Mutations in the Fusion Protein Cleavage Site of Avian Paramyxovirus Serotype 2 Increase Cleavability and Syncytium Formation but Do Not Increase Viral Virulence in Chickens

Abstract

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. 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.

Mutation of the F-Protein Cleavage Site of Avian Paramyxovirus Type 7 Results in Furin Cleavage, Fusion Promotion, and Increased Replication In Vitro but Not Increased Replication, Tissue Tropism, or Virulence in Chickens

“…Therefore, it is likely that other APMV-7 proteins, in addition to the F protein, contribute to the avirulent phenotype of APMV-7, which requires further investigation. Recently, the role of the F protein cleavage site of APMV-2 was investigated (38). The replacement of the F protein cleavage site of wt APMV-2 (KPASR2F) with that of any of the other APMV serotypes (APMV-1 to -9) regardless of whether it was multibasic or not was associated with the ability to form syncytia and plaques and with an increase in virus spread and production.…”

“…A full-length cDNA of the APMV-7 genome was constructed in plasmid pBR322/dr (5,38). The full-length cDNA clone (pAPMV-7) expressing the complete 15,480-nt-long antigenome of APMV-7 was constructed as seven fragments that were generated by reverse transcription-PCR (RT-PCR) of RNA from APMV-7-infected embryonated chicken eggs.…”

“…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 have been determined (15,24,26,34,35,37,41,42). Recently, reverse genetic systems for APMV-2 and -3 have been reported (16,38).…”

Roles of the Fusion and Hemagglutinin-Neuraminidase Proteins in Replication, Tropism, and Pathogenicity of Avian Paramyxoviruses

“…As an initial step toward characterizing other APMV serotypes, complete genome sequences of one or more representative strains of APMV serotypes 2 to 9 have been determined (20,28,30,38,39,43,55,56). Recently, a reverse genetics system for APMV-2 has also been reported (45). The genome of APMV is similar to those of other paramyxoviruses.…”

Past and Present of Reverse Genetics in Animal Virology with Special Reference to Non-Segmented Negative Stranded RNA Viruses: a Review

“…There were reports on contrary to these findings that FPCS as sole determinant of virulence may not be universal, because the ICPI increase in lentogenic chimeric viruses with artificial velogenic cleavage sites were higher but not phenomenal, from 0.0 to 1.13, compared to wildtype velogenic viruses whose ICPI range from 1.6-1.9 (Panda et al, 2004a;Peeters et al, 1999;Romer-Oberdörfer et al, 2006). There had been reports to throw more light on this aspect, suggesting that there would be proteins other than F that contributes to pathogenicity of NDV (Kommers et al, 2003;De Leeuw et al, 2003;Wakamatsu et al, 2006a;Subbiah et al, 2011).…”