title: PPEY motif within the rabies virus (RV) matrix protein is essential for efficient virion release and RV pathogenicity.
abstract: Late (L) domains containing the highly conserved sequence PPXY were first described for retroviruses, and later research confirmed their conservation and importance for efficient budding of several negative-stranded RNA viruses. Rabies virus (RV), a member of the Rhabdoviridae family, contains the sequence PPEY (amino acids 35 to 38) within the N terminus of the matrix (M) protein, but the functions of this potential L-domain in the viral life cycle, viral pathogenicity, and immunogenicity have not been established. Here we constructed a series of recombinant RVs containing mutations within the PPEY motif and analyzed their effects on viral replication and RV pathogenicity. Our results indicate that the first proline at position 35 is the most important for viral replication, whereas P36 and Y38 have a lesser but still noticeable impact. The reduction in viral replication was most likely due to inhibition of virion release, because initially no major impact on RV RNA synthesis was observed. In addition, results from electron microscopy demonstrated that the M4A mutant virus (PPEY-->SAEA) displayed a more cell-associated phenotype than that of wild-type RV. Furthermore, all mutations within the PPEY motif resulted in reduced spread of the recombinant RVs as indicated by a reduction in focus size. Importantly, recombinant PPEY L-domain mutants were highly attenuated in mice yet still elicited potent antibody responses against RV G protein that were as high as those observed after infection with wild-type virus. Our data indicate that the RV PPEY motif has L-domain activity essential for efficient virus production and pathogenicity but is not essential for immunogenicity and thus can be targeted to increase the safety of rabies vaccine vectors.
authors.affiliation: Department of Microbiology and Immunology, Thomas Jefferson University, 531 BLSB, 233 South 10th St., Philadelphia, PA 19107, USA.
authors.forename: Christoph
authors.initials: C
authors.lastname: Wirblich
authors.forename: Gene S
authors.initials: GS
authors.lastname: Tan
authors.forename: Amy
authors.initials: A
authors.lastname: Papaneri
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Godlewski
authors.forename: Jan Marc
authors.initials: JM
authors.lastname: Orenstein
authors.forename: Ronald N
authors.initials: RN
authors.lastname: Harty
authors.forename: Matthias J
authors.initials: MJ
authors.lastname: Schnell
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D017931:DNA Primers / Q000737:chemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry; D011820:Rabies virus / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014763:Viral Matrix Proteins / Q000737:chemistry; D014771:Virion / Q000737:chemistry*
chemical_list: D017931:DNA Primers; D014763:Viral Matrix Proteins; D012313:RNA