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

Finke, Stefan; Granzow, Harald; Hurst, José; Pollin, Reiko; Mettenleiter, Thomas C.

doi:10.1128/jvi.01665-09

Cited by 25 publications

(29 citation statements)

References 46 publications

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“…59 However, other reports have revealed that the M protein of Lyssavirus leads to the intracellular accumulation of virus particles and affects the cell death pathway. 12,60 We aimed at identifying the key protein in the induction of autophagy and found cells infected with GD-SH-01 and rHEP-shM to present a significant conversion of LC3-I to LC3-II, while rHEP-shN, rHEP-shP, and rHEP-shG induce autophagy to a lesser extent than rHEP-shM. These findings reveal that the M protein of the RABV plays a main role in the induction of autophagy.…”

Section: Discussionmentioning

confidence: 99%

Wild-type rabies virus induces autophagy in human and mouse neuroblastoma cell lines

Peng

Sheng-He

et al. 2016

Autophagy

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Different rabies virus (RABV) strains have their own biological characteristics, but little is known about their respective impact on autophagy. Therefore, we evaluated whether attenuated RABV HEP-Flury and wildtype RABV GD-SH-01 strains triggered autophagy. We found that GD-SH-01 infection significantly increased the number of autophagy-like vesicles, the accumulation of enhanced green fluorescent protein (EGFP)-LC3 fluorescence puncta and the conversion of LC3-I to LC3-II, while HEP-Flury was not able to induce this phenomenon. When evaluating autophagic flux, we found that GD-SH-01 infection triggers a complete autophagic response in the human neuroblastoma cell line (SK), while autophagosome fusion with lysosomes was inhibited in a mouse neuroblastoma cell line (NA). In these cells, GD-SH-01 led to apoptosis and mitochondrial dysfunction while triggering autophagy, and apoptosis could be decreased by enhancing autophagy. To further identify the virus constituent causing autophagy, 5 chimeric recombinant viruses carrying single genes of HEP-Flury instead of those of GD-SH-01 were rescued. While the HEP-Flury virus carrying the wild-type matrix protein (M) gene of RABV triggered LC3-I to LC3-II conversion in SK and NA cells, replacement of genes of nucleoprotein (N), phosphoprotein (P) and glycoprotein (G) produced only minor autophagy. But no one single structural protein of GD-SH-01 induced autophagy. Moreover, the AMPK signaling pathway was activated by GD-SH-01 in SK. Therefore, our data provide strong evidence that autophagy is induced by GD-SH-01 and can decrease apoptosis in vitro. Furthermore, the M gene of GD-SH-01 may cooperatively induce autophagy.

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Section: Discussionmentioning

confidence: 99%

Wild-type rabies virus induces autophagy in human and mouse neuroblastoma cell lines

Peng

Sheng-He

et al. 2016

Autophagy

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“…9A) would imply intracellular budding of RABV particles. Indeed, intracellular virus particle formation is known to occur within RABV-infected cells (45,46), and more recent data provided evidence for matrix protein-dependent accumulation of virus-like particles within the rough endoplasmic reticulum (rER) (30,47). Transport of virus particles within secretory Golgi vesicles, however, has not been observed so far.…”

Section: Discussionmentioning

confidence: 98%

“…BSR T7/5 cells (29) were used for virus rescue from recombinant cDNA as described previously (30). Conditional expression of RABV matrix and glycoproteins in MGon cells (31) was used for amplification of G gene-deleted RABV.…”

Section: Methodsmentioning

confidence: 99%

Anterograde Glycoprotein-Dependent Transport of Newly Generated Rabies Virus in Dorsal Root Ganglion Neurons

Bauer

Nolden

Schröter

et al. 2014

J Virol

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Rabies virus (RABV) spread is widely accepted to occur only by retrograde axonal transport. However, examples of anterograde RABV spread in peripheral neurons such as dorsal root ganglion (DRG) neurons indicated a possible bidirectional transport by an uncharacterized mechanism. Here, we analyzed the axonal transport of fluorescence-labeled RABV in DRG neurons by livecell microscopy. Both entry-related retrograde transport of RABV after infection at axon endings and postreplicative transport of newly formed virus were visualized in compartmentalized DRG neuron cultures. Whereas entry-related transport at 1.5 m/s occurred only retrogradely, after 2 days of infection, multiple particles were observed in axons moving in both the anterograde and retrograde directions. The dynamics of postreplicative retrograde transport (1.6 m/s) were similar to those of entry-related retrograde transport. In contrast, anterograde particle transport at 3.4 m/s was faster, indicating active particle transport. Interestingly, RABV missing the glycoproteins did not move anterogradely within the axon. Thus, anterograde RABV particle transport depended on the RABV glycoprotein. Moreover, colocalization of green fluorescent protein (GFP)-labeled ribonucleoproteins (RNPs) and glycoprotein in distal axonal regions as well as cotransport of labeled RNPs with membrane-anchored mCherry reporter confirmed that either complete enveloped virus particles or vesicle associated RNPs were transported. Our data show that anterograde RABV movement in peripheral DRG neurons occurs by active motor protein-dependent transport. We propose two models for postreplicative long-distance transport in peripheral neurons: either transport of complete virus particles or cotransport of RNPs and G-containing vesicles through axons to release virus at distal sites of infected DRG neurons. IMPORTANCERabies virus retrograde axonal transport by dynein motors supports virus spread over long distances and lethal infection of the central nervous system. Though active rabies virus transport has been widely accepted to be unidirectional, evidence for anterograde spread in peripheral neurons supports the hypothesis that in some neurons RABV also enters the anterograde pathway by so-far unknown mechanisms. By live microscopy we visualized fast anterograde axonal transport of rabies virus. The velocities exceeded those of retrograde movements, suggesting that active, most likely kinesin-dependent transport machineries are involved. Dependency of anterograde transport on the expression of virus glycoprotein G and cotransport with vesicles further suggest that complete enveloped virus particles or cotransport of virus ribonucleoprotein and G-containing vesicles occurred. These data provide the first insight in the mechanism of anterograde rabies virus transport and substantially contribute to the understanding of RABV replication and spread of newly formed virus in peripheral neurons.

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“…Recombinant rabies viruses (rRABVs) comprising the nucleotide sequence of attenuated vaccine strain SAD B19 (16) were generated from cDNAs as described previously (17). Mutations in the P and L genes were introduced into full-length cDNA clones by standard techniques.…”

Section: Methodsmentioning

confidence: 99%

“…Then, 1 g of the RNA was used for reverse transcription with oligo(dT) [12][13][14][15][16][17][18] primers (Invitrogen), 40 U/l RNasin, and Superscript III reverse transcriptase (Invitrogen). All steps were performed according to the supplier's instructions.…”

Section: Methodsmentioning

confidence: 99%

A Dynein Light Chain 1 Binding Motif in Rabies Virus Polymerase L Protein Plays a Role in Microtubule Reorganization and Viral Primary Transcription

Bauer

Nolden

Nemitz

et al. 2015

J Virol

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Rabies virus (RABV) polymerase L together with phosphoprotein P forms the PL polymerase complex that is essential for replication and transcription. However, its exact mechanism of action, interactions with cellular factors, and intracellular distribution are yet to be understood. Here by imaging a fluorescently tagged polymerase (mCherry-RABV-L), we show that L accumulates at acetylated and reorganized microtubules (MT). In silico analysis revealed a dynein light chain 1 (DLC1) binding motif in L that could mediate MT binding through dynein motors. As DLC1 binding by polymerase cofactor P is known, we compared the impact of the DLC1-binding motifs in P and L. Viruses with mutations in the respective motifs revealed that both motifs are required for efficient primary transcription, indicating that DLC1 acts as a transcription enhancer by binding to both P and L. Notably, also the levels of cellular DLC1 protein were regulated by both motifs, suggesting regulation of the DLC1 gene expression by both P and L. Finally, disruption of the motif in L resulted in a cell-type-specific loss of MT localization, demonstrating that DLC1 is involved in L-mediated cytoskeleton reorganization. Overall, we conclude that DLC1 acts as a transcription factor that stimulates primary RABV transcription by binding to both P and L. We further conclude that L influences MT organization and posttranslational modification, suggesting a model in which MT manipulation by L contributes to efficient intracellular transport of virus components and thus may serve as an important step in virus replication. IMPORTANCERegulation of rabies virus polymerase complex by viral and cellular factors thus far has not been fully understood. Although cellular dynein light chain 1 (DLC1) has been reported to increase primary transcription by binding to polymerase cofactor phosphoprotein P, the detailed mechanism is unknown, and it is also not known whether the large enzymatic polymerase subunit L is involved. By fluorescence microscopy analysis of fluorescence-tagged rabies virus L, in silico identification of a potential DLC1 binding site in L, and characterization of recombinant rabies virus mutants, we show that a DLC1 binding motif in L is involved in cytoskeleton localization and reorganization, primary transcription regulation by DLC1, and regulation of cellular DLC1 gene expression. By providing evidence for a direct contribution of a DLC1 binding motif in L, our data significantly increase the understanding of rabies virus polymerase regulation and host manipulation by the virus as well. Rhabdoviridae) are neurotropic viruses that enter the central nervous system (CNS) by axonal transport along microtubules (1). The negative-sense genomic viral RNA encodes five proteins, of which the 240-kDa large enzymatic subunit L and the 35-kDa cofactor phosphoprotein P form the PL polymerase complex (2). The viral polymerase is a ribonucleoprotein (RNP)-dependent polymerase that only uses the genomic RNA as a template for RNA synthesis when it is encap...

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

Abstract: Lyssavirus assembly depends on the matrix protein (M).

Cited by 25 publications

References 46 publications

Wild-type rabies virus induces autophagy in human and mouse neuroblastoma cell lines

Wild-type rabies virus induces autophagy in human and mouse neuroblastoma cell lines

Anterograde Glycoprotein-Dependent Transport of Newly Generated Rabies Virus in Dorsal Root Ganglion Neurons

A Dynein Light Chain 1 Binding Motif in Rabies Virus Polymerase L Protein Plays a Role in Microtubule Reorganization and Viral Primary Transcription

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