Interaction of the Rabies Virus P Protein with the LC8 Dynein Light Chain

Abstract

The rabies virus P protein is involved in viral transcription and replication but its precise function is not clear. We investigated the role of P (CVS strain) by searching for cellular partners by using a two-hybrid screening of a PC12 cDNA library. We isolated a cDNA encoding a 10-kDa dynein light chain (LC8). LC8 is a component of cytoplasmic dynein involved in the minus end-directed movement of organelles along microtubules. We confirmed that this molecule interacts with P by coimmunoprecipitation in infected cells and in cells transfected with a plasmid encoding P protein. LC8 was also detected in virus particles. Series of deletions from the N-and C-terminal ends of P protein were used to map the LC8-binding domain to the central part of P (residues 138 to 172). These results are relevant to speculate that dynein may be involved in the axonal transport of rabies virus along microtubules through neuron cells.Rhabdoviruses have a single-stranded negative-sense RNA genome (11 to 15 kb) that is tightly encapsidated by the viral nucleoprotein (N) to form a ribonucleoprotein (RNP). This RNP serves as the template for viral transcription and replication. During transcription, a positive-strand leader RNA and five mRNAs are synthesized. The replication process yields nucleocapsids containing full-length antisense genome RNA which in turn serves as a template for the synthesis of sense genome RNA. The active virus-encoded RNA polymerase complex consists of the large protein (L) and its cofactor, the phosphoprotein (P) (13). The L protein is a multifunctional enzyme and acts as the RNA-dependent RNA polymerase. This polymerase complex carries out all the enzymatic steps of transcription, including the initiation and elongation of transcripts, and cotranscriptional modifications of RNAs, such as capping and polyadenylation (2). The functions of the P protein are not clear. Studies with vesicular stomatitis virus (VSV) have shown that the P protein works as a noncatalytic cofactor of the viral RNA polymerase and as a molecular chaperone helping the viral N protein to bind specifically and correctly to the nascent RNA chain during genome replication. VSV P protein has different phosphorylation states that are believed to bind to the RNP with different affinities and to have different transcription activities (3, 4, 17). The VSV P protein has also been shown to form oligomers, and oligomerization seems to be necessary for binding both to the L protein and to the template (16).By analogy with the VSV P protein, rabies virus P protein is also thought to act as a chaperone and to be a noncatalytic subunit of the viral RNA polymerase. In vitro and in vivo studies have shown that rabies virus P protein forms specific complexes with N and L proteins (10,11,14). We have previously demonstrated the existence of two N protein-binding sites on the P protein: one located between amino acids 69 and 138 and the other in the carboxy-terminal region comprising amino acids 268 to 297 (10). We have shown that the major L binding site re...

Dynein Light Chain Association Sequences Can Facilitate Nuclear Protein Import

“…RPP has also been shown to interact with the DLC LC8 via a DLC-AS containing an archetypal LC8 association motif (KSTQT) in the region 139 -174 using the yeast two-hybrid system (Raux et al, 2000;Poisson et al, 2001). A role in retrograde trafficking of RPP associated with dynein on MT has been suggested (Raux et al, 2000) but evidence for this is lacking, and no direct effect of the DLC-AS on transport into the nucleus has been described.…”

“…Although the sequences responsible for p53, PTHrP, and Rb interaction with MTs/dynein remain largely undefined, short sequences from several other proteins have been identified, using the yeast two-hybrid system and proteomics approaches, which confer interaction with the dynein light chains (DLC), LC8 and Tctex-1. These sequences variously include the consensus motifs KSTQT (single letter amino acid code) and GIQVD for LC8, as well as less well-defined motifs for Tctex-1 interaction, including SKCSR (Raux et al, 2000;Alonso et al, 2001;Poisson et al, 2001;RodriguezCrespo et al, 2001;Mueller et al, 2002;Martinez-Moreno et al, 2003;Sugai et al, 2003;Lo et al, 2005). DLC-ASs have been suggested to mediate cargo association with dynein motor complexes to effect nuclear targeting, so that they could potentially be exploited for delivery of therapeutics to This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E07-01-0030) on June 13, 2007. cell nuclei (Cohen et al, 2005;Mastrobattista et al, 2006a,b); however, this has never been directly demonstrated.…”

“…RPP has also been shown to interact with the DLC LC8 via a DLC-AS containing an archetypal LC8 association motif (KSTQT) in the region 139 -174 using the yeast two-hybrid system (Raux et al, 2000;Poisson et al, 2001). A role in retrograde trafficking of RPP associated with dynein on MT has been suggested (Raux et al, 2000) but evidence for this is lacking, and no direct effect of the DLC-AS on transport into the nucleus has been described.We generated the coding sequences for RPP residues 139-174 (DLC-AS-containing region), 139-297 (DLC-AS-and NLS-containing regions) and 174-297 (NLS-containing region) by PCR and cloned them downstream of GFP in the mammalian expression vector pEGFP-C1 (Figure 1). The constructs were then transfected into COS-7 cells and analyzed by CLSM.…”

“…RPP's nucleocytoplasmic distribution is determined by an NLS and a nuclear export signal (NES). Although a DLC-AS region is present proximal to the NLS-containing domain (residues 139-174 and 174-297, respectively; see Figure 1; Raux et al, 2000;Poisson et al, 2001;Pasdeloup et al, 2005), its role in RPP subcellular localization is unclear. We find here that the DLC-AS, though having no intrinsic NLS activity, can synergize with the NLS to enhance nuclear accumulation both in situ in RPP and when expressed in fusion proteins with heterologous NLSs.…”

Perspectives in Diagnosis and Treatment of Rabies Viral Encephalitis: Insights from Pathogenesis

“…Minor variations in G protein such as substitution of arginine at position 333 affects neuroinvasiveness by use of different neuronal pathways [68]. Interaction between the phosphoprotein of rabies virus, and LC8-the cytoplasmic dynein light chain that regulates microtubule-mediated transport-is implicated in viral transport [69][70][71]. However, mutant viruses with deletion in phosphoprotein encompassing LC8-interacting motif retained their neuroivasiveness and virulence [72].…”