Genome-wide transposon mutagenesis of paramyxoviruses reveals constraints on genomic plasticity

Abstract

The antigenic and genomic stability of paramyxoviruses remains a mystery. Here, we evaluate the genetic plasticity of Sendai virus (SeV) and mumps virus (MuV), sialic acid-using paramyxoviruses that infect mammals from two Paramyxoviridae subfamilies (Orthoparamyxovirinae and Rubulavirinae). We performed saturating whole-genome transposon insertional mutagenesis, and identified important commonalities: disordered regions in the N and P genes near the 3' genomic end were more tolerant to insertional disruptions; but the envelope glycoproteins were not, highlighting structural constraints that contribute to the restricted antigenic drift in paramyxoviruses. Nonetheless, when we applied our strategy to a fusion-defective Newcastle disease virus (Avulavirinae subfamily), we could select for Frevertants and other insertants in the 5' end of the genome. Our genome-wide interrogation of representative paramyxovirus genomes from all three Paramyxoviridae subfamilies provides a family-wide context in which to explore specific variations within and among paramyxovirus genera and species.

Population Variability Generated during Rescue Process and Passaging of Recombinant Mumps Viruses

“…Whether this facilitates persistence and detection of biologically relevant variants or only the ones that are Vero-specific is unknown. Bamford et al [25] and Ikegame et al [18] that obtained results comparable to ours also used Vero as a cellular substrate. Still, it must be emphasized that a variety of factors, including innate immunity reactions, influence the variability of viral populations and the heterogeneities observed during passaging in Vero cells cannot be considered as general quasispecies characteristics of MuV strains.…”

“…Heterogeneous sites we detected were in a nearby downstream region. Ikegame et al [18] also observed that recombinant MuVs preferentially permit mutations (which in their study were insertions) in N and P genes, especially in their noncoding regions. Whether changes in 5 terminal region of N gene can have influence on the nature or quantity of defective interfering particles as has been shown for a point mutation in Sendai virus [57]; or influence mRNA transcription gradient as in measles virus [58] has yet not been shown for any MuV strain.…”

“…Long-term transmission of a minor variant with premature stop codon in envelope glycoprotein has been observed in nature, in dengue virus type 1 populations, possibly offering selective advantage for coinfecting viruses by ensuring the presence of viruses differentially adapted to various cell types, decoying host immune responses, or allowing the production of extra capsid and membrane proteins [54]. For MuV, in in vitro experiments, the existence and continuing replication of viral variants that rely on other variants in the pool to complement their genomic defects has been previously shown [18] but the biological significance of variants with shortened proteins has not been determined. Premature stop codons in M gene of measles virus are characteristic for strains that cause subacute sclerosing panencephalitis, a fatal progressive neurological disorder sometimes caused by wild-type measles strains [55].…”

“…In construction of all our recombinant viruses, two paramyxoviral genomic rules were strictly adhered to: the rule of six and N-phasing. In the article by Ikegame et al [18], only the rule of six was strictly followed. They presented results that show that the replication of MuVs possessing genomes with false N-phasing was not as successful and such viruses were quickly and overwhelmingly outgrown.…”

“…The ability to manipulate MuV genome by reverse genetics systems has been used as a tool (a) for investigation of MuV biology [14][15][16][17][18][19][20][21], (b) for optimization of a mumps vaccine [22], and (c) to investigate possibilities of using MuV as a vector in recombinant vaccines or in gene therapy [4,[23][24][25][26].…”