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Journal of Virology, September 2008, p. 8400-8410, Vol. 82, No. 17
0022-538X/08/$08.00+0     doi:10.1128/JVI.00474-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Loss of the N-Linked Glycan at Residue 173 of Human Parainfluenza Virus Type 1 Hemagglutinin-Neuraminidase Exposes a Second Receptor-Binding Site{triangledown}

Irina V. Alymova,1 Garry Taylor,4 Vasiliy P. Mishin,1 Makiko Watanabe,1 K. Gopal Murti,2,{dagger} Kelli Boyd,3 Pooran Chand,5 Y. Sudhakara Babu,5 and Allen Portner1*

Departments of Infectious Diseases,1 Molecular Biotechnology,2 Animal Resources Center, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, Tennessee 38105-2794,3 Center for Biomolecular Science, University of St. Andrews, North Haugh, St. Andrews, Fife KY16 9ST, Scotland,4 BioCryst Pharmaceuticals, Inc., 2190 Parkway Lake Drive, Birmingham, Alabama 352445

Received 4 March 2008/ Accepted 15 June 2008


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ABSTRACT
 
BCX 2798 (4-azido-5-isobutyrylamino-2,3-didehydro-2,3,4,5-tetradeoxy-d-glycero-d-galacto-2-nonulopyranosic acid) effectively inhibited the activities of the hemagglutinin-neuraminidase (HN) of human parainfluenza viruses (hPIV) in vitro and protected mice from lethal infection with a recombinant Sendai virus whose HN was replaced with that of hPIV-1 (rSeV[hPIV-1HN]) (I. V. Alymova, G. Taylor, T. Takimoto, T. H. Lin., P. Chand, Y. S. Babu, C. Li, X. Xiong, and A. Portner, Antimicrob. Agents Chemother. 48:1495-1502, 2004). The ability of BCX 2798 to select drug-resistant variants in vivo was examined. A variant with an Asn-to-Ser mutation at residue 173 (N173S) in HN was recovered from mice after a second passage of rSeV(hPIV-1HN) in the presence of BCX 2798 (10 mg/kg of body weight daily). The N173S mutant remained sensitive to BCX 2798 in neuraminidase inhibition assays but was more than 10,000-fold less sensitive to the compound in hemagglutination inhibition tests than rSeV(hPIV-1HN). Its susceptibility to BCX 2798 in plaque reduction assays was reduced fivefold and did not differ from that of rSeV(hPIV-1HN) in mice. The N173S mutant failed to be efficiently eluted from erythrocytes and released from cells. It demonstrated reduced growth in cell culture and superior growth in mice. The results for gel electrophoresis analysis were consistent with the loss of the N-linked glycan at residue 173 in the mutant. Sequence and structural comparisons revealed that residue 173 on hPIV-1 HN is located close to the region of the second receptor-binding site identified in Newcastle disease virus HN. Our study suggests that the N-linked glycan at residue 173 masks a second receptor-binding site on hPIV-1 HN.


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INTRODUCTION
 
Human parainfluenza virus (hPIV) types 1 through 3 are a major cause of croup and upper and lower respiratory tract infections in young children, the elderly, and immunocompromised persons (17, 20, 39). These viruses cause annual epidemics, and since reinfection occurs throughout life, there is a need for the development of novel effective antiviral agents.

Previously, we reported the high potency of the novel selective hemagglutinin-neuraminidase (HN) inhibitor BCX 2798 (4-azido-5-isobutyrylamino-2,3-didehydro-2,3,4,5-tetradeoxy-d-glycero-d-galacto-2-nonulopyranosic acid) (Fig. 1B) against parainfluenza virus infections in vitro and in vivo (1) and against the lethal synergism between parainfluenza virus and Streptococcus pneumoniae in a mouse model (2). BCX 2798 is a synthetic drug whose structure was based on binding of the lead compound Neu5Ac2en (2-deoxy-2,3-dehydro-N-acetylneuraminic acid) (Fig. 1A) to the receptor-binding/ neuraminidase (NA) active site (catalytic site) of Newcastle disease virus (NDV) HN (an avian paramyxovirus) (6, 42). The analysis of the Neu5Ac2en-NDV HN complex revealed that amino acid residues forming the catalytic site are highly conserved among all paramyxoviruses. These findings allowed us to use NDV HN as a model in the structure-based design of hPIV HN inhibitors.


Figure 1
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  		FIG. 1. Structure of the lead compound and BCX 2798. (A) Neu5Ac2en (2-deoxy-2,3-dehydro-N-acetylneuraminic acid); (B) BCX 2798 (4-azido-5-isobutyrylamino-2,3-didehydro-2,3,4,5-tetradeoxy-d-glycero-d-galacto-2-nonulopyranosic acid).

The HN of the parainfluenza virus is a multifunctional protein that is involved in the attachment of the virus to sialic acid-containing cell receptors, leading to the promotion of membrane fusion. HN also cleaves sialic acid residues from the cell membrane and new virions via its NA activity, preventing viral self-aggregation and allowing efficient virus spread. BCX 2798 successfully inhibited the HN activities and growth of all hPIVs in cell culture (1), consistent with the conservation of amino acid residues forming the NDV HN catalytic site among all parainfluenza viruses.

While hPIVs infect small animals poorly (5, 21, 28, 43), rSeV(hPIV-1HN), a virus that we developed in which the HN gene of the Sendai virus (SeV) was replaced with that of hPIV-1, causes severe illness and robustly replicates in the lungs of mice. We determined that BCX 2798 was highly effective in prophylaxis against lethal infection with rSeV(hPIV-1HN) in mice (1). This model virus was also successfully used to develop and characterize the lethal synergism between rSeV(hPIV-1HN) and S. pneumoniae in mice and to determine that prophylactic treatment with BCX 2798 prevented this lethal synergism in 80% of dually infected animals (2). Because the novel HN inhibitor demonstrated high efficacy in the animal model (indicating potential for clinical use) and because rSeV(hPIV-1HN) has proved its usefulness for evaluation of test compounds in vivo, we determined the possibility of, and molecular basis for, the emergence of resistant viruses whose HNs carry mutations during BCX 2798 treatment of mice infected with rSeV(hPIV-1HN).

The clinical importance of any antiviral drug can be compromised if readily transmitted drug-resistant variants emerge. Zanamivir and oseltamivir, which were also developed from the NA lead inhibitor Neu5Ac2en (19, 46), are selective high-affinity inhibitors of influenza A and B virus NAs (47), preventing efficient virus release from the cell surface (10). An extensive study indicated that influenza virus resistance to NA inhibitors does not develop easily in tissue culture, animal models, or humans (before recent reports). When resistance does appear, the mechanism depends on the model used for such studies (11, 14, 22, 23, 33). The resistance in tissue culture and mice resulted from mutations in the active site of NA and in the receptor-binding site of the influenza virus hemagglutinin (HA) (a protein responsible for the attachment of the influenza virus to the cell surface), while only substitutions in the active site of NA have been reported (so far) to occur in resistant viruses isolated from drug-treated humans.

One of the influenza virus NA inhibitors, zanamivir, was used as a tool to study the mechanism of parainfluenza virus resistance to sialic acid-based inhibitors in vitro. Zanamivir (at millimolar concentrations) inhibited receptor binding, cell fusion, and NA activities of hPIV-3 (9), which was consistent with the presence of a single (catalytic) site carrying out both binding and NA activities on the HN molecule of hPIV-3 (18). Serial passages of virus in tissue culture cells in the presence of zanamivir resulted in the selection of a virus variant, T193I, with reduced sensitivity to the drug in binding and NA assays (30). The data from that report indicated that changes in the catalytic site of hPIV-3 HN contributed to drug resistance. Increased avidity for the receptors (29) and reduced affinity of the HN molecule catalytic site for zanamivir (35) were proposed as mechanisms for resistance to sialic acid-based inhibitors in vitro.

Although selective parainfluenza virus HN inhibitors were developed based on the three-dimensional structure of the NDV HN catalytic site, BCX 2798 does not inhibit NDV binding, even though it is highly effective in inhibiting virus NA activity (3). These data are consistent with the presence in NDV HN of a second site, which possesses only receptor-binding activity (48) and has no affinity for BCX 2798. The hPIVs are not thought to have a second receptor-binding site on their HNs. However, the possibility that mutant viruses possessing a second receptor-binding site (and being resistant to HN inhibitors) may exist within a natural hPIV population was never excluded (4, 37).

Our report is the first describing the isolation of an rSeV (hPIV-1HN) drug-resistant variant, N173S, in mice after treatment with BCX 2798. We propose that resistance of the N173S mutant virus to the novel inhibitor in binding assays was caused by the appearance of a second receptor-binding site on its HN due to the loss of the N-linked glycan at residue 173. We also hypothesize that N-linked glycans shield preexisting second receptor-binding sites in HNs of all hPIVs. In addition, we characterize the growth and sensitivity of the isolated N173S mutant virus to BCX 2798 in both cell culture and mice and discuss the potential clinical importance of hPIVs possessing a second receptor-binding site.


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MATERIALS AND METHODS
 
HN inhibitor. BCX 2798 (BioCryst Pharmaceuticals, Inc., Birmingham, AL) is the derivative of Neu5Ac2en in which the O-4 hydroxyl group was replaced by an azido group and the methyl group of the acetamino moiety at C-5 was replaced by an isopropyl group. BCX 2798, provided as a lyophilized powder, was solubilized in phosphate-buffered saline (PBS) before use in in vitro and in vivo experiments.

Cell cultures and viruses. LLC-MK2 (monkey kidney epithelium) and 293T (human kidney epithelium) [used for the rescue of rSeV(hPIV-1HN)] cells were grown in Dulbecco's modified Eagle's medium that contained 10% fetal bovine serum.

rSeV(hPIV-1HN), which contains the hPIV-1 HN gene instead of the HN gene of SeV, was rescued from the full-length SeV cDNA genome pSeV(+) (16) by using reverse-genetic methods, as described previously (1). The rescued virus was plaque purified on LLC-MK2 cells and amplified in embryonated chicken eggs. Sequence analysis of the HN gene of rSeV(hPIV-1HN) revealed no mutations.

To rescue the rSeV(hPIV-1HN) containing the N173S mutation, the NotI-AscI fragment in the pSeV(hPIV-1HN) plasmid was replaced with the fragment obtained in reverse transcriptase PCR (RT-PCR), where RNA of the N173S mutant virus was used as a template. The final plasmid, pSeV(hPIV-1HN)/N173S, was used to rescue the virus (1).

The infectivity of the rescued viruses was determined using plaque assays. Briefly, LLC-MK2 cells in six-well plates were inoculated with serial 10-fold virus dilutions. After incubation for 1 h at room temperature (RT), the inoculum was removed, and 1x minimum essential medium containing 2% bovine serum albumin and acetylated trypsin (5 µg/ml) mixed with 1% agarose was added to the plates. After 5 days of incubation at 33°C, the second overlay, which consisted of minimal essential medium with 5% fetal bovine serum mixed with 1% neutral red and agarose, was added to the plates to help visualize plaques.

The rSeV(hPIV-1HN) mutant viruses recovered from the lungs of mice were plaque purified twice in LLC-MK2 cells in the absence of an inhibitor and amplified in embryonated chicken eggs. The titers of virus stocks were determined using plaque assays, and mutations in the HN and fusion (F) genes were verified by sequencing the RT-PCR product.

Animal studies. Experiments using 8-week-old female 129x1/SvJ mice (Jackson Laboratories, Bar Harbor, Maine) were performed in a Biosafety Level 2+ facility in the Animal Resources Center at St. Jude Children's Research Hospital (hereafter referred to as St. Jude). Animals were given general anesthesia that consisted of inhaled isoflurane at 2.5% (Baxter Healthcare Corporation, Deerfield, IL), and all studies were approved by the Animal Care and Use Committee at St. Jude.

For selection of resistant rSeV(hPIV-1HN) variants, mice (15 per group) were inoculated intranasally with one 90% mouse lethal dose (MLD90) (4.2 x 107 PFU per mouse) of virus in a volume of 50 µl in experiment 1. Intranasal treatment with two equally divided doses (dose volume, 50 µl) of BCX 2798 (total of 10 mg/kg of body weight per day) was started 4 h before lethal infection and continued for 5 days. Control animals were infected but were treated only with PBS. Mice were observed daily for 21 days to evaluate for changes in weight and for survival. At day 6 after the start of infection, five mice each from treated and untreated groups were euthanized, and their lungs were removed under sterile conditions for subsequent experiments. Viruses recovered from each lung were assayed for in vitro sensitivity to BCX 2798 and analyzed for the presence of amino acid substitutions in the HN protein.

In experiment 2, pooled virus from the lungs of five treated mice was passaged through mice, which were again treated with 10 mg/kg per day of BCX 2798 as described in the preceding text. Pooled virus from untreated mice was passaged through mice inoculated with PBS. The mean titers of the pooled viruses were 7.6 x 105 PFU/ml and 6.8 x 106 PFU/ml for treated and untreated mice, respectively. In experiment 2, the animals in the treated and untreated groups were infected with virus doses of 7.6 x 104 PFU per mouse. At day 6 after the start of infection, lungs from treated and untreated mice were harvested, and recovered viruses were analyzed as described in preceding text. In each experiment (experiments 1 and 2), 50 virus clones from treated mice were sequenced.

To compare the growths of rSeV(hPIV-1HN) and the N173S mutant virus, mice were infected intranasally with doses of 103 PFU per mouse. At 1, 3, 5, or 7 days after infection, lungs from three mice from each group were harvested to analyze virus titers. The lungs were washed three times with PBS, homogenized, and suspended in PBS (total volume, 1 ml). The suspensions were centrifuged at 2,000 x g for 10 min to clear cellular debris. Virus titers were determined by plaque assays using LLC-MK2 cells as described in the preceding text.

To determine the sensitivity of the N173S mutant virus to BCX 2798 in vivo, mice were infected intranasally with one MLD90 (1.0 x 107 PFU per mouse) and treated with 10 mg/kg per day of the compound as described in the preceding text. Mice were observed daily for 21 days for weight loss, length of survival, and death.

Cell culture assays. Plaque reduction assays for determining the susceptibilities of rSeV(hPIV-1HN) and isolated mutant viruses to BCX 2798 were performed by using six-well plates in which LLC-MK2 cells were inoculated with 100 to 200 PFU of test viruses in the presence of various concentrations of BCX 2798 (range of final concentrations, 0.1 to 100 µM). The concentrations required to inhibit virus replication to 50% of the level of the control (without the compound) (EC50s) were determined.

To evaluate the stability of the N173S substitution, mutant virus was passaged five times without BCX 2798 in LLC-MK2 cells at a multiplicity of infection (MOI) of 0.1. Virus-containing supernatant fluid was collected 48 h after infection and used to inoculate new LLC-MK2 cells. After the fifth passage, PCR products from 10 plaques were sequenced to determine whether mutations in the HN genes were present.

To compare the growths of rSeV(hPIV-1HN) and the N173S mutant virus in vitro, LLC-MK2 cells in 24-well plates were infected with viruses at a low MOI of 0.001. Viruses were adsorbed for 1 h at RT and then removed, and Dulbecco's modified Eagle's medium that contained 0.1% bovine serum albumin and acetylated trypsin (1 µg/ml) was added. At 24, 48, 72, and 96 h after infection, samples were harvested, and virus titers were determined using plaque assays as described in the preceding text.

To compare the releases of rSeV(hPIV-1HN) and the N173S mutant virus, LLC-MK2 cells in 80-mm-diameter dishes were infected with viruses at a high MOI of 50. After 1 h of adsorption at RT, the cells were washed and infection medium was added. After 18 h of infection, cells were pelleted and fixed with 2.5% glutaraldehyde in phosphate buffer for subsequent transmission electron microscopy. The pellets were rinsed in the buffer, postfixed with 1% osmium tetroxide, dehydrated through a graded series of ethanol, and embedded in Spurr low-viscosity medium. Ultrathin sections of cells were cut using a Sorvall MT 6000 ultramicrotome, then stained with lead citrate, and viewed using a JEOL 1200EX II electron microscope as described elsewhere (26).

NA and NI assays. Parainfluenza viruses used in NA and NA inhibition (NI) assays were concentrated and purified through a gradient of 30% to 50% sucrose in PBS, as described previously (45). The activity of each viral NA was measured with a standard fluorometric assay, using 2'-(4-methylumbelliferyl)-{alpha}-d-N-acetylneuraminic acid (MUNANA) (Sigma-Aldrich, Inc., St. Louis, MO) as the substrate (38), as described previously (1).

To compare the NA activities of rSeV(hPIV-1HN) and the N173S mutant virus, we ascertained the pH optimum for development of these activities, the Michaelis constant (Km; provides an approximation of the substrate concentration required for significant catalysis to occur), and the maximum velocity (Vmax; provides an approximation for the NA activity in the relation to the amount of HN protein). The pH optimum was determined for 0.1 M citrate-phosphate buffer, 4 mM CaCl2, and 100 µM of MUNANA. The Km and Vmax values were measured at pH 5.4 for 0.1 M citrate-phosphate buffer, 4 mM CaCl2, and MUNANA, with a final substrate concentration of 0.078 to 10 mM. The reaction was conducted at 37°C with a total volume of 50 µl, and the fluorescence of the released 4-methylumbelliferone was measured every 71 s, 25 times, with a Fluoroskan II instrument (Labsystems, Helsinki, Finland), using excitation and emission wavelengths of 355 and 460 nm, respectively.

The enzyme kinetic data were fit to the Michaelis-Menten equation by using nonlinear regression (GraphPad Prism 4; GraphPad, San Diego, CA) to establish the Km and Vmax of substrate conversion. The NA activities of both viruses were then calculated per 1 µg of the virion HN. To determine the amount of HN in the sample, viral proteins were separated on a 10% sodium dodecyl sulfate (SDS)-polyacrylamide bis-Tris gel (Invitrogen, Carlsbad, CA) and stained with Gel Code blue stain (Pierce Biotechnology, Rockford, IL). After extensive gel washing, imaging was accomplished using the Bio-Rad Gel Doc 2000 system, and the density of the HN band was analyzed with Quantity One software (version 4.2.1; Bio-Rad, Hercules, CA).

In NI assays, BCX 2798 was diluted (ratio, 1:4), and 25 µl of each dilution was incubated for 30 min at RT with 25 µl of diluted virus, whose NA activity was equal to 200 relative fluorescence units. The reaction was started by the addition of substrate and stopped after 1 h of incubation at 37°C. The extent of NI was defined as the concentration of the compound required to reduce the NA activity of the treated virus to 50% of that of the control virus. The 50% inhibitory concentrations (IC50s) were calculated by plotting the percentage of fluorescence inhibition (relative to the control level) versus the log concentrations of the compounds.

Hemagglutination and HI assays. Hemagglutination assays were performed by using 0.5% chicken and turkey red blood cells (RBCs) as described previously (1). For hemagglutination inhibition (HI) assays, BCX 2798 was serially diluted in PBS (ratio, 1:4), and the dilutions were preincubated with four HA units (HAU) of virus for 1 h at RT. Then, the HA tests were performed, and the concentration of the compound that was associated with 50% agglutination was established and considered to be the IC50. In the elution test, each virus was diluted to provide eight HAU after 1 h at 4°C, then the plate was shifted to 37°C, and elution of viruses from RBCs was recorded after 0.5, 3, and 18 h of incubation.

Analysis of viral proteins. LLC-MK2 cell monolayers were infected with rSeV(hPIV-1HN) or the N173S mutant virus at an MOI of 0.1. At 20 h after infection, the cells were metabolically radiolabeled with [35S]methionine-cysteine (500 µCi per 107 cells) (Perkin-Elmer, Boston, MA). At 48 h after infection, the cell culture supernatants were harvested, and the cellular debris was removed with centrifugation at 2,000 x g for 30 min. Then, viruses were subjected to ultracentrifugation through a 35% sucrose cushion at 100,000 x g (SW 28 rotor; Beckman, Fullerton, CA) for 90 min. The pellet containing virus particles was resuspended in 200 µl PBS.

Separation of viral HN and F glycoproteins from the RNP/M complex was carried out as described previously (31). Briefly, 100 µg of concentrated, purified virus was incubated in 200 µl of a solubilization buffer (40 mM Tris-HCl [pH 7.9 at 20°C], 6 mM MgCl2, 0.1% NP-40, and 30 mM NaCl) for 30 min at RT. The reaction mixture was layered on a 35% sucrose cushion and centrifuged in a Beckman SW28 rotor at 100,000 x g for 2 h at 4°C. The solution above the sucrose cushion containing glycoproteins was collected and concentrated using a YM-100 Centricon ultracel unit (Millipore, Bedford, MA) in accordance with the manufacturer's protocol. To remove N-glycosylated moieties, virus glycoproteins were pretreated with 500 units of peptide N-glycosidase F (PNGase F; New England Biolabs, Beverly, MA) for 2 h at 37°C. Whole radiolabeled virus, its glycoproteins, and glycosidase-treated glycoproteins were examined using 10% SDS-polyacrylamide gel electrophoresis (PAGE) under reducing conditions, followed by fluorography.

Sequence analysis and molecular graphics of the viral HN gene. For sequencing of HN genes of viruses isolated from mouse lungs, LLC-MK2 cells in six-well plates were infected with virus from plaques suspended in PBS. Total RNA was isolated from infected cell monolayers or supernatants; cDNA synthesis and PCR amplification were done using the one-step RT-PCR system (Qiagen, Valencia, CA). Sequencing was performed by the Hartwell Center for Bioinformatics and Biotechnology at St. Jude. Information about the primers is available upon request. In addition to the HN gene, the F gene in the N173S mutant virus was also sequenced. The sequence revealed no amino acid changes in the F protein.

Superposition of the HN dimer structures of NDV and hPIV-3 and image creation were done using PyMOL (7); in silico mutations were created using the program Coot (8).

Statistical analysis. Comparison of survival between groups of mice was done by using the Mantel-Cox chi-square test to analyze the Kaplan-Meier survival data. Viral titers in the mouse lungs and tissue culture were compared using repeated-measure analysis of variance. A comparison of weight loss and mean number of days to death between groups was determined by using the unpaired Student t test. The mean number of days to death was estimated as the number of days that the mice survived after viral infection. A P value of <0.05 was considered significant.


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RESULTS
 
Isolation of the rSeV(hPIV-1HN) variants. In an attempt to isolate BCX 2798-resistant variants, mice were infected with one MLD90 (4.2 x 107 PFU per mouse) of rSeV(hPIV-1HN) intranasally and treated intranasally with BCX 2798 (10 mg/kg per day) for 5 days starting 4 h before infection. Control animals were infected but were treated only with PBS. In our previous experiments, this treatment scheme protected 100% of mice from death after infection with rSeV(hPIV-1HN) (1).

Similar to our previously published results, 10 mg/kg per day of BCX 2798 protected 8 of 10 (80%) infected animals from death, whereas 100% of the mice in the control group died. The protection of mice from lethal challenge with rSeV(hPIV-1HN) corresponded to the reduction (about 10-fold) of virus titers in the lungs (data not shown).

To further characterize rSeV(hPIV-1HN) passaged through mice, viruses recovered from the lungs of treated animals (taken at day 6 after infection) were analyzed for their sensitivity to BCX 2798 in plaque reduction assays. Concurrently, the HN genes were examined to determine whether amino acid substitutions had occurred. The sensitivities of viruses recovered from each lung of treated mice did not differ from those of viruses recovered from the lungs of control animals, and the mean EC50s for both groups ranged from 1.3 to 2.0 µM (data not shown). The sequences of HN genes isolated from 50 virus clones did not reveal any amino acid changes in the HN gene of rSeV(hPIV-1HN) passaged through treated animals.

In the next animal experiment, we pooled viruses from the lungs of treated mice and passaged the viruses again through mice treated with 10 mg/kg per day of BCX 2798. Similarly, pooled viruses from untreated mice were passaged again through mice treated with PBS only. Because the mean titers of the pooled viruses in treated and untreated groups (7.6 x 105 PFU/ml and 6.8 x 106 PFU/ml, respectively) were not sufficient to induce lethal infection in mice in a second-passage infection experiment, the animals were infected with the highest achievable challenge virus doses, 7.6 x 104 PFU per mouse. Infection of mice in the second round resulted in minimal weight loss and moderate replication of viruses in the mouse lungs (Table 1). The mean lung virus titers were 5.4 x 104 PFU/ml and 7.1 x 105 PFU/ml for treated and control mice, respectively. The susceptibilities of viruses recovered from the lungs of treated mice to BCX 2798 did not differ from those of viruses recovered from the lungs of control animals.


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  		TABLE 1. Effect of prophylactic treatmenta with BCX 2798 on rSeV(hPIV-1HN) second-passageb infection in mice

The sequences of the HN genes from 50 virus clones of rSeV(hPIV-1HN) from treated mice revealed single amino acid changes—Thr553 to Ala (T553A), Pro29 to Gln (P29Q), Asn173 to Ser (N173S), and Asn23 to Ser (N23S)—in four virus clones that were not seen in the HN genes of viruses recovered from the lungs of untreated control mice.

Sensitivities of cloned rSeV(hPIV-1HN) mutant viruses to BCX 2798 in in vitro assays. We characterized selected mutant viruses in HI, NI, and plaque reduction assays with BCX 2798. BCX 2798 inhibited binding, NA activity, and growth in LLC-MK2 cells of the N23S, P29Q, and T553A mutant viruses at doses similar to those used in experiments with rSeV(hPIV-1HN) (hereafter referred to as the parent virus) (Table 2). These results suggested that the amino acid substitutions N23S, P29Q, and T553A did not result in a drug-resistant virus phenotype, and the reason for selection of these virus variants is not clear. The data from NI assays with the N173S mutant virus showed that the concentration of BCX 2798 inhibiting mutant virus NA activity by 50% did not differ from that of the parent virus. However, the binding of the N173S mutant virus to chicken (abundant in NeuAc{alpha}2,3Gal sialic acid-containing receptors) or turkey (abundant in NeuAc{alpha}2,6Gal sialic acid-containing receptors) (15, 44) RBCs was not inhibited by BCX 2798, even at a dose of 1,000 µM (as determined by HI tests). Two types of RBCs were used in these HI assays with the N173S mutant virus to exclude the contribution of hPIV-1 receptor specificity (41) to these results. The growth of the N173S mutant virus in cells was inhibited by BCX 2798 at a dose approximately fivefold higher (EC50 of 6.5 µM) than that of the parent virus (as determined by plaque reduction assays). Thus, the results from HI and plaque reduction assays indicated that the N173S mutant virus had a drug-resistant phenotype.


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  		TABLE 2. Sensitivities of parent and mutant viruses to BCX 2798 in in vitro assays

Our data from HI assays (Table 2) indicated that the N173S mutant virus has increased binding ability. The influence of the N173S substitution on mutant virus release was evaluated in elution assays at an elevated temperature with different RBCs and by electron microscopic analysis of infected cells. The parent virus was completely eluted from chicken RBCs after 0.5 h and from turkey RBCs after 3 h of incubation at 37°C (Fig. 2). In contrast, the N173S mutant virus remained attached to both types of RBCs (at the lowest dilutions), even 18 h after incubation. Similarly, the N173S mutant virus appeared to remain at the surfaces of infected LLC-MK2 cells (as shown by electron micrographs) (Fig. 3B), while the parent virus showed normal budding and release (Fig. 3A). The data from these experiments indicated that the N173S substitution in the HN of rSeV(hPIV-1HN) uncoupled the binding and NA activities of the HN molecule, thus resulting in a decreased ability for the mutant virus to release.


Figure 2
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  		FIG. 2. Elution of the parent and the N173S mutant virus from RBCs. In HA assays, both viruses were diluted to provide eight HAU with 0.5% chicken (upper rows) and turkey (lower rows) RBCs after 1 h at 4°C. Then, the plate was shifted to 37°C, and elution of viruses from RBCs was recorded after 0.5, 3, and 18 h of incubation.


Figure 3
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  		FIG. 3. Electron micrographs of LLC-MK2 cells infected with the parent (A) and the N173S mutant virus (B). Cells in 80-mm-diameter dishes were infected with viruses at a high MOI of 50 and processed for subsequent transmission electron microscopy 18 h after infection. Note the few virions (arrows) associated with the cell membrane, which occurs during normal release (A), and a large number of membrane-associated virions in mutant-infected cells (B). Bar = 1 µm.

Our data from NI assays (Table 2) pointed out that the N173S mutation did not affect the NA function of the HN molecule. We confirmed this conclusion by comparing the Km, Vmax, and pH optimum levels for NA activities of the parent and the N173S mutant virus by using a standard fluorometric assay with MUNANA as the substrate. We found that the NA activity of the N173S mutant virus did not differ from that of the parent virus (P < 0.05) (Table 3).


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  		TABLE 3. NA activities of parent and N173S mutant viruses

The results from the NA and NI assays suggested that loss of susceptibility to BCX 2798 in HI tests and delayed elution from RBCs or infected LLC-MK2 cells by the N173S mutant virus were due not to the changes in the catalytic site but to a different mechanism.

We previously demonstrated that NDV has an additional receptor-binding site on its HN (48) that renders the virus resistant to the HN inhibitor BCX 2798 in HI tests (3). The crystal structure of the hPIV-1 HN has not been determined. To evaluate the location of Asn173 on hPIV-1 HN, we superposed the HN dimer structures of NDV (48) and hPIV-3 (18) (Fig. 4A). The structure of hPIV-3 HN is expected to be a better model for hPIV-1 HN than for NDV HN around the location of Asn173, as there are no gaps or insertions between the two sequences around this region. Superposition of the crystal structures of the NDV HN (Protein Data Bank [PDB] code 1usr) (48) and hPIV-3 HN (PDB code 1v2i) (18) shows that within the second receptor-binding site, a Leu174 in hPIV-3 HN superimposes with Phe156 in NDV HN, a residue that forms part of the hydrophobic binding pocket for the acetamido group of the receptor sialic acid (48). This leucine is conserved in all hPIV HNs and is Leu176 in hPIV-1 HN.


Figure 4
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  		FIG. 4. The relative locations of the catalytic and second receptor-binding sites (A) and possible conservation of a second receptor-binding site in hPIV-1 HN (B). (A) Shown are the superimposed dimers of NDV HN (green; PDB code 1usr) and hPIV-3 HN (cyan; PDB code 1v2i) with the thiosialoside (yellow bonds) that identified the second receptor-binding site in the NDV HN structure and the relative locations of the catalytic and second receptor-binding sites. (B) Focus on the second receptor-binding site at the dimer interface. The residue numbering is for hPIV-1/NDV HN. Where hPIV-1 HN has a different amino acid relative to hPIV-3 HN, the structure of hPIV-3 HN has been mutated to show the possible location of the hPIV-1 HN residue. This was the case for Asn173 (Lys in hPIV-3), Arg520 (Ala in hPIV-3), and Leu521 (Ile in hPIV-3).

Indeed, as shown in Fig. 4B, there appears to be a conserved hydrophobic pocket in hPIV-1 HN formed from Leu176, Leu555, Phe563, and Leu521 (which would correspond to Phe156, Phe553, Leu561, and Val517 of NDV HN). The key residue Arg516 in NDV HN, which interacts with the sialic acid carboxylate group, is conserved in hPIV-1 HN (Arg520). Asn173 in hPIV-1 HN would therefore probably occupy the same position as Lys171 in hPIV-3 HN, which is only ~8 Å from the second receptor-binding site in the superimposed NDV HN structure. Thus, a structural analysis suggests the presence of a putative second receptor-binding site very close to Asn173 of hPIV-1 HN. Computer modeling data also show that Asn173 is remote from the proposed location of the catalytic site on hPIV-1 HN (Fig. 4A).

The hPIV-1 HN has nine potential N-linked glycosylation sites, and one of them, Asn-Ile-Ser, begins at amino acid residue 173 (12, 13). The Asn-to-Ser substitution at residue 173 in the mutant virus could result in the loss of this glycosylation site. To evaluate the loss of the N-linked glycosylation site in the N173S mutant virus, we metabolically labeled the parent and the N173S mutant virus with [35S]methionine-cysteine and analyzed concentrated purified viruses, their glycoproteins, and PNGase F-treated glycoproteins under reducing conditions, using 10% SDS-PAGE (Fig. 5). The HN and P proteins of the parent virus migrated as two distinct bands (lane 1), while the HN and P proteins of the N173S mutant virus migrated as a single band (lane 2). This result indicated an increased electrophoretic mobility for the HN protein of the mutant virus in comparison with that of the HN protein of the parent virus. The migration patterns of separated glycoproteins of the parent virus (lane 3) and the N173S mutant virus (lane 4) also demonstrated an increased electrophoretic mobility for the HN of the N173S mutant virus, corresponding to a molecular mass loss of approximately 3,000 Da (as determined by ImageQuant TL, version 2003.02; Amersham Bioscience), compared to the level for the HN of the parent virus. After PNGase F treatment, HNs of the parent virus (lane 5) and the N173S mutant virus (lane 6) migrated to identical positions, thus confirming that the altered mobility of the HN of the mutant virus described above was due to the absence of the N-linked glycan at residue 173.


Figure 5
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  		FIG. 5. Electrophoretic mobility of HN for the parent and the N173S mutant virus. Concentrated purified [35S]methionine-cysteine-labeled viruses (lanes: 1, parent; 2, N173S), glycoproteins (lanes: 3, parent; 4, N173S), and PNGase F-treated glycoproteins (lanes: 5, parent; 6, N173S) were analyzed under reducing conditions with 10% SDS-PAGE. Separation of viral glycoproteins is described in Materials and Methods. PNGase F-treated HN and F are indicated by asterisks. Molecular mass standards in kilodaltons are indicated at right. Below is shown an enlargement of lanes 3 to 6 to show more clearly the mobilities of HN for the parent and the N173S mutant viruses.

Here, we propose that the loss of the glycan shield in the N173S mutant virus has exposed a preexisting second receptor-binding site on rSeV(hPIV-1HN) HN.

We recognize that unequivocal evidence for the second receptor-binding site on N173S mutant virus HN can be provided only by a crystallographic study. However, the results strongly support our hypothesis: BCX 2798, a selective inhibitor of parainfluenza virus HN, effectively inhibited the HA and NA activities of the parent virus and the NA activity of the N173S mutant virus (i.e., the mutation did not influence the catalytic site) but was unable to block the HA function of the N173S mutant virus (Table 2); while the NA activity of the N173S mutant virus was not changed (Table 3), this virus was not efficiently eluted from RBCs at an elevated temperature (Fig. 2) and released from the surfaces of infected cells (Fig. 3B) (most probably because it binds to cells through two sites); a structural analysis suggests the presence of a putative second receptor-binding site very close to Asn173 and its remoteness of location from the proposed catalytic site on hPIV-1 HN (Fig. 4); gel electrophoresis analysis was consistent with the loss of a glycosylation site at residue 173 in the mutant virus (Fig. 5), proposing the hypothesis that the N-linked glycan shields a second receptor-binding site on hPIV-1 HN.

Growth kinetics of the N173S mutant virus. To determine how the presence of the second receptor-binding site on the HN of the N173S mutant virus influences virus growth, we performed a series of in vitro and in vivo experiments.

First, we evaluated the stability of the N173S substitution. The N173S mutant virus was passaged five times in LLC-MK2 cells at an MOI of 0.1 in the absence of BCX 2798. The sequences of the HN genes from 10 virus clones confirmed the presence of the N173S mutation in the HN molecule. This finding indicated that this amino acid substitution is stable.

To compare the growths of the parent and the N173S mutant virus in tissue culture cells, we infected LLC-MK2 cells with both viruses at an MOI of 0.001. Virus titers were evaluated using plaque assays at 24, 48, 72, and 96 h after infection. The titers of the N173S mutant virus were significantly lower (from seven- to ninefold) than those of the parent virus at 24 and 48 h after infection but did not differ from those of the parent virus at later time points (P < 0.05) (Fig. 6A).


Figure 6
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  		FIG. 6. Growth kinetics of the parent and the N173S mutant virus. (A) LLC-MK2 cells were infected with the parent (•) and the N173S mutant virus ({blacktriangleup}) at an MOI of 0.001. Virus titers were determined for culture supernatant fluids by plaque assays at the times indicated on the x axis. Values presented are the means of results from three independent experiments, plotted with error bars indicating the standard errors of the means (SEM). (B) The 129x1/SvJ mice were infected with the parent (•) and the N173S mutant virus ({blacktriangleup}) at a dose of 103 PFU per mouse. Lungs were collected at 1, 3, 5, and 7 days after infection, and the lung virus titers were determined by plaque assays. Values are the mean titers of virus from three animals, plotted with error bars indicating the SEM. An asterisk indicates a significant difference in titers for the N173S mutant virus at that time point compared to the parent virus (P < 0.05).

In the N173S mutant virus isolated in mice, we sequenced only the HN and F genes (with no mutations found in the F molecule). To exclude the contribution of amino acid changes (other than N173S) in the entire virus genome toward the compromised growth of the N173S mutant virus in LLC-MK2 cells, we rescued the rSeV(hPIV-1HN) possessing the N173S mutation by using reverse-genetic methods. The growth curves of the parent and the rescued N173S mutant virus were identical (data not shown) to that described in the preceding paragraph. These data indicated that the presence of the second receptor-binding site at residue 173 does not enhance the growth of recombinant SeV carrying hPIV-1 HN in LLC-MK2 cells.

To examine the growth of the N173S mutant virus in mice, we infected 129x1/SvJ mice with a low dose (103 PFU per mouse) of the parent and the N173S mutant virus and determined (using plaque assays) the mean virus titers in lung homogenates at days 1, 3, 5, and 7 after infection. This dose of viruses did not lead to weight loss or death in infected animals. Our data indicated that the titers of virus from the lungs of mice infected with the N173S mutant virus were significantly higher (from 6- to 12-fold) than those determined for the parent virus at days 3, 5, and 7 after infection (P < 0.05) (Fig. 6B). Similar mouse data (not shown) were obtained with the rescued N173S mutant virus. This result indicated an increased ability for the N173S mutant virus to grow in 129x1/SvJ mice.

Sensitivity of the N173S mutant virus to BCX 2798 in mice. Our in vitro data described in the preceding text indicated that despite the high level (more than 10,000-fold) of resistance of the N173S mutant virus to BCX 2798 in HI tests, the general susceptibility of the mutant virus to the compound (determined in plaque reduction assays) was not dramatically decreased. The EC50 for the N173S mutant virus was only fivefold greater than that of the parent virus (Table 2). Therefore, it was of interest to determine the susceptibility of the mutant virus to BCX 2798 in the mouse model.

Previously, we determined the efficacy of BCX 2798 against a 90% lethal infection of 129x1/SvJ mice with the parent virus (1). It was shown that 10 mg/kg per day of the compound administered to mice starting 4 h before infection for five consecutive days resulted in a significant reduction of weight loss and complete protection from death. This is the only scheme of drug administration currently available for evaluation of BCX 2798 against parainfluenza virus infections in mice. Similarly to experiments determining the sensitivity of the parent virus to BCX 2798, we inoculated 129x1/SvJ mice with one MLD90 (1.0 x 107 PFU per mouse) of the N173S mutant virus and treated them intranasally with 10 mg/kg per day of the compound or with PBS, as described in the preceding text.

Comparison of the survival curves of the N173S mutant virus-infected mice showed that while 100% of PBS-treated infected mice died, treatment with BCX 2798 (10 mg/kg per day) protected 100% of infected mice from death (P < 0.05) (Fig. 7A). The mean number of days to death for mice in the control group was 11.2 days. Unlike mice in the PBS-treated group, who lost a maximum of 35.1% of their initial weight by day 11, mice treated with BCX 2798 lost a maximum of 18.2% of their initial weight by day 7 and began to regain weight by day 9 (Fig. 7B). This result indicated that the N173S mutant virus retained its susceptibility to BCX 2798 in the mouse model. The susceptibility of the N173S mutant virus to BCX 2798 in mice did not differ from that previously established for the parent virus (1).


Figure 7
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  		FIG. 7. Effect of pretreatment with BCX 2798 on survival (A) and weight loss (B) of mice infected with a lethal dose of the N173S mutant virus. BCX 2798 (10 mg/kg per day; {blacktriangleup}) was intranasally administered to mice (10 per group) for 5 days; administration began 4 h before infection with one MLD90 of the N173S mutant virus. Infected control mice were treated only with PBS (•). Mean values for weight loss are plotted with error bars indicating the standard errors of the means (SEM). An asterisk indicates a significant difference in survival or weight change for the drug-treated group compared to the untreated control group (P < 0.05).


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DISCUSSION
 
BCX 2798 is a selective inhibitor of the parainfluenza virus HN and represents a new class of promising antiviral compounds for the management of hPIV infections. As with any antiviral agent, the emergence of drug-resistant variants is possible. To assess the potential for selection of hPIVs which are resistant to BCX 2798 due to mutations in HN, we passaged rSeV(hPIV-1HN)—a virus in which the HN gene of SeV was substituted with that of hPIV-1—in mice in the presence of the compound.

In a second-passage experiment, we isolated from treated mice one rSeV(hPIV-1HN) variant with an Asn-to-Ser substitution at amino acid residue 173 of HN that was highly resistant to BCX 2798 in HI assays. Even at the highest tested dose, 1,000 µM, BCX 2798 did not inhibit binding of the N173S mutant virus to RBCs, whereas the binding of the parent virus was blocked at 0.11 µM (IC50) BCX 2798. In addition to substantial resistance to the inhibitor in HI assays, the N173S mutant virus was unable to be either eluted from RBCs at 37°C or released from the surfaces of infected LLC-MK2 cells as efficiently as the parent virus. The level of NA activity and the concentration of BCX 2798 required to inhibit it for the N173S mutant virus did not differ from those determined for the parent virus, thus indicating that an Asn-to-Ser substitution does not affect the catalytic site of the mutant virus.

For many years, there was confusion about whether the HN molecule of paramyxoviruses possessed single or separate active sites for HA and NA activities. Different studies suggested either a single switchable site responsible for both functions (24, 40) or two separate sites for HA and NA activities (27). Cocrystallization of NDV HN with thiosialoside showed the presence of an additional receptor-binding site that consisted of residues from both monomers of the HN dimeric molecule (48). This discovery elucidated why BCX 2798, whose design was based on the three-dimensional structure of the catalytic site of NDV HN, does not inhibit NDV binding but is highly effective against its NA (3).

A structural comparison of NDV HN and hPIV-3 HN suggested that residue 173 of hPIV-1 is located very close to a region that for hPIV-1 has all the hallmarks of a second receptor-binding site. Structural analysis and data from biological assays indicated the possibility that the N173S mutant virus isolated from mice treated with BCX 2798 possesses a second receptor-binding site on its HN, which is of hPIV-1 origin.

Our result from gel electrophoresis analysis was consistent with the loss of the N-linked glycan in the N173S mutant virus. Here, we suggest that the loss of the glycan shield at position 173 uncovers a preexisting second receptor-binding site on the HN protein of hPIV-1.

Studies with influenza virus indicate that glycan removal in the region of the HA receptor-binding site by itself can affect virus receptor-binding specificity, elution from RBCs (32), and sensitivity to NA inhibitors (25) because of unbalanced binding and NA activities. The distance of the 173 residue from the catalytic site in hPIV-1 is not consistent with this possibility.

The amino acid alignment and computer modeling data of hPIV-2, hPIV-3, and NDV HNs (6) suggest that residues 522 of hPIV-2 and 523 of hPIV-3 HNs are potential N-linked glycosylation sites closely located to the region identified as a second binding site on NDV HN (48). Indeed, the crystal structure of hPIV-3 HN shows a glycan attached to Asn 523 that is very close to the proposed location of the putative second receptor-binding site (PDB code 1v2i) (18). These observations indicate that, similar to hPIV-1, second receptor-binding sites on hPIV-2 and hPIV-3 may be shielded with N-linked glycans.

The locations of the second receptor-binding sites on hPIV-1 and hPIV-3 HNs were proposed in two previous publications (4, 37). The mutagenesis study with the hPIV-1 HN claimed that the Asn-to-Asp substitution at residue 523 (located in the Arg516 region of NDV HN, forming the second receptor-binding site) creates a second receptor-binding site, resulting in the inability of BCX 2798 to inhibit binding of the mutant virus to cell receptors, and delays mutant virus elution from RBCs (4). When we reproduced the experiments with virus obtained from the authors, we identified the presence of at least two viruses in the sample. One virus had three mutations in hPIV-1 HN: Asn to Thr at residue 173, Asn to Ser at residue 361, and Asn to Asp at residue 523. Another mutant virus possessed analogous substitutions only at residues 361 and 523. Our experiments determined that only the mutant virus variant containing a substitution at residue 173 (among others) has characteristics of the virus possessing the second receptor-binding site. These data suggested that the mutation at residue 173 is responsible for the second receptor-binding site virus phenotype. The data from this study also indicated that it is not the type of amino acid substitution at residue 173 (Asn to Ser or Asn to Thr) but the loss of the N-linked glycosylation site that uncovers a second receptor-binding site affecting the virus' ability to elute from cells and its sensitivity to the HN inhibitor.

Another report suggested the presence of the second receptor-binding site in the region of residue 552 of the H552Q variant mutant hPIV-3 virus (34, 37). Superposition of the HN dimer structures of NDV (48) and hPIV-3 (18) does not support the suggestion for the presence of the second binding site at residue 552 of the H552Q mutant virus, because H552 is distant from the location of the hydrophobic pocket that forms the recognition site for the acetamido methyl group of the receptor (Fig. 4B). Thus, the effects (such as increased binding avidity and resistance to zanamivir) observed with the H552Q mutant virus (34) might be due to different mechanisms. However, only a crystallographic study can determine the locations of second receptor-binding sites on hPIV HNs.

The N173S mutant virus showed reduced growth in cell culture during the first 48 h after infection, compared to the parent virus. The lack of efficient release of the N173S mutant virus from the cell surface might be among the possible reasons for the reduction of virus yield in cell culture. The growth of the N173S mutant virus in the lungs of infected mice was significantly higher than that of the parent virus at days 3, 5, and 7 after infection. We do not know the exact factors contributing to the differences between these in vitro and in vivo results. It is possible that the second receptor-binding site of N173S recognizes additional sialic acid receptors in airway cells of mice that are not present in LLC-MK2 cells. This may increase the amount of mutant virus adsorbed to airway cells and, as a consequence, lead to the higher N173S mutant virus yield in the mouse lungs.

The inability of BCX 2798 to inhibit the N173S mutant virus binding (at dosages up to 1,000 µM) did not abolish virus susceptibility to the compound. These results suggest that while a second binding site of the N173S mutant virus was not blocked by the compound, inhibition of the virus catalytic site continued to provide an antiviral effect. However, it required five times more of the compound to reach the level of inhibition similar to that of the parent virus.

While the general susceptibility of the N173S mutant virus to BCX 2798 in cultured cells was slightly (about fivefold) reduced, the sensitivity of the N173S mutant virus to BCX 2798 in mice did not differ from that of the parent virus when 10 mg/kg per day of the compound was administered to mice for five consecutive days starting 4 h before infection. We do not exclude the possibility that experiments on resistance with lower (than 10 mg/kg per day) dosages of BCX 2798 in nonlethal mouse models (which are currently not available) may reveal a decrease in the N173S mutant virus drug susceptibility. Comprehensive resistance studies will clarify the mechanism of the N173S mutant virus emergence and predict how the presence of hPIV-1 viruses possessing a second receptor-binding site may influence the course of HN inhibitor use.

Similar to influenza virus NA inhibitors (14, 23), resistance to parainfluenza HN inhibitors does not develop easily in a mouse model. Of the 50 virus clones isolated in LLC-MK2 cells from the lungs of treated mice in a second-passage experiment, only one clone possessed the N173S substitution. These data indicate the possibility for a low rate of drug-resistant virus emergence during treatment with HN inhibitors. However, additional studies are needed to understand whether the frequency of resistance emergence is truly low during treatment with HN inhibitors.

We do not know if hPIV-1 viruses possessing the second receptor-binding site circulate in nature and what advantages or disadvantages the second binding site may provide these viruses. Analysis of the virus in human samples will help to determine the frequency of this virus population. Future studies focusing on the biological properties of the N173S mutant virus' second receptor-binding site, such as receptor-binding specificity, its ability to cooperate or compete with the catalytic site (as was shown for NDV [36]), or its influence on the ability of HN to interact with F protein, will resolve questions concerning the biological significance of the second receptor-binding site on hPIV-1.


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ACKNOWLEDGMENTS
 
This work was supported by research grant 160244010 from BioCryst Pharmaceuticals, Inc. (Birmingham, AL), by Cancer Center Support Grant CA 21765 from the National Cancer Institute, by the American Lebanese Syrian Associated Charities (ALSAC), and by the Wellcome Trust.

We thank Ruth Ann Scroggs and Pamela Freiden for technical assistance and Julia Cay Jones and Donald Samulack for editorial assistance.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Infectious Diseases, Mail Stop 330, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105-2794. Phone: (901) 495-3408. Fax: (901) 523-2622. E-mail: allen.portner{at}stjude.org Back

{triangledown} Published ahead of print on 25 June 2008. Back

{dagger} Retired. Back


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Journal of Virology, September 2008, p. 8400-8410, Vol. 82, No. 17
0022-538X/08/$08.00+0     doi:10.1128/JVI.00474-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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