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

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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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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The Deoptimization of Rabies Virus Matrix Protein Impacts Viral Transcription and Replication

Viruses ◽

10.3390/v12010004 ◽

2019 ◽

Vol 12 (1) ◽

pp. 4 ◽

Cited By ~ 1

Author(s):

Jun Luo ◽

Yue Zhang ◽

Qiong Zhang ◽

Yuting Wu ◽

Boyue Zhang ◽

...

Keyword(s):

Rabies Virus ◽

Matrix Protein ◽

Early Stage ◽

Viral Proteins ◽

Codon Pair Bias ◽

Codon Pair ◽

Codon Deoptimization ◽

Cell Spread ◽

Transcription And Replication

Rabies virus (RABV) matrix (M) protein plays several important roles during RABV infection. Although previous studies have assessed the functions of M through gene rearrangements, this interferes with the position of other viral proteins. In this study, we attenuated M expression through deoptimizing its codon usage based on codon pair bias in RABV. This strategy more objectively clarifies the role of M during virus infection. Codon-deoptimized M inhibited RABV replication during the early stages of infection, but enhanced viral titers at later stages. Codon-deoptimized M also inhibited genome synthesis at early stage of infection and increased the RABV transcription rates. Attenuated M through codon deoptimization enhanced RABV glycoprotein expression following RABV infection in neuronal cells, but had no influence on the cell-to-cell spread of RABV. In addition, codon-deoptimized M virus induced higher levels of apoptosis compared to the parental RABV. These results indicate that codon-deoptimized M increases glycoprotein expression, providing a foundation for further investigation of the role of M during RABV infection.

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Study on the recovery of CTN-1V5 strain rabies virus with reverse genetics system

Journal of Applied Virology ◽

10.21092/jav.v3i3.40 ◽

2014 ◽

Vol 3 (3) ◽

pp. 90

Author(s):

Yunpeng Wang ◽

Shouchun Cao ◽

Leitai Shi ◽

Jia Li ◽

Guanmu Dong

Keyword(s):

Rna Polymerase Ii ◽

Rabies Virus ◽

Reverse Genetics ◽

Hammerhead Ribozyme ◽

Vero Cells ◽

Virus Genome ◽

Eukaryotic Expression ◽

Full Length ◽

Direct Immunofluorescence ◽

Cmv Promoter

Use molecular cloning techniques. The CTN-1V5 strains of rabies virus genome is divided into six fragments in order to cloned into Eukaryotic expression vector PVAX 1.0(+). Recombinant full length genomic cDNA was flanked by a hammerhead ribozyme and hepatitis delta virus ribozyme. four helper plasmids encoding the nucleoprotein, phosphoprotein,Glycoprotein and the large protein were constructed and co-transfected with a plasmid containing the full-length CTN viral genome into Vero cells. Recombinant CTN-1V5 virus was successfully recovered from the cloned cDNA under the control of a CMV promoter driven by RNA polymerase II. The recovered CTN-1V5 virus was identified by direct Immunofluorescence technique with anti-nucleoprotein monoclonal-antibody. A group of mice was challenged with the recombinant-strain by intracerebral inoculation, resulting in 100% morbidity.and the titer of the recombinant CTN-1V5 virus was 5.0 log LD50/mL.

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

Journal of Virology ◽

10.1128/jvi.77.22.12074-12082.2003 ◽

2003 ◽

Vol 77 (22) ◽

pp. 12074-12082 ◽

Cited By ~ 73

Author(s):

Stefan Finke ◽

Karl-Klaus Conzelmann

Keyword(s):

Rabies Virus ◽

Rna Synthesis ◽

Matrix Protein ◽

Particle Density ◽

Virus Assembly ◽

Protein Composition ◽

Virus Rna ◽

M Proteins ◽

Protein Functions ◽

Synthesis Activity

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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Proteome analysis of virus–host cell interaction: rabies virus replication in Vero cells in two different media

Applied Microbiology and Biotechnology ◽

10.1007/s00253-013-4939-1 ◽

2013 ◽

Vol 97 (12) ◽

pp. 5493-5506 ◽

Cited By ~ 13

Author(s):

Sabine Kluge ◽

Samia Rourou ◽

Diana Vester ◽

Samy Majoul ◽

Dirk Benndorf ◽

...

Keyword(s):

Host Cell ◽

Rabies Virus ◽

Virus Replication ◽

Proteome Analysis ◽

Vero Cells ◽

Cell Interaction ◽

Host Cell Interaction

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A new system to measure and compare hepatitis C virus replication capacity using full-length, replication competent viruses

Journal of Virological Methods ◽

10.1016/j.jviromet.2013.08.009 ◽

2013 ◽

Vol 194 (1-2) ◽

pp. 82-88

Author(s):

Britta Lassmann ◽

Vaithilingaraja Arumugaswami ◽

Kara W. Chew ◽

Martha J. Lewis

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Virus Replication ◽

Full Length ◽

Replication Capacity ◽

New System

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Role of Small Envelope Protein in Sustaining the Intracellular and Extracellular Levels of Hepatitis B Virus Large and Middle Envelope Proteins

Viruses ◽

10.3390/v13040613 ◽

2021 ◽

Vol 13 (4) ◽

pp. 613

Author(s):

Jing Zhang ◽

Yongxiang Wang ◽

Shuwen Fu ◽

Quan Yuan ◽

Qianru Wang ◽

...

Keyword(s):

Envelope Proteins ◽

Full Length ◽

M Protein ◽

Intracellular Level ◽

S Protein ◽

L Protein ◽

M Proteins ◽

B Virus ◽

Subviral Particle ◽

Genotype A

Hepatitis B virus (HBV) expresses co-terminal large (L), middle (M), and small (S) envelope proteins. S protein drives virion and subviral particle secretion, whereas L protein inhibits subviral particle secretion but coordinates virion morphogenesis. We previously found that preventing S protein expression from a subgenomic construct eliminated M protein. The present study further examined impact of S protein on L and M proteins. Mutations were introduced to subgenomic construct of genotype A or 1.1mer replication construct of genotype A or D, and viral proteins were analyzed from transfected Huh7 cells. Mutating S gene ATG to prevent expression of full-length S protein eliminated M protein, reduced intracellular level of L protein despite its blocked secretion, and generated a truncated S protein through translation initiation from a downstream ATG. Truncated S protein was secretion deficient and could inhibit secretion of L, M, S proteins from wild-type constructs. Providing full-length S protein in trans rescued L protein secretion and increased its intracellular level from mutants of lost S gene ATG. Lost core protein expression reduced all the three envelope proteins. In conclusion, full-length S protein could sustain intracellular and extracellular L and M proteins, while truncated S protein could block subviral particle secretion.

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shRNA targeting nonstructural protein inhibits the replication of severe fever with thrombocytopenia syndrome virus

Future Virology ◽

10.2217/fvl-2020-0186 ◽

2021 ◽

Author(s):

Lili Dou ◽

Xiaoli Tao ◽

Wei Zhao ◽

Guofeng Zheng ◽

Ying Lu ◽

...

Keyword(s):

Tissue Culture ◽

Western Blotting ◽

Virus Replication ◽

Nonstructural Protein ◽

Vero Cells ◽

Antiviral Effects ◽

Infective Dose ◽

Shrna Plasmid ◽

Dose Dependent ◽

Severe Fever

Aim: To explore whether shRNA targeting nonstructural protein (NSs) of severe fever with thrombocytopenia syndrome virus (SFTSV) could inhibit SFTSV replication in Vero cells. Materials & methods: SFTSV used in this experiment was propagated in Vero cells and stored at -20°C. shRNA plasmid against NSs of SFTSV was transfected to Vero cells and infected with SFTSV, after which western blotting and tissue culture infective dose (TCID50) were used to measure the virus titers. Results: shRNA against NSs protein decreased the expression of NSs and inhibited the replication of SFTSV. Conclusion: The constructed SFTSV NSs-shRNA plasmid could inhibit the replication of SFTSV. It was concluded that SFTSV NSs-shRNA could inhibit virus replication for at least 72 h. shRNA-mediated antiviral effects were dose-dependent.

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