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 (PPEY3SAEA) 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.Rabies virus (RV) is a nonsegmented negative-stranded RNA of the genus Lyssavirus within the Rhabdoviridae family (19). Rhabdoviruses, such as RV and vesicular stomatitis virus (VSV), are simple enveloped RNA viruses which encode only five proteins: a nucleoprotein (N), which encapsidates the genomic and antigenomic RNA and forms the ribonucleoprotein (RNP); a phosphoprotein (P), which is the noncatalytic subunit of the RNA polymerase complex; a matrix protein (M); a transmembrane glycoprotein (G); and the viral polymerase (L) (54). Whereas the RNP is the template for viral transcription and replication (4), the RV G and M are the key players in viral budding and assembly (38-40, 43, 46). In the case of RV G, it has been shown that this protein is not essential for viral budding, but the amount of virions is reduced about 30-fold if RV G is deleted (39). In addition, Mebatsion et al. showed that the RV G cytoplasmic domain (CD) specifically interacts with the RV M protein and that a CD-deleted glycoprotein reduces RV budding about sixfold (39,40). This is in contrast to VSV, for which it has been shown that only a CD is required and not a specific amino acid sequence (46). However, similar to RV, VSV can also bud without its own glycoprotein but with also a reduced efficiency. ...

Intergenotypic Replacement of Lyssavirus Matrix Proteins Demonstrates the Role of Lyssavirus M Proteins in Intracellular Virus Accumulation

“…Intracellular accumulation of RABV RNPs or retention of almost complete virions could be a result of defects in late stages of virus assembly after RNP condensation by M. Indeed, examples for such late domain-defective RABV and other rhabdovirus species have been described (20,21,48). Since the late domain PPEY motif in the M proteins of lyssaviruses is conserved, we did not expect a typical late domain-defective phenotype with virus accumulations at the surface of the infected cells.…”

A Leucine Residue in the C Terminus of Human Parainfluenza Virus Type 3 Matrix Protein Is Essential for Efficient Virus-Like Particle and Virion Release

“…In addition, even in the M proteins containing the same late domain, the critical residue for viral budding may also be different. For example, RV and VSV both contain the PPxY L domain; for RV, the first proline of PPEY is most important for efficient budding, whereas for VSV, the tyrosine of the PPPY L domain is most critical for efficient budding (55,56). Although the HPIV3 M protein does not contain a known L domain, we found that a critical leucine (L302) residue localizing at the C-terminal 80 aa is required for VLP production via regulation of the ubiquitination of the M protein (Fig.…”

Conserved Motifs within Ebola and Marburg Virus VP40 Proteins Are Important for Stability, Localization, and Subsequent Budding of Virus-Like Particles

“…Like many enveloped viruses such as rhabdovirus (11) and arenaviruses (44), Ebola virus encodes late-assembly or L domains, which are sequences required for the membrane fission event that separates viral and cellular membranes to release nascent virion particles (1,5,7,10,12,18,25,27,34). Thus far, four classes of L domains have been identified which were defined by their conserved amino acid core sequences: the Pro-Thr/ Ser-Ala-Pro (PT/SAP) motif (25,27), the Pro-Pro-x-Tyr (PPxY) motif (11,12,18,19,41,53), the Tyr-x-x-Leu (YxxL) motif (3,15,27,37), and the Phe-Pro-Ile-Val (FPIV) motif (39). Both PTAP and the PPxY motifs are essential for efficient particle release for eVP40 (25,27,48,49), whereas mVP40 contains only a PPxY motif.…”