Rhabdovirus Matrix Protein Structures Reveal a Novel Mode of Self-Association

Viral hemorrhagic septicemia virus (VHSV) is a pathogenic fish rhabdovirus found in discrete locales throughout the Northern Hemisphere. VHSV infection of fish cells leads to upregulation of the host's virus detection response, but the virus quickly suppresses interferon (IFN) production and antiviral gene expression. By systematically screening each of the six VHSV structural and nonstructural genes, we identified matrix protein (M) as the virus' most potent antihost protein. Only M of VHSV genotype IV sublineage b (VHSV-IVb) suppressed mitochondrial antiviral signaling protein (MAVS) and type I IFN-induced gene expression in a dose-dependent manner. M also suppressed the constitutively active simian virus 40 (SV40) promoter and globally decreased cellular RNA levels. Chromatin immunoprecipitation (ChIP) studies illustrated that M inhibited RNA polymerase II (RNAP II) recruitment to gene promoters and decreased RNAP II C-terminal domain (CTD) Ser2 phosphorylation during VHSV infection. However, transcription directed by RNAP I to III was suppressed by M. To identify regions of functional importance, M proteins from a variety of VHSV strains were tested in cell-based transcriptional inhibition assays. M of a particular VHSV-Ia strain, F1, was significantly less potent than IVb M at inhibiting SV40/ luciferase (Luc) expression yet differed by just 4 amino acids. Mutation of D62 to alanine alone, or in combination with an E181-to-alanine mutation (D62A E181A), dramatically reduced the ability of IVb M to suppress host transcription. Introducing either M D62A or D62A E181A mutations into VHSV-IVb via reverse genetics resulted in viruses that replicated efficiently but exhibited less cytotoxicity and reduced antitranscriptional activities, implicating M as a primary regulator of cytopathicity and host transcriptional suppression.IMPORTANCE Viruses must suppress host antiviral responses to replicate and spread between hosts. In these studies, we identified the matrix protein of the deadly fish novirhabdovirus VHSV as a critical mediator of host suppression during infection. Our studies indicated that M alone could block cellular gene expression at very low expression levels. We identified several subtle mutations in M that were less potent at suppressing host transcription. When these mutations were engineered back into recombinant viruses, the resulting viruses replicated well but elicited less toxicity in infected cells and activated host innate immune responses more robustly. These data demonstrated that VHSV M plays an important role in mediating both virusinduced cell toxicity and viral replication. Our data suggest that its roles in these two processes can be separated to design effective attenuated viruses for vaccine candidates.

Section: Discussionmentioning

confidence: 99%

Role of Viral Hemorrhagic Septicemia Virus Matrix (M) Protein in Suppressing Host Transcription

Qin

Weaver

Pore

et al. 2017

“…Because positively charged amino acids are located in the N-terminal region of the lyssavirus M proteins, membrane binding is thought to occur similarly to vesicular stomatitis virus (VSV) M. For VSV M, it has been shown that the N terminus is required for membrane binding and, in addition, a second membrane-binding region in the globular domain of VSV M has been described (10). Interestingly, amino acids within this second membrane binding region are also involved in binding of Nterminal residues during the process of self-association, which has been suggested to enhance membrane binding of both, VSV M and lyssavirus M proteins (19). Since both positively charged N-terminal residues and those residues that are involved in M self-association are highly conserved (see Fig.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, RABV M regulates viral RNA synthesis (14,17) and has been described to contribute to TRAIL-dependent induction of apoptosis (25) and to mitochondrial dysfunction (18). Recently, the X-ray crystal structure analysis of M of genotype 2 Lagos bat lyssavirus demonstrated a high conservation of M structures within the rhabdovirus family, despite the lack of sequence conservation between the lyssavirus and vesiculovirus genera (19).…”

mentioning

confidence: 99%

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

Finke

Granzow

Hurst

et al. 2010

“…As in the case of the matrix proteins of other enveloped RNA viruses such as rhabdovirus M and filovirus VP40 (17,19), bacterially expressed Z proteins from the Old World arenaviruses lymphocytic choriomeningitis virus (LCMV) and Lassa fever virus (LASV) were reported to exhibit self-assem-bly properties in vitro (25). Self-interaction of matrix proteins is believed to be critical for their function as viral buddingdriving factors.…”

mentioning

confidence: 99%

Molecular Determinants of Arenavirus Z Protein Homo-Oligomerization and L Polymerase Binding

Loureiro

Wilda

Macleod

et al. 2011

The arenavirus Z is a zinc-binding RING protein that has been implicated in multiple functions during the viral life cycle. These roles of Z involve interactions with viral and cellular proteins that remain incompletely understood. In this regard, Z inhibits viral RNA transcription and replication through direct interaction with the viral L polymerase. Here, we defined the L-binding domain of Tacaribe virus (TCRV) Z protein and the structural requirements mediating Z homo-oligomerization. By using site-directed mutagenesis, coimmunoprecipitation, and functional assays, we showed that residues R37, N39, W44, L50, and Y57, located around the zinc coordination site I, play a critical role in the Z-L interaction. We also found that Z protein from either TCRV or the pathogenic Junin virus (JUNV) self-associates into oligomeric forms in mammalian cells. Importantly, mutation of the myristoylation site, the strictly conserved residue G at position 2, severely impaired the ability of both TCRV Z and JUNV Z to self-interact as well as their capacity to accumulate at the plasma membrane, strongly suggesting that Z homo-oligomerization is associated with its myristoylation and cell membrane targeting. In contrast, disruption of the RING structure or substitution of W44 or N39, which are critical for L protein recognition, did not affect Z self-binding. Overall, the data presented here indicate that homo-oligomerization is not a requirement for Z-L interaction or Z-mediated polymerase activity inhibition.