The Matrix Protein of a Plant Rhabdovirus Mediates Superinfection Exclusion by Inhibiting Viral Transcription

Superinfection exclusion (SIE) is a widespread phenomenon in which an established virus infection prevents reinfection by closely related viruses. Understanding the mechanisms governing SIE will not only advance our basic knowledge of virus infection cycles but may also lead to improved design of antiviral measures. Despite the significance of SIE, our knowledge about viral SIE determinants and their modes of actions remain limited. In this study, we show that sonchus yellow net virus (SYNV) SIE is mediated by the viral matrix (M) protein. During primary infections, accumulation of M protein in infected nuclei results in coiling of genomic nucleocapsids and suppression of viral transcription. Consequently, nucleocapsids released by potential superinfectors are sequestered and are unable to initiate new infections. Our data suggest that SYNV SIE is caused by M protein-mediated transition from replication to virion assembly and that this process prevents secondary infections. ABSTRACT Superinfection exclusion (SIE) or cross-protection phenomena have been documented for plant viruses for nearly a century and are widespread among taxonomically diverse viruses, but little information is available about SIE of plant negative-strand RNA viruses. Here, we demonstrate that SIE by sonchus yellow net nucleorhabdovirus virus (SYNV) is mediated by the viral matrix (M) protein, a multifunctional protein involved in transcription regulation, virion assembly, and virus budding. We show that fluorescent protein-tagged SYNV variants display mutual exclusion/cross-protection in Nicotiana benthamiana plants. Transient expression of the SYNV M protein, but not other viral proteins, interfered with SYNV local infections. In addition, SYNV M deletion mutants failed to exclude superinfection by wild-type SYNV. An SYNV minireplicon reporter gene expression assay showed that the M protein inhibited viral transcription. However, M protein mutants with weakened nuclear localization signals (NLS) and deficient nuclear interactions with the SYNV nucleocapsid protein were unable to suppress transcription. Moreover, SYNV with M NLS mutations exhibited compromised SIE against wild-type SYNV. From these data, we propose that M protein accumulating in nuclei with primary SYNV infections either coils or prevents uncoiling of nucleocapsids released by the superinfecting SYNV virions and suppresses transcription of superinfecting genomes, thereby preventing superinfection. Our model suggests that the rhabdovirus M protein regulates the transition from replication to virion assembly and renders the infected cells nonpermissive for secondary infections. IMPORTANCE Superinfection exclusion (SIE) is a widespread phenomenon in which an established virus infection prevents reinfection by closely related viruses. Understanding the mechanisms governing SIE will not only advance our basic knowledge of virus infection cycles but may also lead to improved design of antiviral measures. Despite the significance of SIE, our knowledge about viral SIE determinants and their modes of actions remain limited. In this study, we show that sonchus yellow net virus (SYNV) SIE is mediated by the viral matrix (M) protein. During primary infections, accumulation of M protein in infected nuclei results in coiling of genomic nucleocapsids and suppression of viral transcription. Consequently, nucleocapsids released by potential superinfectors are sequestered and are unable to initiate new infections. Our data suggest that SYNV SIE is caused by M protein-mediated transition from replication to virion assembly and that this process prevents secondary infections.