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Avian paramyxovirus serotype 2 (APMV-2) is one of the nine serotypes of APMV, which infect a wide variety of avian species around the world. In this study, we constructed a reverse genetics system for recovery of infectious recombinant APMV-2 strain Yucaipa (APMV-2/Yuc) from cloned cDNA. The rescued recombinant virus (rAPMV-2) resembled the biological virus in growth properties in vitro and in pathogenicity in vivo. The reverse genetics system was used to analyze the role of the cleavage site of the fusion (F) protein in viral replication and pathogenesis. The cleavage site of APMV-2/Yuc (KPASR↓F) contains only a single basic residue (position −1) that matches the preferred furin cleavage site [RX(K/R)R↓]. (Underlining indicates the basic amino acids at the F protein cleavage site, and the arrow indicates the site of cleavage.) Contrary to what would be expected for this cleavage sequence, APMV-2 does not require, and is not augmented by, exogenous protease supplementation for growth in cell culture. However, it does not form syncytia, and the virus is avirulent in chickens. A total of 12 APMV-2 mutants with F protein cleavage site sequences derived from APMV serotypes 1 to 9 were generated. These sites contain from 1 to 5 basic residues. Whereas a number of these cleavage sites are associated with protease dependence and lack of syncytium formation in their respective native viruses, when transferred into the APMV-2 backbone, all of them conferred protease independence, syncytium formation, and increased replication in cell culture. Examination of selected mutants during a pulse-chase experiment demonstrated an increase in F protein cleavage compared to that for wild-type APMV-2. Despite the gains in cleavability, replication, and syncytium formation, analysis of viral pathogenicity in 9-day-old embryonated chicken eggs, 1-day-old chicks, and 2-week-old chickens showed that the F protein cleavage site mutants did not exhibit increased pathogenicity and remained avirulent. These results imply that structural features in addition to the cleavage site play a major role in the cleavability of the F protein and the activity of the cleaved protein. Furthermore, cleavage of the F protein is not a determinant of APMV-2 pathogenicity in chickens.
Paramyxoviruses are pleomorphic, enveloped viruses containing a nonsegmented, negative-sense, single-stranded RNA genome. These viruses have been isolated from a wide variety of mammalian and avian species around the world (20). All paramyxoviruses isolated from avian species are classified into the genus Avulavirus, representing the avian paramyxoviruses (APMV), and the genus Metapneumovirus, representing the avian metapneumoviruses in the family Paramyxoviridae. APMV have been divided into nine different serotypes (APMV-1 to -9) based on hemagglutination inhibition (HI) and neuraminidase inhibition (NI) assays (1). APMV-1, which includes all strains of Newcastle disease virus (NDV), has been characterized extensively because virulent NDV strains cause severe disease in chickens. The complete genome sequence and reverse genetics systems are available for the following representative NDV strains: lentogenic strains LaSota (28, 30) and B1 (24), mesogenic strains Beaudette C (18) and Anhinga (13), and velogenic strains Hert/33 (11), ZJ1 (21), and Texas GB (unpublished data). As an initial step toward characterizing the other APMV serotypes, complete genome sequences of one or more representative strains of APMV serotypes 2 to 9 were recently determined (8, 19, 25, 26, 33, 34, 36, 38).
APMV-2 was first isolated from a diseased chicken in 1956 in Yucaipa, CA (7). Since then, many APMV-2 strains have been isolated from domestic poultry and from free-range, captive, and wild birds around the world (2). APMV-2 infections have been reported in chickens in the United States, Canada, Russia, Japan, Israel, India, Saudi Arabia, and Costa Rica and in turkeys in the United States, Canada, Israel, France, and Italy (4). The infection is more prevalent in turkeys than in chickens (6). APMV-2 infection was shown to affect hatchability and poult yield in turkeys (5). APMV-2 was found to cause a drop in egg production in commercial layer and broiler breeder farms in Scotland (37). APMV-2 strains have also frequently been isolated from passerine and psittacine birds. Surveillance of wild birds has indicated that APMV-2 infections are more frequent in passerines (3, 35).
The genome of APMV-2 strain Yucaipa (APMV-2/Yuc) is 14,904 nucleotides (nt) in length and contains a 55-nt leader sequence at the 3′ end and a 154-nt trailer sequence at the 5′ end. The genome consists of genes that encode nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase protein (HN), and large protein (L). The genes are flanked on either side by highly conserved transcription start and stop sequences and have intergenic sequences of various lengths. Similarly to that for other paramyxoviruses, the P gene contains a putative editing site for the production of V and W proteins (36). Thus, the APMV-2 genome is broadly similar to that of NDV.
The envelope of paramyxoviruses contains two surface glycoproteins, the hemagglutinin-neuraminidase (HN) protein, which is responsible for attachment to the host cell, and the fusion (F) protein, which mediates fusion of the viral envelope with the cell membrane. The F protein is synthesized as an inactive precursor (F0) and is cleaved into two biologically active, disulfide bonded F2-F1 subunits by host cell protease (20). Cleavage of the F protein is necessary for virus entry and cell-to-cell fusion. The F protein cleavage site is a well-characterized determinant of NDV pathogenicity in chickens (17, 23, 27, 28). The F protein of mesogenic and velogenic strains of NDV typically contains a polybasic cleavage site [(R/K)RQ(R/K)R↓F)] that contains the preferred recognition site for furin [RX(K/R)R↓], which is an intracellular protease present in a wide range of cells and tissues. (Underlining indicates the basic amino acids at the F protein cleavage site, and the arrow indicates the site of cleavage.) Consequently, the F protein of these strains can be cleaved in different tissues, making it possible for virulent strains to spread systemically. In contrast, avirulent NDV strains typically have basic residues at the −1 and −4 positions in the cleavage site [(G/E)(K/R)Q(G/E)R↓L)] and depend on secretory protease (or, in cell culture, added trypsin or chicken egg allantoic fluid) for cleavage. This limits the replication of avirulent strains to the respiratory and enteric tracts, where the secretory protease is found.
The putative F cleavage site of APMV-2/Yuc (93DKPASR↓F99) has two basic residues (underlined), of which only the basic residue in the −1 position conforms to the preferred furin cleavage site. Conversely, the F1 subunit of strain Yucaipa begins with a phenylalanine residue, as is characteristic of virulent NDV strains, rather than a leucine residue, as is seen in most avirulent NDV strains (9). APMV-2 strain Yucaipa replicates in a wide range of cells without the addition of exogenous protease, and the inclusion of protease does not improve the efficiency of replication (36). This is incongruent with the lack of the preferred furin motif, although it was not known whether efficient intracellular cleavage indeed occurred. Interestingly, APMV-2 produces single-cell infections and does not cause syncytium formation, a hallmark of paramyxovirus cytopathic effect (CPE). Also, APMV-2/Yuc is highly attenuated in chickens, which is incongruent with its independence from exogenous protease. Thus, questions remained about the cleavability of the F protein of APMV-2/Yuc and its role in infectivity and pathogenicity.
To investigate the role of the F protein cleavage site in replication and pathogenesis of APMV-2, we have developed a reverse genetics system for APMV-2/Yuc. The full-length APMV-2 cDNA clone was used to generate 10 APMV-2 mutants whose F protein cleavage sites were derived from APMV-1 to -9, as well as an 11th mutant in which the only difference from the recombinant virus (rAPMV-2) was a change in the terminal residue of the F1 subunit from phenylalanine to leucine. All of the F protein cleavage site mutants replicated efficiently and maintained the mutations after propagation in embryonated chicken eggs. The 10 mutants containing cleavage sites derived from the other APMV serotypes exhibited protease independence, syncytium formation, and increased replication in vitro. However, the mutations did not change the avirulent nature of APMV-2/Yuc as determined by mean death time (MDT) in 9-day-old embryonated chicken eggs, by intracerebral pathogenicity index (ICPI) in 1-day-old chicks, and by natural route of infection in 2-week-old chickens. These results suggest that the cleavage site sequence is not a major determinant for cleavability of the F protein of APMV-2 and virulence of APMV-2 in chickens.
APMV-2 strain Yucaipa (APMV-2/Yuc) was obtained from the National Veterinary Services Laboratory, Ames, IA. The virus was grown in the allantoic cavities of 9-day-old embryonated, specific-pathogen-free (SPF) chicken eggs. The allantoic fluid was collected 3 days postinoculation (dpi), and the hemagglutination (HA) titer was determined using 0.5% chicken erythrocytes (RBC) at room temperature. The modified vaccinia virus strain Ankara expressing T7 RNA polymerase (MVA) was grown in primary chicken embryo fibroblasts (DF1). DF1, human epidermoid carcinoma (HEp-2), and Madin-Darby canine kidney (MDCK) cell lines were grown in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and maintained in DMEM with 5% FBS. The African green monkey kidney Vero cell line was grown in Eagle's minimal essential medium (EMEM) containing 10% FBS and maintained in EMEM with 5% FBS. In experiments that required supplementation of exogenous protease for cleavage of the F protein, either 1 μg/ml of acetyl trypsin (Invitrogen) or 5% chicken egg allantoic fluid was used. The ability of the viruses to produce plaques was tested in DF1, Vero, and MDCK cells under 0.8% methylcellulose overlay. Plaques were visualized by immunoperoxidase staining using a polyclonal antiserum raised against APMV-2/Yuc in chickens.
Support plasmids pN, pP, and pL were constructed to individually express the N, P, and L proteins, respectively. cDNAs bearing the open reading frames (ORFs) of the N, P, and L genes (positions 141 to 1514, 1681 to 2880, and 7938 to 14666 in the complete genomic sequence, respectively) were cloned under the control of the T7 RNA polymerase promoter in vector pGEM7Z (N and P genes) or pTM1 (L gene). A full-length cDNA of the APMV-2 genome was constructed in plasmid pBR322/dr/Yuc. This plasmid is a modified form of plasmid pBR322 in which the fragment between the EcoRI and PstI sites was removed and replaced by a 72-nt oligonucleotide linker. An 84-nt hepatitis delta virus (HDV) antigenome ribozyme sequence and a T7 RNA polymerase transcription termination signal (10) were inserted into the downstream end of this linker. The full-length cDNA clone (pAPMV-2) expressing the complete 14,904-nt-long antigenome of APMV-2/Yuc was constructed as six fragments that were generated by reverse transcription-PCR (RT-PCR) of RNA from APMV-2/Yuc-infected cells. To facilitate construction, a total of five unique restriction enzyme sites were created by mutating 10 nt without changing any amino acids (Fig. 1). These fragments were sequentially cloned into the pBR322/dr/Yuc plasmid between the T7 promoter and the HDV antigenome ribozyme sequence (Fig. 1). In the full-length cDNA, the F ORF is flanked by unique NotI and PacI enzyme sites, which made it possible to readily substitute mutated F genes. A total of 11 F protein cleavage site mutants (Table 1) were generated by overlapping PCR. The mutated fragments were digested with NotI and PacI enzymes and were used to replace the corresponding fragments in the full-length cDNA of pAPMV-2. The full-length cDNAs of all fusion protein cleavage site mutants were sequenced in their entirety using an ABI 3130xl genetic analyzer (Applied Biosystems).
Infectious virus was recovered from the cDNAs as previously described (10, 14, 18). Briefly, HEp-2 cells were grown overnight to 70% confluence in six-well culture plates and were cotransfected with 5 μg of the respective full-length cDNA plasmid, 3 μg of pN, 2 μg of pP, and 1 μg of pL by using 15 μl of Lipofectamine 2000 (Invitrogen). Along with the transfection mixture, 1 focus-forming unit per cell of recombinant vaccinia virus expressing T7 RNA polymerase (MVA) was added. The transfection mixture was replaced after 6 h with DMEM containing 5% FBS. Two days after transfection, the HEp-2 cells were scraped into the medium and frozen and thawed three times, and the resulting supernatant was inoculated into the allantoic cavities of 9-day-old embryonated, SPF chicken eggs. The allantoic fluid was harvested 3 dpi and tested for HA activity. Allantoic fluid with a positive HA titer was used for the isolation of the viral RNA followed by sequence confirmation of the F protein cleavage site. The recovered parental and mutant viruses were passaged three times in 9-day-old embryonated chicken eggs, and their stability was verified by sequencing the complete F ORF of each mutant virus. Additionally, the HN ORF of each mutant virus was sequenced after three passages to ensure that there were no mutations in the HN protein.
The growth characteristics of the parental and mutant viruses were evaluated in DF1, Vero, and MDCK cells with and without 5% allantoic fluid supplementation in the medium. The ability of the mutant viruses to produce plaques was tested in DF1, Vero, and MDCK cells under 0.8% methylcellulose overlay. The plaques were immunostained using anti-APMV-2/Yuc polyclonal serum in DF1 and Vero cells 3 dpi or in MDCK cells 7 dpi depending on the onset of plaques. The ability of mutant viruses to produce syncytia was tested in DF1 cells. The cells were fixed with cold methanol 3 dpi.
The growth kinetics of the wild-type, parental, and mutant viruses was determined in DF1 cells. Briefly, DF1 cells grown in six-well plates were infected with each virus at a multiplicity of infection (MOI) of 1 with or without 5% allantoic fluid supplementation. After a 1-h adsorption, the infected cells were washed with phosphate-buffered saline (PBS), the medium was replaced, and the cultures were incubated at 37°C. At 24, 48, 72, 96, and 120 h postinfection, 200 μl of culture supernatants was collected and stored at −70°C for virus titration, and an equal volume of fresh medium was added back. Virus titers of the samples were determined by serial endpoint assay on DF1 cells in 96-well plates, with duplicate wells per virus per dilution. The infected cells were stained by an immunoperoxidase method using the polyclonal antiserum raised against APMV-2/Yuc in chickens. The virus titers (50% endpoint tissue culture infectious dose [TCID50]/ml) were calculated using the method of Reed and Muench (29).
A keyhole limpet hemocyanin (KLH)-conjugated synthetic peptide (27 residues), representing amino acids 510 to 536 of the cytoplasmic tail of the F protein of APMV-2 strain Yucaipa, was custom synthesized (Invitrogen). A rabbit was injected subcutaneously with 0.5 mg of peptide in Freund's complete adjuvant. After 2 weeks, a booster immunization was given with 0.5 mg peptide in Freund's incomplete adjuvant, and 2 weeks later, the hyperimmune serum was collected. Western blotting was performed using infected-cell lysates to confirm the specificity of antiserum to the Yucaipa F protein (data not shown).
Radioimmunoprecipitations were performed as described previously (16). Briefly, DF1 cells were infected with parental or mutant APMV-2 at an MOI of 10 for 12 h at 37°C, after which time the cells were switched to methionine- and cysteine-free medium (starvation medium). After 2 h, the medium was replaced by starvation medium containing 100 μCi per ml of [35S]methionine-cysteine (EasyTag EXPRESS35S protein labeling mix; PerkinElmer), and the cells were incubated at 37°C for 30 min (pulse). Subsequently, the medium was replaced by medium containing an excess of unlabeled methionine and cysteine, and the cells were further incubated for 0, 20, 40, and 60 min (chase). Cell lysates were prepared by lysing the cells in radioimmunoprecipitation assay (RIPA) buffer (0.005 M Tris-HCl, 0.15 M NaCl, 1% sodium deoxycholate, 1% NP-40, 1 mM phenylmethylsulfonyl fluoride [PMSF]), and the APMV-2 F protein was precipitated from the lysates by incubation with antiserum specific for the APMV-2 F cytoplasmic tail and analyzed by 10% SDS-polyacrylamide gel electrophoresis. Labeled proteins were visualized by autoradiography. Autoradiographs were scanned, and densitometric analysis of the F0 and F1 protein bands was performed by using the Photoshop program.
The pathogenicity of the mutant viruses was determined by two internationally accepted pathogenicity tests (4). These were (i) the mean death time (MDT) test in 9-day-old embryonated chicken eggs and (ii) the intracerebral pathogenicity index (ICPI) test in 1-day-old chicks.
Briefly, for the MDT test, a series of 10-fold (10−6 to 10−9) dilutions of fresh allantoic fluid from infected eggs was made in sterile phosphate-buffered saline (PBS), and 0.1 ml of each dilution was inoculated into the allantoic cavities of five 9-day-old embryonated chicken eggs per dilution and incubated at 37°C. Each egg was examined three times daily for 7 days, and the time of embryo death, if any, was recorded. The minimum lethal dose (MLD) is the highest virus dilution that causes all embryos inoculated with that dilution to die. The MDT is the mean time in hours for the MLD to kill all of the inoculated embryos. The MDT has been used to classify APMV-1 strains into the following categories: velogenic strains (less than 60 h), mesogenic strains (60 to 90 h), and lentogenic strains (more than 90 h).
For the ICPI test, 0.05 ml of a 1:10 dilution of fresh infective allantoic fluid for each virus was inoculated into groups of 10 1-day-old SPF chicks via the intracerebral route. The birds were observed for clinical signs and mortality once every 8 h for a period of 10 days. At each observation, the birds were scored as follows: 0 if normal, 1 if sick, and 2 if dead. The ICPI is the mean score per bird for all observations over the 10-day period. Highly virulent velogenic viruses give values approaching 2, and avirulent or lentogenic strains give values close to 0.
The effect of the number of basic amino acid residues at the F protein cleavage site on viral pathogenesis and tissue tropism was determined by experimentally infecting 2-week-old SPF chickens with mutant viruses that differed from one another by the number of basic amino acids at their F protein cleavage site. The viruses compared were rAPMV-2 (type 4), rAPMV-2, rAPMV-2 (type 3), rAPMV-2 (type 1v), and rAPMV-2 (type 5), which contain 1, 2, 3, 4, and 5 basic amino acids, respectively, at their F protein cleavage sites. Briefly, groups of six 2-week-old SPF chickens were inoculated with 0.2 ml of 28 HA units of each virus by the oculonasal route. The birds were observed daily and scored for any clinical signs for 7 dpi. Three birds from each group were euthanized 3 and 7 dpi, and oral and cloacal swabs were taken. The following tissues were collected 3 dpi for virus isolation and immunohistochemistry: brain, lung, trachea, spleen, kidney, and cecal tonsils. At 7 dpi, only brain, trachea, and lungs were collected for virus isolation. The tissue samples were processed in three ways: (i) homogenates were prepared for virus titration, (ii) samples were frozen for subsequent immunohistochemistry, and (iii) samples were fixed in formalin for subsequent histopathology.
The oral and cloacal swabs were collected in 1 ml of PBS containing antibiotics (2,000 units/ml penicillin G, 200 μg/ml of gentamicin sulfate, and 4 μg/ml of amphotericin B; Sigma Chemical Co., St. Louis, MO). The swab-containing tubes were centrifuged at 1,000 × g for 20 min, and the supernatants were removed for virus isolation. Virus isolation was performed by inoculating the supernatant into the allantoic cavities of 9-day-old embryonated chicken eggs, and 3 dpi, the allantoic fluid was tested for HA activity. The HA-positive samples were further confirmed by HI tests with APMV-2-specific antiserum. The virus titers in the tissue samples were determined by the following method. Briefly, the tissue samples were homogenized, and the supernatant was serially diluted and used to infect DF1 cells, with duplicate wells per dilution. Infected wells were identified by immunostaining, and the TCID50/ml was calculated using the method of Reed and Muench (29).
The frozen tissue samples collected 3 dpi were sectioned at Histoserve, Inc. (MD). Briefly, the frozen sections were rehydrated in three 10-min changes of PBS. The sections were fixed in ice-cold acetone for 15 min at −80°C and then washed three times in PBS and blocked with 2% bovine serum albumin for 1 h at room temperature inside a humidified chamber. The sections were incubated with a 1:500 dilution of the primary polyclonal antiserum for 2 h at room temperature. After three washes with PBS, the sections were incubated further with horseradish peroxidase (HRP)-conjugated goat anti-chicken antibody for 30 min. After a final wash cycle, the sections were incubated with 3-3′-diaminobenzidine tetrahydrochloride (DAB; Vector Laboratories) for 2 min, washed with distilled water, and counterstained with hematoxylin (Vector Laboratories). Sections were mounted with mounting medium and examined under a light microscope (Zeiss Axiovert 200M), and microphotographs were taken. For histopathology, tissue samples were collected from the brain, trachea, lung, spleen, cecal tonsils, and kidney 3 dpi and fixed in 10% neutral buffered formalin. The fixed tissues were processed, embedded with paraffin, sectioned, and then stained by hematoxylin and eosin (H&E).
A cDNA clone expressing the antigenome of APMV-2/Yuc was constructed from six cDNA segments that were synthesized by RT-PCR from virion-derived genomic RNA (Fig. 1). The cDNA segments were cloned in a sequential manner into the low-copy-number plasmid pBR322/dr/Yuc between a T7 promoter and the hepatitis delta virus ribozyme sequence. The resulting APMV-2 cDNA in the plasmid pBR322/dr/Yuc is a faithful copy of the published APMV-2/Yuc genome consensus sequence (36) except for 10 silent nucleotide changes that were introduced to create five new unique restriction enzyme sites used in the construction (Fig. 1). This construct contains a T7 promoter that initiates a transcript with three extra G residues at its 5′ end, which increases the efficiency of T7 polymerase transcription and does not interfere with recovery (18).
Eleven mutant derivatives with mutations in the F protein cleavage site were constructed (Table 1). Specifically, the wild-type APMV-2 F protein cleavage site (KPASR↓F), which contains two basic amino acids, was replaced with the naturally occurring F protein cleavage sites of APMV serotypes 1 to 9 (Table 1), which contain from one to five basic amino acids. The serotype 1 substitution was represented by three different sequences: the “virulent” sequence RRQKR↓F (type 1v), the “avirulent” sequence GRQGR↓L (type 1av), and the highly basic virulent African strain sequence RRRRR↓F (type 1 Africa) (Table 1). In addition, one construct, rAPMV-2 (F-L) (Table 1), contained the APMV-2 cleavage site in which the phenylalanine residue at the N-terminal end of the F1 subunit, which is associated with improved cleavage and virulence in APMV-1, was replaced by leucine, which is found in avirulent strains of APMV-1. Of the panel of 11 cleavage site mutants, leucine was present at the N terminus of the F1 subunit in five cases: the rAPMV-2 (F-L) mutant noted above, the “avirulent” APMV-1 mutant noted above, and the type 3, 6, and 8 mutants (Table 1). These mutants were readily constructed using the unique NotI and PacI sites flanking the F gene (Fig. 1).
The rAPMV-2 wild-type parent and the 11 F cleavage site mutants were recovered by transfection of the respective antigenome plasmids into HEp-2 cells together with plasmids encoding the N, P, and L proteins, necessary for viral RNA replication and transcription. Plasmid transcription was driven by T7 RNA polymerase supplied by T7 recombinant vaccinia virus strain MVA. The supernatants from the transfected HEp-2 monolayers were inoculated into the allantoic cavities of 9-day-old embryonated chicken eggs. Allantoic fluid was harvested 3 days after infection and tested for HA activity. Allantoic fluid with a positive HA titer was used as a preliminary viral stock. Part of this material was used to isolate viral genomic RNA, which was subjected to RT-PCR and partial sequence analysis of the F gene to confirm the sequence of the F protein cleavage site. The parental virus and the F protein cleavage site mutants were readily recovered from full-length cDNA clones. None of these recombinant viruses required the addition of exogenous protease during transfection and recovery. Each of these viruses was passaged three times in 9-day-old embryonated chicken eggs, and the sequences of the F and HN ORFs were confirmed, showing that the introduced mutations were maintained without any adventitious mutations.
All 12 recombinant viruses replicated in DF1, Vero, and MDCK cells, and the supplementation by exogenous proteases or egg allantoic fluid as a source of protease did not enhance growth of any of the viruses. In general, all of the viruses replicated well in DF1 and Vero cells, while their growth pattern was comparatively slow in MDCK cells. The parental recombinant virus resembled its biological parent in growth characteristics, causing single-cell infection in all three cell lines rather than forming syncytia. Also, neither biological nor recombinant APMV-2 produced plaques under methylcellulose overlay in the presence or absence of exogenous proteases. In contrast, nearly all of the F protein cleavage site mutant viruses, with the sole exception of rAPMV-2 (F-L), gained the ability to produce syncytia and plaques under methylcellulose overlay in the DF1 and Vero cells by 3 dpi and in MDCK cells by 7 dpi (Fig. 2A). Among the 10 F cleavage site mutants that had gained the ability to form syncytia and plaques, there was no difference in morphology and size of plaques in DF1 and Vero cells. However, rAPMV-2 (type 1v), rAPMV-2 (type 3), rAPMV-2 (type 4), rAPMV-2 (type 5), and rAPMV-2 (type 9) produced slightly larger plaques than did other mutants in MDCK cells. There was no apparent difference in morphology of these plaques. Further, the syncytia produced by rAPMV-2 (type 1 Africa), rAPMV-2 (type 3), and rAPMV-2 (type 9) were larger in size than those produced by other mutants (Fig. 2B).
The replication kinetics of parental and F protein cleavage site mutant viruses were compared in a multistep growth cycle. Monolayers of DF1 cells were infected with the viruses, the cells were washed 1 h later, and samples from the medium overlay were collected at 24-h intervals and quantified in DF1 cells by the TCID50 method. This analysis showed that all of the syncytium-forming mutant viruses grew to a 10-fold-higher titer than biological or recombinant wild-type APMV-2 by 3 dpi, whereas the single nonsyncytial mutant, rAPMV-2 (F-L), replicated similarly to the wild-type parent (Fig. 3).
To examine whether the alterations in the amino acid sequence of the F protein cleavage site in the various mutants indeed affected cleavage of the F0 protein, DF1 cells were infected with parental rAPMV-2, which produces single-cell infection, and some of the syncytium-forming cleavage site mutants, specifically, rAPMV-2 (type 1v), rAPMV-2 (type 5), and rAPMV-2 (type 1 Africa). The viral proteins in DF1-infected cells were pulse-labeled for 30 min with Trans35S-label and chased for 0, 20, 40, and 60 min in nonradioactive medium. Cell lysates were prepared and subjected to immunoprecipitation with an antiserum specific for the cytoplasmic tail of the APMV-2/Yuc F protein. Immunoprecipitates were analyzed by SDS-PAGE under reducing conditions (Fig. 4A). Autoradiographs of polyacrylamide gels were scanned by densitometry, and the percentage of F protein cleavage at each time point was calculated by dividing the amount of F1 protein by the total of F1 plus F0 proteins (Fig. 4B). The most efficient cleavage of F protein occurred in rAPMV-2 (type 1 Africa), which contained five arginine (R) residues at the cleavage site (RRRRR↓F). Even at 0 min of chase, the efficiency of F protein cleavage was 100% (Fig. 4A and B). The relative efficiencies of F protein cleavage in rAPMV-2 (type 1v) (RRQKR↓F) and rAPMV-2 (type 5) (KRKKR↓F) were 56 and 27% after 20 min of chase, compared to 2% in parental rAPMV-2 (KPASR↓F). The cleavage efficiencies in rAPMV-2 (type 1v) and rAPMV-2 (type 5) increased to 94 and 75% after 60 min of chase, compared to 43% in parental rAPMV-2. These results showed that cleavage of the F0 protein was more efficient in these syncytium-forming APMV-2 mutants than in the nonsyncytial rAPMV-2 parent. Furthermore, the results showed that not only the number but also the type of basic residue influences the cleavage of F0 protein. For example, rAPMV-2 (type 1 Africa) and rAPMV-2 (type 5) both contain five basic residues, but they vary greatly in cleavage efficiency. rAPMV-2 (type 1 Africa) contains five R residues, whereas rAPMV-2 (type 5) contains lysine (K) in the −2, −3, and −5 positions. These results suggest that R is preferred over K for cleavage by host cell proteases.
The pathogenicity of the biological wild-type APMV-2, recombinant wild-type rAPMV-2, and 11 F protein cleavage site mutant viruses was determined by the mean death time (MDT) test in 9-day-old embryonated chicken eggs and by the intracerebral pathogenicity index (ICPI) test in 1-day-old chicks (data not shown). Similarly to biological wild-type APMV-2, rAPMV-2 and the mutant viruses did not cause death of chicken embryos within the standard 7-day (168-h) time limit for the assay, and thus the MDT for all of the viruses is scored as >168 h. However, it was observed that, when the incubation was extended to 10 days, the syncytial mutant viruses caused embryo deaths at lower dilutions (10−2 to 10−3) than did the biological wild-type APMV-2, rAPMV-2, and rAPMV-2 (F-L) viruses (data not shown). This suggested that, while the syncytial mutant viruses remained avirulent by the standard MDT assay, they were marginally more virulent than the wild-type viruses or the rAPMV-2 (F-L) mutant in embryonated chicken eggs during extended incubation and at lower dilution. All of the recombinant viruses had ICPI values of zero, resembling wild-type APMV-2. Both tests suggested that the cleavage site of the F protein and the ability to form syncytia did not significantly affect the pathogenicity of APMV-2 in chickens.
The effect of the F protein cleavage site on viral pathogenesis was further studied by experimentally infecting 2-week-old SPF chickens with selected mutant viruses that varied in number of basic amino acids at the F protein cleavage site and, in one case, by the presence of L as the first residue of the F1 subunit. The following viruses were chosen for comparison: rAPMV-2 (type 4) (DIQPR↓F), rAPMV-2 (KPASR↓F), rAPMV-2 (type 3) (RPRGR↓L), rAPMV-2 (type 1v) (RRQKR↓F), and rAPMV-2 (type 5) (KRKKR↓F). These contained 1, 2, 3, 4, and 5 basic amino acid residues, respectively, at the F protein cleavage site, and the rAPMV-2 (type 3) mutant contained L at the terminus of F1. The birds were infected with 0.2 ml of 28 HA units of infective fresh allantoic fluid by the oculonasal route. The birds were observed daily for 7 days postinfection. Three birds from each group were sacrificed on day 3 and the remaining on day 7. Oral and cloacal swabs were taken upon sacrifice and analyzed for viral shedding. In addition, tissue samples were taken on day 3 from the brain, lung, trachea, spleen, kidney, and cecal tonsil and on day 7 from the brain, lung, and trachea. These were analyzed for virus titer, immunohistochemistry of viral antigens, and histopathology.
There were no apparent clinical signs of illnesses in any of the infected groups throughout the study period. The oral or cloacal viral shedding was inconsistent due to low titer, and there was no significant difference in viral shedding between the parental and F protein cleavage site mutant viruses either 3 or 7 dpi (data not shown). Histopathological examinations of tissue samples collected 3 dpi revealed similar microscopic findings in all of the tested recombinant viruses. This is illustrated with representative viruses in Fig. 5. Specifically, the trachea showed mild lymphocytic tracheitis, with epithelial attenuation and regeneration (Fig. 5b). In the lungs, mild to moderate multifocal lymphohistiocytic, perivascular, and interstitial pneumonia was observed (Fig. 5d), and in the spleen, there was minimal lymphoid depletion (Fig. 5f). Microscopic lesions were not found in any of the other tissues.
The replication of parental and mutant viruses was quantified in the tissue samples taken 3 (Fig. 6A) and 7 (Fig. 6B) dpi. None of the viruses was detected in the brain either 3 or 7 dpi. The titers of virus in the other tissues showed no consistent pattern with regard to the F protein cleavage site. At 3 dpi, for example, rAPMV-2 (type 4), with a single basic residue, was reduced in titer compared to the parental rAPMV-2 virus, which has two basic residues, in the lungs, trachea, spleen, and kidney. However, the same was true of rAPMV-2 (type 1v), which has four basic amino acids at the F protein cleavage site. These three viruses had similar titers in the cecal tonsils. At 3 dpi, the replication of rAPMV-2 (type 3), with three unpaired basic amino acids at the F protein cleavage site, showed a slightly increased virus titer in lungs, trachea, spleen, and kidney compared to that for the rAPMV-2 parent but no significant difference in titer in the cecal tonsil. At 3 dpi, the replication of the rAPMV-2 (type 5) mutant, with five basic amino acids at the F protein cleavage site, showed an increased virus titer in trachea compared to that for the rAPMV-2 parent, but there was no significant difference in titer in the spleen and kidney, and the viral titers in the lungs and cecal tonsil were reduced (Fig. 6A). At 7 dpi, rAPMV-2 (type 4), rAPMV-2 (type 1v), and rAPMV-2 (type 5) had reduced virus titers in the upper respiratory tract (trachea) and lower respiratory tract (lungs), whereas rAPMV-2 (type 3) had a reduced virus titer in trachea, without any significant difference of titer in lung (Fig. 6B). Thus, there was no clear relationship between virus titer and sequence of the F protein cleavage site. Viral antigens were detected by immunohistochemistry in tissue samples that were also positive by virus titration, which is shown for rAPMV-2 in Fig. 7. This confirmed that the detection of virus in harvested tissue indeed was associated with infection of the organ. All infected birds were seropositive by 7 dpi as observed by the HI test, with no differences associated with specific viruses (data not shown).
In paramyxoviruses, proteolytic processing of the F protein is a prerequisite for the generation of mature infectious virus. There are nine serotypes of APMV within the genus Avulavirus in the family Paramyxoviridae. Virulent strains of APMV-1 (NDV) have multibasic cleavage sites that are recognized and cleaved by furin, a ubiquitous intracellular protease whose preferred cleavage sequence is RX(R/K)R↓. These viruses are able to replicate in most cell types and cause systemic infection. The avirulent strains of APMV-1 have one or occasionally two unpaired basic amino acids that lack the furin motif and are cleaved by trypsin-like extracellular proteases. Hence, these viruses are restricted mostly to the respiratory and gastrointestinal tracts, and they require supplementation with exogenous proteases for in vitro growth. It has been shown that the F protein cleavage site is a major determinant of APMV-1 virulence in chickens (27, 28, 31). In contrast, the contribution of the F protein cleavage site to the pathogenicity of APMV-2 to -9 is unknown. Each of these APMV serotypes has been isolated from a different avian species. The natural host(s) of these viruses is not clearly defined. APMV-2 is endemic among passerines and causes severe respiratory illness in parrots but only mild respiratory illness in chickens. APMV-5 causes high mortality in budgerigars but is apathogenic in chickens. APMV serotypes 3, 4, 6, 7, 8, and 9 also cause mild or inapparent disease in chickens. It is not known whether the avirulence of APMV-2 to -9 in chickens is due to the F protein cleavage site sequence of these serotypes.
The F protein cleavage site sequences vary widely among the APMV serotypes. The F protein cleavage sites of APMV-4 (DIQPR↓F) and APMV-7 (LPSSR↓F) contain a single basic amino acid residue, and that of APMV-2 (KPASR↓F) has two unpaired basic amino acids. However, in each case, only the R residue in the −1 position matches the furin cleavage sequence. These three serotypes contain phenylalanine at the F1 terminal end and do not require exogenous protease supplementation for growth in cell culture (25, 36, 38). APMV-5 (KRKKR↓F) contains five basic residues in the F cleavage site and does not require exogenous protease supplementation for growth in cell culture (34). The requirement of exogenous protease supplementation for APMV-6 (APEPR↓L) varies with strains. The other APMV serotypes, APMV-3 (RPRGR↓L), APMV-8 (YPQTR↓L), and APMV-9 (IREGR↓I), require exogenous protease supplementation for in vitro growth. It is noteworthy that the cleavage sites of APMV-3, -6, and -8 have a leucine instead of a phenylalanine as the first residue of the F1 subunit, which is also found in avirulent strains of APMV-1. The presence of leucine at this position has been associated with reduced cleavability of the APMV-1 F protein (22).
The goal of this study was to evaluate the role of amino acid sequence at the F protein cleavage site of APMV-2 in replication, formation of syncytia and plaques, and pathogenicity. In order to mutate the F protein cleavage site, a reverse genetics system for APMV-2 was developed for the first time. This system will be useful to study the function of the APMV-2 structural features and macromolecules in replication and pathogenesis. We generated 12 recombinant APMV-2 F protein cleavage site mutants: 11 mutants containing the F protein cleavage site of naturally occurring APMV serotypes, and 1 mutant where the phenylalanine at the N-terminal end of the F1 subunit was replaced by a leucine residue.
We were able to recover all of the above-mentioned F protein cleavage site mutants without exogenous protease supplementation, suggesting that the mutations did not adversely affect the functions of F protein. Irrespective of the F protein cleavage site or the type of cell line used, none of the APMV-2 F protein cleavage site mutants required exogenous protease supplementation for in vitro growth, resembling the wild-type APMV-2 virus. This was particularly surprising in the case of mutants containing cleavage sites from the APMV-1 avirulent strain, APMV-3, APMV-8, and APMV-9, because these cleavage sites require exogenous protease when present in their respective natural F proteins. This suggests that APMV-2 contains one or more structural features outside the cleavage site that promotes cleavage by intracellular protease. This might also account for the ability of wild-type APMV-2 to replicate even though its F protein cleavage site contains only two basic residues, one of which is consistent with the preferred furin cleavage site.
A second phenotype was the ability to form syncytia. Wild-type APMV-1, -3, and -5 produce syncytia in infected cells, whereas APMV-2, -4, and -6 to -9 cause single-cell infection. Similarly to wild-type APMV-2, the rAPMV-2 and rAPMV-2 (F-L) mutants caused single-cell infections and did not form syncytia or plaques. Surprisingly, however, all of the other F protein cleavage site mutants produced syncytia and plaques in DF1, Vero, and MDCK cells, even if the cleavage site involved (e.g., APMV-4, -6, and -9) does not confer syncytium or plaque formation in its native virus. In particular, the cleavage sites of APMV-4, -6, -7, and -8, which contain only a single basic amino acid, and the cleavage sites of avirulent APMV-1 and -9, which contain two basic unpaired amino acids, also caused syncytium formation in the absence of exogenous protease when present within the APMV-2 F protein. One possibility for these results could be an alteration of conformation of APMV-2 F protein around the cleavage site due to change in amino acid, leading to more efficient recognition by intracellular proteases and hence cleavage and exposure of the amino-terminal F1 subunit for cell-to-cell fusion. Alternatively, the F protein does not need much help from HN to mediate fusion, and it may not require as much cleaved F to get the virus to fuse with cells and subsequently cause infection. This could also explain the difference in syncytium formation due to the good cleavage mutants having sufficient F protein present to push past a threshold needed to induce cell-cell fusion. It is known that cell-cell fusion differs from virus-cell fusion in the amount of fusion activity needed to get the two pairs of membranes to merge. To verify that the amino acid modifications at the F protein cleavage site indeed led to cleavage of F0 protein, pulse-chase experiments with the parental and a few selected syncytium-forming mutant viruses were performed. The results showed that the F protein of parental rAPMV-2 is cleaved relatively slowly but that the efficiency of cleavage is greatly enhanced in the mutants that were examined. Further, our growth kinetics results showed that syncytium formation increased the replication of the mutant viruses 10-fold, suggesting that the enhanced growth was probably due to increased cell-to-cell spread of the virus.
Thus, incorporation of the cleavage sites of the other APMV serotypes into APMV-2 resulted in growth that was independent of added protease, was increased 10-fold, and conferred the ability to form syncytia and plaques. The effects were similar for the various cleavage sites irrespective of the number of basic residues or the presence of phenylalanine versus leucine at the N terminus of the F1 subunit. These results led to the expectation that virulence in vivo would be enhanced, based on the well-known example of APMV-1, for which the sequence at the F protein cleavage site is a major determinant of virulence (27, 28, 31). Surprisingly, however, all of the viruses retained the avirulent phenotype of the APMV-2 parent. In all cases, the MDT values of APMV-2 F protein cleavage site mutants were more than 168 h and the ICPI values were zero, similar to that of the wild-type APMV-2. This suggests that the F protein cleavage site does not play an important role in the virulence of APMV-2.
In addition, the effect of number of basic amino acids at the F protein cleavage site on pathogenesis was studied in 2-week-old chickens. We chose five mutants that varied from one another in number of basic amino acid residues at the F protein cleavage site, i.e., rAPMV-2 (type 4), rAPMV-2, rAPMV-2 (type 3), rAPMV-2 (type 1v), and rAPMV-2 (type 5), with 1, 2, 3, 4, and 5 basic amino acids, respectively, at the F protein cleavage site. Our results did not show any significant difference in viral replication and tissue tropism among the F protein cleavage site mutants, suggesting no direct correlation between the number of basic amino acids at the F protein cleavage site and the pathogenicity of APMV-2. Again, these results are consistent with the interpretation that the F protein cleavage site is not a major determinant of virulence in APMV-2.
We have shown previously that altering the F protein cleavage site of an avirulent strain to that of a neurovirulent strain of NDV did not convert the avirulent strain into a neurovirulent strain after a natural route of infection (27). Although the biological activities of the fusion protein and growth characteristics of the virus in vivo improved over those for the avirulent parental strain, the complete spectrum of virulence phenotype could not be achieved by modifying the F cleavage site. Further, it has been shown that other proteins of NDV, such as the HN, V, NP, P, and L proteins, are responsible for virulence along with the F protein (12, 15, 27, 32). Therefore, it is possible that all of these proteins along with the F protein might contribute to the virulence of APMV-2, which requires further investigation.
In conclusion, we found that replacement of the F protein cleavage site of APMV-2 with that of any of the other APMV serotypes was associated with replication independent of added protease, the ability to form syncytia and plaques, and increased viral replication. It may be that these substitutions caused a conformational change leading to increased efficiency of F protein cleavage and function. However, none of these changes increased the virulence or changed the tropism of the virus. Thus, the cleavage and function of the F protein do not appear to be important factors in the virulence of APMV-2.
We thank Daniel Rockemann for his technical assistance and help. We thank Arthur Samuel for his help with handling of chickens during the animal experiments and Heather Shive (NIH) for her help with the interpretation of H&E-stained slides. We also thank Bernie Moss (NIAID, NIH) for providing the T7 recombinant vaccinia virus.
This research was supported by NIAID contract no. N01A060009 (85% support) and the NIAID NIH Intramural Research Program (15% support).
The views expressed herein do not necessarily reflect the official policies of the Department of Health and Human Services, nor does mention of trade names, commercial practices, or organizations imply endorsement by the U.S. Government.
Published ahead of print on 30 March 2011.