Dissociation of Rabies Virus Matrix Protein Functions in Regulation of Viral RNA Synthesis and Virus Assembly

ABSTRACT Recently, we have shown that the rabies virus (RV) matrix (M) protein regulates the balance of virus RNA synthesis by shifting synthesis activity from transcription to replication (S. Finke, R. Mueller-Waldeck, and K. K. Conzelmann, J. Gen. Virol. 84:1613-1621, 2003). Here we describe the identification of an M residue critical for regulation of RV RNA synthesis. By analyzing the phenotype of heterotypic RV M proteins with respect to RNA synthesis of RV SAD L16, we identified the M proteins of the RV ERA and PV strains as deficient. Comparison of M sequences suggested that a single residue, arginine 58, was critical. A recombinant virus having this amino acid exchanged with a glycine, SAD M(R58G), has lost the abilities to downregulate RV transcription and to stimulate replication. This resulted in an increase in the transcription rate of more than 15-fold, as previously observed for M deletion mutants. Most importantly, the efficiencies of virus assembly and budding were equal for wild-type M and M(R58G), as determined in assays studying the transient complementation of an M- and G-deficient RV construct, NPgrL. In addition, virus particle density, protein composition, and specific infectivity of SAD L16 and SAD M(R58G) viruses were identical. Thus, we have identified mutations that affect the function of M only in regulation of RNA synthesis, but not in assembly and budding, providing evidence that these functions are genetically separable.

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PPEY Motif within the Rabies Virus (RV) Matrix Protein Is Essential for Efficient Virion Release and RV Pathogenicity

Journal of Virology ◽

10.1128/jvi.00889-08 ◽

2008 ◽

Vol 82 (19) ◽

pp. 9730-9738 ◽

Cited By ~ 56

Author(s):

Christoph Wirblich ◽

Gene S. Tan ◽

Amy Papaneri ◽

Peter J. Godlewski ◽

Jan Marc Orenstein ◽

...

Keyword(s):

Viral Replication ◽

Rabies Virus ◽

Rna Synthesis ◽

Matrix Protein ◽

Virus Production ◽

Wild Type Virus ◽

Wild Type ◽

Conserved Sequence ◽

Virion Release ◽

The Matrix

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.

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Replication of a Nipah Virus Encoding a Nuclear-Retained Matrix Protein

The Journal of Infectious Diseases ◽

10.1093/infdis/jiz440 ◽

2019 ◽

Vol 221 (Supplement_4) ◽

pp. S389-S394 ◽

Cited By ~ 2

Author(s):

Marc Ringel ◽

Laura Behner ◽

Anja Heiner ◽

Lucie Sauerhering ◽

Andrea Maisner

Keyword(s):

Plasma Membrane ◽

Nuclear Export ◽

Matrix Protein ◽

Virus Assembly ◽

Nipah Virus ◽

Nuclear Export Signal ◽

Cell Types ◽

Surface Transport ◽

M Proteins ◽

Infected Cells

Abstract Nipah virus (NiV) matrix protein (NiV M) plays a major role in virus assembly. It undergoes nuclear transit before accumulating at the plasma membrane and recruiting nucleocapsids to the budding sites. Because nuclear NiV M cannot be detected in all cell types, we wondered whether it can reach the cell surface by bypassing the nucleus. Using an M mutant with a defective nuclear export signal (MNESmut), however, we revealed that the nuclear import of M is ubiquitous, because MNESmut was retained in the nuclei of all cell types tested. Because a functional nuclear transit is a general prerequisite for M surface transport, we wanted to characterize the effect of nuclear-retained M protein in a full viral context and generated a recombinant NiV-MNESmut. Mutant NiV-MNESmut caused increased cell-cell fusion and produced lower virus titers. As expected for an assembly defective NiV, perinuclear inclusions (IBperi) were formed, but inclusions at the plasma membrane (IBPM), which probably represent the viral assembly platforms, were not found. It is interesting to note that the transport-defective MNESmut was recruited to IBperi. This probably prevents overaccumulation of nonfunctional M proteins in the cytoplasm and nuclei of NiV-infected cells and thus provides first evidence that IBperi are functionally relevant aggresome-like compartments.

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The ATPase ATP6V1A facilitates rabies virus replication by promoting virion uncoating and interacting with the viral matrix protein

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.014190 ◽

2020 ◽

pp. jbc.RA120.014190

Author(s):

Xing Liu ◽

Fang Li ◽

Jiwen Zhang ◽

Lulu Wang ◽

Jinliang Wang ◽

...

Keyword(s):

Rabies Virus ◽

Virus Replication ◽

Matrix Protein ◽

Early Stage ◽

Vero Cells ◽

Full Length ◽

Interaction Domain ◽

Glutamic Acid Residue ◽

Proteomic Approach ◽

M Proteins

Rabies virus (RABV) matrix protein (M) plays crucial roles in viral transcription, replication, assembly, and budding; however, its function during the early stage of virus replication remains unknown. Here, we mapped the protein interactome between RABV M and human host factors using a proteomic approach, finding a link to the V-type proton ATPase (V-ATPase) catalytic subunit A (ATP6V1A) which is located in the endosomes where RABV first enters. By downregulating or upregulating ATP6V1A expression in HEK293T cells, we found that ATP6V1A facilitated RABV replication. We further found that ATP6V1A was involved in the dissociation of incoming viral M proteins during viral uncoating. Co-immunoprecipitation demonstrated that M interacted with the full length or middle domain of ATP6V1A, which was dependent on the lysine residue at position 256 and the glutamic acid residue at position 279. RABV growth and uncoating in ATP6V1A-depleted cells was restored by trans-complementation with the full length or interaction domain of ATP6V1A. Moreover, stably overexpressed ATP6V1A enhanced RABV growth in Vero cells which are used for the production of rabies vaccine. Our findings identify a new partner for RABV M proteins and establish a new role of ATP6V1A by promoting virion uncoating during RABV replication.

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

Journal of Virology ◽

10.1128/jvi.01665-09 ◽

2009 ◽

Vol 84 (4) ◽

pp. 1816-1827 ◽

Cited By ~ 17

Author(s):

Stefan Finke ◽

Harald Granzow ◽

Jose Hurst ◽

Reiko Pollin ◽

Thomas C. Mettenleiter

Keyword(s):

Endoplasmic Reticulum ◽

Matrix Protein ◽

Virus Assembly ◽

Virus Release ◽

Host Tropism ◽

Adaptive Mutations ◽

M Proteins ◽

The Matrix ◽

Virus Genomes

ABSTRACT Lyssavirus assembly depends on the matrix protein (M). We compared lyssavirus M proteins from different genotypes for their ability to support assembly and egress of genotype 1 rabies virus (RABV). Transcomplementation of M-deficient RABV with M from European bat lyssavirus (EBLV) types 1 and 2 reduced the release of infectious virus. Stable introduction of the heterogenotypic M proteins into RABV led to chimeric viruses with reduced virus release and intracellular accumulation of virus genomes. Although the chimeras indicated genotype-specific evolution of M, rapid selection of a compensatory mutant suggested conserved mechanisms of lyssavirus assembly and the requirement for only few adaptive mutations to fit the heterogenotypic M to a RABV backbone. Whereas the compensatory mutant replicated to similar infectious titers as RABV M-expressing virus, ultrastructural analysis revealed that both nonadapted EBLV M chimeras and the compensatory mutant differed from RABV M expressing viruses in the lack of intracellular viruslike structures that are enveloped and accumulate in cisterna of the degranulated and dilated rough endoplasmic reticulum compartment. Moreover, all viruses were able to bud at the plasma membrane. Since the lack of the intracellular viruslike structures correlated with the type of M protein but not with the efficiency of virus release, we hypothesize that the M proteins of EBLV-1 and RABV differ in their target membranes for virus assembly. Although the biological function of intracellular assembly and accumulation of viruslike structures in the endoplasmic reticulum remain unclear, the observed differences could contribute to diverse host tropism or pathogenicity.

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The two-stage interaction of Ebola virus VP40 with nucleoprotein results in a switch from viral RNA synthesis to virion assembly/budding

Protein & Cell ◽

10.1007/s13238-020-00764-0 ◽

2020 ◽

Author(s):

Linjuan Wu ◽

Dongning Jin ◽

Dan Wang ◽

Xuping Jing ◽

Peng Gong ◽

...

Keyword(s):

Rna Synthesis ◽

Matrix Protein ◽

Ebola Virus ◽

Virus Assembly ◽

Rna Virus ◽

Critical Role ◽

Viral Rna ◽

Hydrophobic Core ◽

Virion Assembly ◽

N Terminus

AbstractEbola virus (EBOV) is an enveloped negative-sense RNA virus and a member of the filovirus family. Nucleoprotein (NP) expression alone leads to the formation of inclusion bodies (IBs), which are critical for viral RNA synthesis. The matrix protein, VP40, not only plays a critical role in virus assembly/budding, but also can regulate transcription and replication of the viral genome. However, the molecular mechanism by which VP40 regulates viral RNA synthesis and virion assembly/budding is unknown. Here, we show that within IBs the N-terminus of NP recruits VP40 and is required for VLP-containing NP release. Furthermore, we find four point mutations (L692A, P697A, P698A and W699A) within the C-terminal hydrophobic core of NP result in a stronger VP40–NP interaction within IBs, sequestering VP40 within IBs, reducing VP40–VLP egress, abolishing the incorporation of NC-like structures into VP40–VLP, and inhibiting viral RNA synthesis, suggesting that the interaction of N-terminus of NP with VP40 induces a conformational change in the C-terminus of NP. Consequently, the C-terminal hydrophobic core of NP is exposed and binds VP40, thereby inhibiting RNA synthesis and initiating virion assembly/budding.

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The role of ATP in in vitro vaccinia virus RNA synthesis effects of AMP-PNP and ATP gamma S.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)70800-x ◽

1980 ◽

Vol 255 (11) ◽

pp. 5396-5403

Author(s):

S. Shuman ◽

E. Spencer ◽

H. Furneaux ◽

J. Hurwitz

Keyword(s):

Vaccinia Virus ◽

Rna Synthesis ◽

Virus Rna ◽

Gamma S

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Detection of Rabies Virus RNA Isolated from Several Species of Animals in Brazil by RT-PCR.

Journal of Veterinary Medical Science ◽

10.1292/jvms.63.1309 ◽

2001 ◽

Vol 63 (12) ◽

pp. 1309-1313 ◽

Cited By ~ 16

Author(s):

Mikako ITO ◽

Takuya ITOU ◽

Takeo SAKAI ◽

Marli F.C. SANTOS ◽

Yohko T. ARAI ◽

...

Keyword(s):

Rabies Virus ◽

Rt Pcr ◽

Virus Rna

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Two Overlapping Domains of a Lyssavirus Matrix Protein That Acts on Different Cell Death Pathways

Journal of Virology ◽

10.1128/jvi.00761-10 ◽

2010 ◽

Vol 84 (19) ◽

pp. 9897-9906 ◽

Cited By ~ 19

Author(s):

Florence Larrous ◽

Alireza Gholami ◽

Shahul Mouhamad ◽

Jérôme Estaquier ◽

Hervé Bourhy

Keyword(s):

Cell Death ◽

Amino Acid ◽

Matrix Protein ◽

Full Length ◽

M Protein ◽

Genotype 3 ◽

Cell Death Pathways ◽

Acid Fragment ◽

M Proteins ◽

Cellular Apoptosis

ABSTRACT The lyssavirus matrix (M) protein induces apoptosis. The regions of the M protein that are essential for triggering cell death pathways are not yet clearly defined. We therefore compared the M proteins from two viruses that have contrasting characteristics in terms of cellular apoptosis: a genotype 3 lyssavirus, Mokola virus (MOK), and a genotype 1 rabies virus isolated from a dog from Thailand (THA). We identified a 20-amino-acid fragment (corresponding to positions 67 to 86) that retained the cell death activities of the full-length M protein from MOK via both the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and inhibition of cytochrome c oxidase (CcO) activity. We found that the amino acids at positions 77 and 81 have an essential role in triggering these two cell death pathways. Directed mutagenesis demonstrated that the amino acid at position 77 affects CcO activity, whereas the amino acid at position 81 affects TRAIL-dependent apoptosis. Mutations in the full-length M protein that compromised induction of either of these two pathways resulted in delayed apoptosis compared with the time to apoptosis for the nonmutated control.

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