviral HNs, such as Newcastle Disease Virus (NDV) and parainfluenza virus 5 (PIV5). For instance, PAC1 HN contains an arginine triad (R173, R415 and R503), which specifically interacts with the carboxyl group of sialic acid. In addition to neuraminidase activity, PAC1 HN harbors the critical residues responsible for binding (hemagglutinating) activity: E400, R415 and Y531 (Crennell et al., 2000; Yuan et al., 2005). These and other important functional amino acids are shown in Table 1.

Activity

PorPV

PIV5

NDV

Major receptor binding site

L 219 I 221 A 223 E 400 R 415 Y 523 R 173 D 197 E 400 R 415 R 503 Y 531 E 552

F 209 R 211 L 213 E 390 R 405 Y 523 R 163 D 187 E 390 R 405 R 495 Y 523 E 544

F 220 S 222 L 224 E 401 R 416 Y 526 R 174 D 198 E 401 R 416 R 498 Y 526 E 547

Neuraminidase

HN amino acid residues (single letter code and position) of PorPV-PAC1 (KP229773) compared with the equivalent positions in crystallized HN of parainfluenza virus 5 (PIV5, K02870) and Newcastle disease virus (NDV, AF212323). Non-conserved residues are in bold type.

3.3. Neuraminidase activity The specificity of PAC1 neuraminidase was investigated using several sialylated molecules. The substrates cleaved by HN were fetuin and sialyl(α2,3)lactose (3SL) while sialyl(α2,6)lactose (6SL) and colominic acid were not susceptible to cleavage. Therefore, only the first two substrates were used for kinetic analyses. The kinetic parameters and specific activity of HN proteins were measured in the range of 0.25 to 8.5 mg/ml for fetuin and 0.025 to 0.65 mg/ml for 3SL. HN showed typical Michaelis-Menten kinetics with fetuin with Km of 0.029 μM (1.4 μg/ml) and Vmax of 522.8 nmol min− 1 mg− 1 (Fig. 3A and C). When 3SL was used as substrate, a sigmoidal behavior was found, suggesting cooperativity. The kinetic parameters were: S50 of 0.15 μM and Vmax of 154.3 nmol min− 1 mg− 1. The calculated Hill constant (nH) was 1.77, close to 2, (Fig. 3D) which suggests that at least two

3.2. Purification of the HN protein After the purification procedure, a sample of HN protein with a purification factor of 369 and an activity yield of 8.4%, was obtained (Table 2). Samples of the last purification stage, which consisted of treatment with Triton X-114 and ultracentrifugation at 100,000 × g/ 1 h, were subjected to SDS-PAGE, resulting in a single protein band (Fig. 2A). The calculated molecular mass of ~66 kDa correlated with the expected Mr. of the PorPV HN protein. Hemagglutinating activity was followed during the purification process and a specific activity in the last purification stage of 10,159 UHA/mg protein was obtained, which corresponds to 290 times the initial value in the viral supernatant (35 UHA/mg). 220

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Table 2 Balance of purification process. Stage

Vol (ml)

Protein (mg)

Enzymatic activity (nmol min− 1)

Specific activity (nmoles/min mg)

Activity yield (%)

Purification fold

Virus supernatant Pellet 31,000 × g Sucrose gradient Triton X-114

150 3 4 5

2770.50 20.91 7.4 0.63

2351.64 384.62 277.27 197.56

0.85 18.39 37.47 313.59

100.00 16.35 11.79 8.40

1.00 21.64 44.08 368.93

0.14 μM instead of 0.15 μM. On the other hand, ATP inhibited neuraminidase activity (Fig. 5), increasing the lag phase of the enzymatic curve and decreasing the value of Vmax to 61.6 nmol min− 1 mg− 1, with S50 of 0.12 μM. To determine the possible mechanism of inhibition of ATP on neuraminidase activity we performed an inhibition study using fetuin as substrate, which is plotted in Fig. 6. ATP inhibited PAC1 neuraminidase in a competitive manner with a Ki of 0.026 mg/ml. 3.6. Inhibition of PorPV neuraminidase by ATP depends on phosphorylation In order to determine the importance of nucleotide/adenosine phosphorylation on enzymatic inhibition, several assays using tri-, diand monophosphate molecules of adenosine were used. Fig. 7 shows that 90% inhibition on the neuraminidase activity of PorPV was reached with 10 mM ATP. This inhibitory effect was not observed with ADP or AMP at the same concentration. Similar inhibition results were obtained using CTP and GTP, although GTP showed a relative lower effect than ATP. 4. Discussion PorPV is an important virus of veterinary interest reported in Mexico (Escobar-Lopez et al., 2012; Sanchez-Betancourt et al., 2012; Stephan et al., 1988). A major viral constituent is the HN glycoprotein, a multifunctional protein and the most exposed and antigenic component of virions (Hernandez et al., 1998). In the present work we purified and characterized the neuraminidase activity of PorPV HN. We selected PAC1, a viral isolate related with systemic infection in pigs of all ages (Borraz-Arguello et al., 2008). We found that the PAC1 HN sequence is highly similar to the available PorPV HN sequences, in particular to those from group A (Fig. 1). Three or fewer amino acids were different in the HN proteins from the first (1984) to the last (2009) isolates, suggesting that the PorPV virus has a slow mutation rate. The low variation rate in the HN of PorPV has also been demonstrated in a recent study, where several isolates from 2007 to 2013 were included (Cuevas-Romero et al., 2016b). Since residues important for neuraminidase activity in paramyxoviral HNs are conserved in the PorPV isolates, all these HN proteins may have similar activities. However, it is necessary to analyze each virus experimentally and consider characteristics such as HN glycosylation patterns and their tridimensional structures. Neuraminidase activity of PorPV was tested using four different potential substrates: fetuin, 3SL, 6SL and colominic acid. We tested all these molecules since they are all present in the CNS (Schnaar et al., 2014), which is a target site of PorPV infection. Our results show that the specificity of the PorPV neuraminidase is restricted to 3SL. Because fetuin contains N-linked sialic acid alpha (2,3) and alpha (2,6) (Baenziger and Fiete, 1979) it is used as a universal substrate for neuraminidases and it is susceptible to hydrolysis by enzymes with either 3SL or 6SL specificity. The typical viral neuraminidase possesses specificity towards particular sialylated molecules such as 3SL or 6SL but other types of sialylated molecules may be recognized, such as colominic acid [sialic acid (alpha2,8) linked] (Corfield et al., 1983; Corfield et al., 1982). Other viruses such as Newcastle disease virus and influenza viruses are able to cleave colominic acid (Corfield et al.,

Fig. 2. SDS-PAGE of purified PorPV HN protein. Lane 1, HN after the last stage of purification. Lane 2, Molecular weight marker.

catalytic sites exist, thus the active enzyme could be a dimer. 3.4. Inhibition of PorPV neuraminidase by zanamivir In order to identify possible inhibitors for the NA activity of PorPV HN, the usual influenza neuraminidase inhibitor zanamivir was incubated with purified HN. A dose-dependent inhibition with an IC50 of 0.24 μM was determined. The inhibition curve is shown in Fig. 4. 3.5. Activation of PorPV neuraminidase by Ca2 + and inhibition by NTPs To identify putative modulators of the HN protein, neuraminidase activity assays were performed in the presence of Ca2 + or ATP using 3SL as a substrate. These compounds drastically modified the saturation curves and the kinetic parameters. As appreciated in Fig. 5, Ca2 + enhanced enzyme activity and eliminated/decreased sigmoidicity resulting in hyperbolic kinetics. The Vmax increased from 154.3 to 368 nmol min− 1 mg− 1, whereas S50 presented a slight modification, 221

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Fig. 3. Kinetic analysis of neuraminidase activity of PorPV HN. Saturation curves using fetuin (A) or sialyl(α2,3)lactose (B) as substrates and the linear analysis for each curve by Lineweaver-Burk plot (C) or Hill coefficient (D).

Fig. 5. Effect of Ca2 + and ATP on PorPV neuraminidase activity using sialyl(α2,3)lactose as substrate. Saturation curves were carried out in the presence of increasing concentrations of sialyl(α2,3)lactose and constant ATP (0.1 mM) or CaCl2 (7.5 mM), which are compared with no ATP nor CaCl2 control.

Fig. 4. PorPV neuraminidase is inhibited by zanamivir. Fetuin was used as substrate and increasing concentrations of zanamivir was pre-incubated with purified HN.

1982). However, in our experiments, colominic acid was not hydrolyzed by PorPV HN. In previous reports, our group determined that the specificity of PorPV hemagglutinating activity (strain LPMV-1984) is restricted to 3SL residues (Reyes-Leyva et al., 1993) and that this molecule has a significant role during the infection in cell culture (Reyes-Leyva et al., 1997). These data agree with the present findings on the enzymatic activity of the PAC1 HN, which specifically recognizes 3SL. In other work, Vallejo et al. (2000) showed that a differential expression of sialic acid on porcine organs during the maturation process may be involved in the permissiveness to PorPV infection. Neu5Acα(2–3)Gal is present in the newborn's CNS, which may be exploited by the virus. Maturation of the CNS induces a decrease of NeuAcα(2–3)Gal residues (Vallejo et al., 2000) that may explain the absence of CNS infection in adult pigs. On the other hand, cooperative kinetics typical of allosteric enzymes were found for the PAC1 HN, Mahon et al., 1995 reported a paramyx-

Fig. 6. Competitive inhibition of ATP on PorPV neuraminidase activity. Ki was calculated using two different concentrations of ATP in addition to the control with no ATP added.

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bind divalent ions with relative high affinity (O'Sullivan and Perrin, 1964; Wilson and Chin, 1991), which could interfere with the interaction of Ca 2 + ions in the HN structure and result in decreased enzymatic activity. Nevertheless, these interactions should be tested and analyzed at a structural level, such as with crystallographic studies or by in silico structure prediction analyses. Conflict of interest statement The authors declare that they have no competing interests. Acknowledgements Fig. 7. Effect of triphosphated nucleotides on the neuraminidase activity of PorPV HN. Nucleotides were pre-incubated 30 min and fetuin was then added to initiate the determination of neuraminidase activity. Two different concentrations were used of each nucleotide and residual enzymatic activity was determined. Only the nucleotides of adenine were used in its di- and monophosphated versions.

This study was funded by Redes Temáticas-SEP 2012-2015 México (Benemérita Universidad Autónoma de Puebla) and Funds for Scientific Infrastructure from the Instituto Mexicano del Seguro Social (CTFIS/ 10RD/12/2011). SF and BP wish to acknowledge the support from the ICMG Chemistry Nanobio NMR and Mass spectrometry Platforms and Labex Arcane (ANR-11-LABX-0003-01). We thank Dr. Paulina CortésHernández for critical review and suggestions to this article.

oviral (Newcastle disease virus) HN, which possesses cooperative activity when inserted in the viral envelope (Mahon et al., 1995). In another study, the cooperativity of mumps virus neuraminidase with an oligomerization factor of 2, was reported, which suggests that the active enzyme is a dimer (Reyes-Leyva et al., 2007). HN proteins as other membrane viral proteins may be associated in membranes as oligomers, i.e. dimeric, trimeric or tetrameric complexes. Paramyxoviral HNs are well known to exist as tetramers composed by two dimers (Doms et al., 1993; Markwell and Fox, 1980; Waxham et al., 1986). The allosteric properties of neuraminidases can favor the successive activation of each monomer in an oligomer, with increasing affinity as more sialic acid residues are available on the tissues. This property of virions supports the idea that neuraminidase represents a spreading factor, which allows viruses to reach other sialic acids molecules as receptors at different sites for replication (Shaw and Palese, 2013). Influenza NA inhibitors, oseltamivir and zanamivir, are available and zanamivir was tested on PorPV NA activity. Under our assay conditions, PorPV NA activity was not fully inhibited by zanamivir, remaining around 25% even at the maximum zanamivir concentration tested. The IC50 was calculated at 240 nM, which is higher than for susceptible influenza A or B viruses (Nguyen et al., 2010; OkomoAdhiambo et al., 2010). There is no consensus on the cut-off (threshold) for influenza NA resistance to zanamivir and the discrimination of resistance/susceptibility is determined using reference strains. IC50 values above 2 nM are generally considered resistant, although some isolates may display values higher than 100 nM (Gubareva et al., 2001; Okomo-Adhiambo et al., 2010). Therefore, the estimation of an IC50 value of zanamivir for PorPV NA has limited relevance. PorPV neuraminidase inhibition should be tested in infection assays, as has been widely characterized and discussed for influenza viruses. As an approach to identify some enzyme modulators, we tested Ca2 + and ATP on PorPV neuraminidase activity. Ca2 + enhanced enzyme activity two-fold and converted the enzyme to MichaelisMenten behavior/favored hyperbolic behavior. Calcium ions are an integral part of the HN protein of paramyxoviruses, as demonstrated by crystallization studies for the Newcastle disease virus (Crennell et al., 2000), parainfluenza virus 5 (Yuan et al., 2005) and parainfluenza virus 3 (Lawrence et al., 2004). Surprisingly PorPV neuraminidase activity was competitively inhibited by NTPs, whereas NDP and MNP had only marginal inhibitory effects. There is no previous information on nucleotides as NA inhibitors and our data open the interesting possibility that this modulation may exist for other viral neuraminidases such as those of influenza viruses. NTPs possess a negative charge at physiological pH, similar to neuraminic acid, which is specifically recognized by neuraminidases. The overall negative charge of mono- and di-nucleotide phosphate is lower than that of NTPs which may be related to their reduced ability to inhibit neuraminidases. Moreover, NTPs are able to

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