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

Abstract

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...

Phenotypic Consequences In vivo and In vitro of Rearranging the P Gene of RABV HEP-Flury

“…Leader RNA can bind La protein which may inhibit cellular RNA synthesis ( Kurilla et al, 1984 ), thus inhibited the viral replication. And L protein of an attenuated vaccine strain SAD B19 can bind to a dynein light chain 1 (DLC1) acted as a transcription enhancer ( Bauer et al, 2015 ). Both of them decreased the viral replication and stimulated the viral transcription.…”

Molecular Function Analysis of Rabies Virus RNA Polymerase L Protein by Using an L Gene-Deficient Virus

“…Furthermore, it was previously reported that the balance of transcription and replication in RABVinfected cells is regulated by the M protein (6), suggesting direct or indirect interaction between L and M proteins. Interaction of the RABV L protein with host cellular molecules also plays an important role in the propagation cycle: Bauer et al (7) recently reported that the RABV L protein has a dynein light chain 1 binding motif that mediates the interaction of the L protein with cytoskeletal microtubules and also that the interaction is involved in the efficient primary transcription by RABV.…”

Overexpression of MAP2 and NF-H Associated with Dendritic Pathology in the Spinal Cord of Mice Infected with Rabies Virus

“…This protein is involved in the movement of negative organelles down the length of the microtubules [ 33 ]. Similarly, the rabies viral polymerase L forms a binding complex with dynein light chain 1 (DLC1), which is involved in the linking, modification, and post-translational modification of microtubules and in the regulation of the primary transcription of rabies virus [ 34 ]. This demonstrates that the dynein can be involved in the axonal transport of rabies virus along the length of the microtubules through the neuronal cells [ 33 ].…”

“…A link has been demonstrated between DLC1 and the MAP proteins in the direct binding to tubulin dimers [ 35 ]. This finding suggests that the virus not only uses the microtubule network during entry but also actively manipulates the cytoskeleton and associated proteins to achieve rapid and efficient transport [ 34 ]. It is possible that overexpression of MAP2 and NF-H is induced by DLC1 or by expression of a viral protein, as occurs in the post-translational modification associated with DLC1-polymerase L [ 34 ], DLC8, and P protein [ 33 ], and in the transport mediated by p75NTR [ 32 ].…”