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

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.

Virus Population Bottlenecks: What Are They Telling Us?

“…The very fact that the each of the cell clusters contained a single variant betrays highly stringent population bottlenecks that permitted just one variant to infect a group of connected cells in an SL. Strikingly similar observations have since been made with other viruses, including WSMV, apple latent spherical virus (ALSV), tobacco etch virus (TEV), and a negative-strand RNA virus known as sonchus yellow net virus (SYNV) [29][30][31][32][33] , underlining the highly conserved nature of intracellular bottlenecking encountered by virus populations.…”

“…It is also well known that established infections exclude secondary invasions by the same virus 7,[38][39][40] . Finally, such exclusion/repression has been shown to be actively enforced by virus-encoded proteins in several cases 30,31,34 . Why would viruses actively block most copies of their own genomes from replicating in the same cell?…”

Recent advances in understanding the biological roles of the plant nuclear envelope

“…After entry into the cell, viral particles release nucleocapsid cores which are transported into the nucleus through NPCs 79 . Following primary transcription, nuclear export, and translation, viral proteins are imported back into the nucleus to participate in replication, formation of new nucleocapsid cores, and subsequent budding 79,80 . and thus older than the previously reported origin in moss 90 .…”

“…Additionally, M proteins have been shown to target NPCs to inhibit the export of host-cell mRNAs and function in super infection exclusion (SIE) [ 80 , 88 ]. SIE occurs when the primary virus infection prevents the infected cells from subsequent infection by the same or related virus.…”

“…SIE occurs when the primary virus infection prevents the infected cells from subsequent infection by the same or related virus. Deletion of the M protein, as well as mutating the NLS, resulted in the failure of the primary infection to inhibit transcription and super infection [ 80 ]. The current hypothesis is that M protein from the primary infection is either re-coiling or preventing the uncoiling of the nucleocapsids released by secondary invader, subsequently suppressing transcription and preventing super infection [ 80 ].…”