LC8 Binding Site Research Articles

Multivalency in 53BP1-LC8 interactions suggest new binding mode in LC8 interactions.

Tumor protein p53 binding protein (53BP1) is a 1972 residue long protein with a critical role in DNA damage repair. 53BP1 forms large, phase-separated nuclear bodies at sites of DNA double strand breaks. An oligomerization domain and two LC8 binding sites have been shown to be crucial for robust recruitment of 53BP1 to sites of DNA damage. LC8 is a small, dimeric hub protein which is known to dimerize intrinsically disordered client proteins. We recently discovered a third LC8 binding site in 53BP1 using a free online algorithm, LC8Pred, and this new site may alter interpretations of previous experiments in which 53BP1 was mutated to eliminate LC8 binding. We provide a biophysical characterization of the LC8 binding domain and oligomerization domains of 53BP1, focusing on differences in LC8 binding in the presence and absence of the oligomerization domain. We also find that the oligomerization domain of 53BP1 is trimeric—a first for LC8 binding clients. Isothermal titration calorimetry shows divergent binding mechanisms for different LC8 binding sites in 53BP1 in the presence and absence of the oligomerization domain, highlighting the importance of multivalency in 53BP1-LC8 interactions.

Multivalent binding of the hub protein LC8 at a newly discovered site in 53BP1

Tumor suppressor p53 binding protein 1 (53BP1) is a scaffolding protein involved in poly-ADP ribose polymerase inhibitor hypersensitivity in BRCA1-negative cancers. 53BP1 plays a critical role in the DNA damage response and relies on its oligomerization to create foci that promote repair of DNA double-strand breaks. Previous work shows that mutation of either the oligomerization domain or the dynein light chain 8 (LC8)-binding sites of 53BP1 results in reduced accumulation of 53BP1 at double-strand breaks. Mutation of both abolishes focus formation almost completely. Here, we show that, contrary to current literature, 53BP1 contains three LC8-binding sites, all of which are conserved in mammals. Isothermal titration calorimetry measuring binding affinity of 53BP1 variants with LC8 shows that the third LC8-binding site has a high affinity and can bind LC8 in the absence of other sites. NMR titrations confirm that the third site binds LC8 even in variants that lack the other LC8-binding sites. The third site is the closest to the oligomerization domain of 53BP1, and its discovery would challenge our current understanding of the role of LC8 in 53BP1 function.

LC8 drives higher order oligomerization in 53BP1

53BP1 is a scaffold protein involved in Poly-ADP Ribose Polymerase inhibitor (PARPi) hypersensitivity in BRCA1 negative cancers. 53BP1 foci accumulate at DNA double strand breaks and protect them from end resection, favoring nonhomologous end joining for repair. 53BP1 has an oligomerization domain and two LC8 binding sites that are necessary for ideal focus formation. LC8 is a small dimeric protein that binds over 100 client proteins at intrinsically disordered regions (IDRs), leading to their dimerization.

A Novel Interaction Reveals LC8 as a Potential Hijacking Point for a Respiratory Virus

Lower respiratory tract infections (LRTIs) are a major cause of death among immunocompromised patients. Human parainfluenza viruses (hPIVs) are non‐segmented, negative‐stranded RNA viruses that frequently give rise to these infections. In order to transcribe genes or replicate their genomes, these viruses encode three major genes: a nucleocapsid (N), a phosphoprotein (P) and a large RNA‐dependent RNA polymerase (L). P is indispensable for transcription and replication as it positions L at the replication/transcriptional start site and recruits accessory proteins. Phosphoprotein structure has been characterized in related viruses such as measles and mumps, where P has been shown to be an oligomer, featuring an intrinsically disordered region as well as a flexible multimerization domain. However, P has not been well studied in the hPIVs and its oligomerization state and dynamics remain unclear. In this study, we show that the phosphoprotein of hPIV type 3 (hPIV3‐P) interacts with host ‘hub’ protein LC8 resulting in structural changes. LC8 is known to bind over a hundred different proteins in eukaryotic cells, facilitating a functional structure. LC8 preferentially binds to intrinsically disordered regions within binding partners which ultimately results in self association, support, oligomerization or other structural rearrangements required for proper function.We have identified two conserved potential LC8 binding sites within hPIV3‐P using the LC8Pred algorithm. We recombinantly expressed and purified a construct featuring the coiled coil domain of the protein and the LC8 binding site in closest proximity to it. LC8 binding was confirmed via pulldown assays, isothermal titration calorimetry (ITC) and size exclusion chromatography (SEC). Further, we characterized the stability of the coiled coil domain using circular dichroism spectroscopy (CD) and the oligomerization state of the protein bound and unbound to LC8 using analytical ultracentrifugation (AUC). Our results show a well‐defined novel interaction that might be indispensable for phosphoprotein activity and overall viral viability. This adds to the evidence that LC8 is a common target for viral hijacking due to its structural properties.Support or Funding InformationThis work is funded by NSF MCB 1617019

The dynein light chain 8 (LC8) binds predominantly “in-register” to a multivalent intrinsically disordered partner

Dynein light chain 8 (LC8) interacts with intrinsically disordered proteins (IDPs) and influences a wide range of biological processes. It is becoming apparent that among the numerous IDPs that interact with LC8, many contain multiple LC8-binding sites. Although it is established that LC8 forms parallel IDP duplexes with some partners, such as nucleoporin Nup159 and dynein intermediate chain, the molecular details of these interactions and LC8's interactions with other diverse partners remain largely uncharacterized. LC8 dimers could bind in either a paired "in-register" or a heterogeneous off-register manner to any of the available sites on a multivalent partner. Here, using NMR chemical shift perturbation, analytical ultracentrifugation, and native electrospray ionization MS, we show that LC8 forms a predominantly in-register complex when bound to an IDP domain of the multivalent regulatory protein ASCIZ. Using saturation transfer difference NMR, we demonstrate that at substoichiometric LC8 concentrations, the IDP domain preferentially binds to one of the three LC8 recognition motifs. Further, the differential dynamic behavior for the three sites and the size of the fully bound complex confirmed an in-register complex. Dynamics measurements also revealed that coupling between sites depends on the linker length separating these sites. These results identify linker length and motif specificity as drivers of in-register binding in the multivalent LC8-IDP complex assembly and the degree of compositional and conformational heterogeneity as a promising emerging mechanism for tuning of binding and regulation.

LC8 Motif Recognition: Insights from the LC8-Chica Complex

LC8 is a dimeric hub protein involved in a large number of interactions central to cell function. It binds short linear motifs - usually containing a Thr-Gln-Thr (TQT) triplet - in intrinsically disordered regions of its binding partners, some of which have several LC8 recognition motifs in tandem. Hallmarks of the motif are a high variability of sequence and of LC8 binding affinity. Human Chica, a spindle adaptor protein, contains a 70-residue segment with four putative recognition motifs but only three reported LC8 binding sites. Here, NMR-derived secondary chemical shifts and relaxation properties show that the Chica LC8 binding domain is essentially disordered with a dynamically restricted segment in one linker between motifs. Calorimetry of LC8 binding to synthetic motif-mimicking peptides shows that the first motif dominates LC8 recruitment. Crystal structures of LC8 bound to each of two motif peptides show highly ordered and invariant TQT-LC8 interactions and more flexible and conformationally variable non-TQT-LC8 interactions. These data add a new and complex LC8 binding partner to the LC8 knowledgebase and provide new insights into the determinants of motif specificity. We propose that LC8 recognition is based on inflexible interactions between LC8 and TQT residues that act as an anchor, coupled with inherent flexibility of interactions between LC8 and non-TQT residues. The ‘anchored flexibility’ model combines highly rigid as well as highly flexible regions of the same binding groove on the surface of LC8.

Dynein Light Chain LC8 Is Required for RNA Polymerase I-Mediated Transcription in Trypanosoma brucei, Facilitating Assembly and Promoter Binding of Class I Transcription Factor A.

Dynein light chain LC8 is highly conserved among eukaryotes and has both dynein-dependent and dynein-independent functions. Interestingly, LC8 was identified as a subunit of the class I transcription factor A (CITFA), which is essential for transcription by RNA polymerase I (Pol I) in the parasite Trypanosoma brucei. Given that LC8 has never been identified with a basal transcription factor and that T. brucei relies on RNA Pol I for expressing the variant surface glycoprotein (VSG), the key protein in antigenic variation, we investigated the CITFA-specific role of LC8. Depletion of LC8 from mammalian-infective bloodstream trypanosomes affected cell cycle progression, reduced the abundances of rRNA and VSG mRNA, and resulted in rapid cell death. Sedimentation analysis, coimmunoprecipitation of recombinant proteins, and bioinformatic analysis revealed an LC8 binding site near the N terminus of the subunit CITFA2. Mutation of this site prevented the formation of a CITFA2-LC8 heterotetramer and, in vivo, was lethal, affecting assembly of a functional CITFA complex. Gel shift assays and UV cross-linking experiments identified CITFA2 as a promoter-binding CITFA subunit. Accordingly, silencing of LC8 or CITFA2 resulted in a loss of CITFA from RNA Pol I promoters. Hence, we discovered an LC8 interaction that, unprecedentedly, has a basal function in transcription.

The Mechanism of Dynein Light Chain LC8-mediated Oligomerization of the Ana2 Centriole Duplication Factor

Centrioles play a key role in nucleating polarized microtubule networks. In actively dividing cells, centrioles establish the bipolar mitotic spindle and are essential for genomic stability. Drosophila anastral spindle-2 (Ana2) is a conserved centriole duplication factor. Although recent work has demonstrated that an Ana2-dynein light chain (LC8) centriolar complex is critical for proper spindle positioning in neuroblasts, how Ana2 and LC8 interact is yet to be established. Here we examine the Ana2-LC8 interaction and map two LC8-binding sites within the central region of Ana2, Ana2M (residues 156-251). Ana2 LC8-binding site 1 contains a signature TQT motif and robustly binds LC8 (KD of 1.1 μm), whereas site 2 contains a TQC motif and binds LC8 with lower affinity (KD of 13 μm). Both LC8-binding sites flank a predicted ~34-residue α-helix. We present two independent atomic structures of LC8 dimers in complex with Ana2 LC8-binding site 1 and site 2 peptides. The Ana2 peptides form β-strands that extend a central composite LC8 β-sandwich. LC8 recognizes the signature TQT motif in the first LC8 binding site of Ana2, forming extensive van der Waals contacts and hydrogen bonding with the peptide, whereas the Ana2 site 2 TQC motif forms a uniquely extended β-strand, not observed in other dynein light chain-target complexes. Size exclusion chromatography coupled with multiangle static light scattering demonstrates that LC8 dimers bind Ana2M sites and induce Ana2 tetramerization, yielding an Ana2M4-LC88 complex. LC8-mediated Ana2 oligomerization probably enhances Ana2 avidity for centriole-binding factors and may bridge multiple factors as required during spindle positioning and centriole biogenesis.

Structural Features of LC8-Induced Self-Association of Swallow

Cell functions depend on the collective activity of protein networks within which a few proteins, called hubs, participate in a large number of interactions. Dynein light chain LC8, first discovered as a subunit of the motor protein dynein, is considered to have a role broader than that of dynein, and its participation in diverse systems fits the description of a hub. Among its partners is Swallow with which LC8 is essential for proper localization of bicoid mRNA at the anterior cortex of Drosophila oocytes. Why LC8 is essential in this process is not clear, but emerging evidence suggests that LC8 functions by promoting self-association and/or structural organization of its diverse binding partners. This work addresses the energetics and structural features of LC8-induced Swallow self-association distant from LC8 binding. Mutational design based on a hypothetical helical wheel, intermonomer nuclear Overhauser effects assigned to residues expected at interface positions, and circular dichroism spectral characteristics indicate that the LC8-promoted dimer of Swallow is a coiled coil. Secondary chemical shifts and (15)N backbone relaxation identify the boundaries and distinguishing structural features of the coiled coil. Thermodynamic analysis of Swallow polypeptides designed to decouple self-association from LC8 binding reveals that the higher binding affinity of the engineered bivalent Swallow is of purely entropic origin and that the linker separating the coiled coil from the LC8 binding site remains disordered. We speculate that the LC8-promoted coiled coil is critical for bicoid mRNA localization because it favors structural organization of Swallow, which except for the central LC8-promoted coiled coil is primarily disordered.

The versatile molecular complex component LC8 promotes several distinct steps of flagellar assembly

LC8 is present in various molecular complexes. However, its role in these complexes remains unclear. We discovered that although LC8 is a subunit of the radial spoke (RS) complex in Chlamydomonas flagella, it was undetectable in the RS precursor that is converted into the mature RS at the tip of elongating axonemes. Interestingly, LC8 dimers bound in tandem to the N-terminal region of a spoke phosphoprotein, RS protein 3 (RSP3), that docks RSs to axonemes. LC8 enhanced the binding of RSP3 N-terminal fragments to purified axonemes. Likewise, the N-terminal fragments extracted from axonemes contained LC8 and putative spoke-docking proteins. Lastly, perturbations of RSP3's LC8-binding sites resulted in asynchronous flagella with hypophosphorylated RSP3 and defective associations between LC8, RSs, and axonemes. We propose that at the tip of flagella, an array of LC8 dimers binds to RSP3 in RS precursors, triggering phosphorylation, stalk base formation, and axoneme targeting. These multiple effects shed new light on fundamental questions about LC8-containing complexes and axoneme assembly.

Multivalency in the Assembly of Intrinsically Disordered Dynein Intermediate Chain

Dynein light chains are thought to increase binding efficiency of dynein intermediate chain to both dynein heavy chain and dynactin, but their exact role is not clear. Isothermal titration calorimetry and x-ray crystallography reported herein indicate that multivalency effects underlie efficient dynein assembly and regulation. For a ternary complex of a 60-amino acid segment of dynein intermediate chain (IC) bound to two homodimeric dynein light chains Tctex1 and LC8, there is a 50-fold affinity enhancement for the second light chain binding. For a designed IC construct containing two LC8 sites, observed the 1000-fold enhancement reflects a remarkably pure entropic chelate effect of a magnitude commensurate with theoretical predictions. The lower enhancement in wild-type IC is attributed to unfavorable free energy changes associated with incremental interactions of IC with Tctex1. Our results show assembled dynein IC as an elongated, flexible polybivalent duplex, and suggest that polybivalency is an important general mechanism for constructing stable yet reversible and functionally versatile complexes.

Interaction with LC8 Is Required for Pak1 Nuclear Import and Is Indispensable for Zebrafish Development

Pak1 (p21 activated kinase 1) is a serine/threonine kinase implicated in regulation of cell motility and survival and in malignant transformation of mammary epithelial cells. In addition, the dynein light chain, LC8, has been described to cooperate with Pak1 in malignant transformation of breast cancer cells. Pak1 itself may aid breast cancer development by phosphorylating nuclear proteins, including estrogen receptor alpha. Recently, we showed that the LC8 binding site on Pak1 is adjacent to the nuclear localization sequence (NLS) required for Pak1 nuclear import. Here, we demonstrate that the LC8-Pak1 interaction is necessary for epidermal growth factor (EGF)-induced nuclear import of Pak1 in MCF-7 cells, and that this event is contingent upon LC8-mediated Pak1 dimerization. In contrast, Pak2, which lacks an LC8 binding site but contains a nuclear localization sequence identical to that in Pak1, remains cytoplasmic upon EGF stimulation of MCF-7 cells. Furthermore, we show that severe developmental defects in zebrafish embryos caused by morpholino injections targeting Pak are partially rescued by co-injection of wild-type human Pak1, but not by co-injection of mutant Pak1 mRNA disrupting either the LC8 binding or the NLS site. Collectively, these results suggest that LC8 facilitates nuclear import of Pak1 and that this function is indispensable during vertebrate development.

Evaluation of an LC8-Binding Peptide for the Attachment of Artificial Cargo to Dynein

The limited cytoplasmic mobility of nonviral gene carriers is likely to contribute to their low transfection efficiency. This limitation could be overcome by mimicking the viral strategy of recruiting the dynein motor complex for efficient transport toward the host cell nucleus. A promising approach for attaching artificial cargo to dynein is through an adaptor peptide that binds the 8 kDa light chain (LC8) found in the cargo-binding region of the dynein complex. Several viral proteins that bind LC8 have in common an LC8-binding motif defined by (K/R)XTQT. Short peptides containing this motif have also been shown to bind recombinant LC8 in vitro. However, since the majority of intracellular LC8 exists outside of the dynein complex, it remains unclear whether peptides displaying this LC8-binding motif can access and bind to dynein-associated LC8. In this study, we employed biochemical analysis to investigate the feasibility of attaching artificial cargo to the dynein motor complex using a peptide displaying the well-characterized LC8-binding motif. We report that free intracellular LC8 bound specifically to an LC8-binding (TQT) peptide and not to a control peptide with a mutated LC8-binding motif. However, a similar binding interaction between the TQT peptide and intracellular dynein was not detected. To determine whether dynein binding of the TQT peptide was prevented by competition with free intracellular LC8 or due to the inability of the peptide to access its LC8 binding site in the dynein complex, the TQT peptide was evaluated for its ability to bind either purified LC8 or purified dynein. Our results demonstrate that, while the TQT peptide readily binds free LC8, it cannot bind to dynein-associated LC8. The results emphasize the need to identify functional dynein-binding peptides and highlight the importance of designing peptides that bind to the intact dynein motor complex.

Comparative pathogenesis of the SAD-L16 strain of rabies virus and a mutant modifying the dynein light chain binding site of the rabies virus phosphoprotein in young mice

Recent reports have suggested that rabies virus phosphoprotein (P) interaction with dynein minus-end-directed microtubule motor proteins may be of fundamental importance in the axonal transport of rabies virus. A deletion of 11 amino acids was introduced into recombinant rabies virus SAD-L16 (L16) that modified the dynein light chain (LC8) binding site of the rabies virus P, producing mutant L-ΔP11. This mutant is a useful tool for determining the role of P–LC8 interaction in viral spread and pathogenesis. Seven-day-old ICR mice were inoculated into a hindlimb thigh muscle with L16 or L-ΔP11. Histopathological and immunohistochemical analyses of their brains were performed at serial time points in order to determine the pattern of viral spread. L16 spread to the brain and caused a severe encephalitis with apoptotic neuronal changes. L-ΔP11 infected specific brain areas (brainstem and hippocampus) 1–2 days later than L16 and involved a smaller number of neurons in some brain regions. However, the neuronal apoptotic changes produced by both viruses were similar in most brain regions. Following peripheral inoculation, deletions modifying the LC8 binding site had an effect on delaying viral spread, but did not significantly alter the pattern of rabies virus encephalitis. The precise role of the rabies virus P–dynein interaction in the axonal transport of rabies virus, particularly the importance of this interaction during natural infection, merits further study.

The Intermediate Chain of Cytoplasmic Dynein Is Partially Disordered and Gains Structure upon Binding to Light-Chain LC8

The N-terminal domain of dynein intermediate chain, IC(1-289), is highly disordered, but upon binding to dynein light-chain LC8, it undergoes a significant conformational change to a more ordered structure. Using circular dichroism and fluorescence spectroscopy, we demonstrate that the change in conformation is due to an increase in the helical structure and to enhanced compactness in the environment of tryptophan 161. An increase in helical structure and compactness is also observed with trimethylamine-N-oxide (TMAO), a naturally occurring osmolyte used here as a probe to identify regions with a propensity for induced folding. Global protection of IC(1-289) from protease digestion upon LC8 binding was localized to a segment that includes residues downstream of the LC8-binding site. Several smaller constructs of IC(1-289) containing the LC8-binding site and one of the predicted helix or coiled-coil segments were made. IC(1-143) shows no increase in helical structure upon binding, while IC(114-260) shows an increase in helical structure similar to what is observed with IC(1-289). Binding of IC(114-260) to LC8 was monitored by fluorescence and native gel electrophoresis and shows saturation of binding, a stoichiometry of 1:1, and moderate binding affinity. The induced folding of IC(1-289) upon LC8 binding suggests that LC8 could act through the intermediate chain to facilitate dynein assembly or regulate cargo-binding interactions.

Proteomic identification of brain proteins that interact with dynein light chain LC8.

Cytoplasmic dynein is a large minus end-directed microtubule motor that translocates cargos towards the minus end of microtubules. Light chain 8 of the dynein machinery (LC8) has been reported to interact with a large variety of proteins that possess K/RSTQT or GIQVD motifs in their sequence, hence permitting their transport in a retrograde manner. Yeast two-hybrid analysis has revealed that in brain, LC8 associates directly with several proteins such as neuronal nitric oxide synthase, guanylate kinase domain-associated protein and gephyrin. In this work, we report the identification of over 40 polypeptides, by means of a proteomic approach, that interact with LC8 either directly or indirectly. Many of the neuronal proteins that we identified cluster at the post-synaptic terminal, and some of them such as phosphofructokinase, lactate dehydrogenase or aldolase are directly involved in glutamate metabolism. Other pool of proteins identified displayed the LC8 consensus binding motif. Finally, recombinant LC8 was produced and a library of overlapping dodecapeptides (pepscan) was employed to map the LC8 binding site of some of the proteins that were previously identified using the proteomic approach, hence confirming binding to the consensus binding sites.

Interactions of LC8 with N-terminal segments of the intermediate chain of cytoplasmic dynein.

LC8, a highly conserved 10-kDa light chain, and IC74, a 74-kDa intermediate chain, are presumed to promote the assembly of the cytoplasmic dynein motor protein complex and to be engaged in the controlled binding and release of cargo. The interactions of LC8 from Drosophila melanogaster with constructs of IC74 were characterized in vitro by affinity methods, limited proteolysis, and circular dichroism spectroscopy. Previously, we have performed limited proteolysis on the N-terminal domain of IC74, IC(1-289), when free and when bound to dynein light chains LC8 and Tctex-1[1]. We have also shown that upon addition of LC8, IC(1-289) undergoes a significant conformational change from a largely unfolded to a more ordered structure. The purpose of the work presented here is to determine whether residues 1-30 in IC74, predicted to be in a coiled coil, are involved in the stabilization of the protein upon binding to LC8. Constructs of IC74, IC(1-143), and IC(30-143) that include the LC8 binding site but with and without the first 30 residues were prepared, and their binding and protection patterns were compared to our previous results for IC(1-289). The results suggest that coiled coil residues 1-30 are not responsible for the increase in structure we observe when IC(1-289) binds to LC8.

The Roadblock Light Chain Binds a Novel Region of the Cytoplasmic Dynein Intermediate Chain

Cytoplasmic dynein is the major minus-end directed microtubule-based motor in eukaryotic cells. It is composed of a number of different subunits including three light chain families: Tctex1, LC8, and roadblock. The incorporation of the roadblock light chains into the cytoplasmic dynein complex had not been determined. There are two roadblock genes in mammals, ROBL-1 and ROBL-2. We find that both members of the roadblock family bind directly to all of the intermediate chain isoforms of mammalian cytoplasmic dynein. This was determined with three complementary approaches. A yeast two-hybrid assay demonstrated that both roadblock light chains interact with intermediate chain isoforms from the IC74-1 and IC74-2 genes in vivo. This was confirmed in vitro with both a solid phase blot overlay assay and a solution-binding assay. The roadblock-binding domain on the intermediate chain was mapped to an approximately 72 residue region. The binding domain is downstream of each of the two alternative splice sites in the intermediate chains. This location is consistent with the finding that both roadblock-1 and roadblock-2 show no binding specificity for a single IC74-1 or IC74-2 intermediate chain isoform. In addition, this roadblock-binding domain is significantly downstream from both the Tctex1- and LC8-binding sites, supporting the hypothesis that multiple light chain family members can bind to the same intermediate chain.

Extensive attenuation of rabies virus by simultaneously modifying the dynein light chain binding site in the P protein and replacing Arg333 in the G protein.

Rabies virus (RV) is a highly neurotropic virus that migrates from the portal of entry to the central nervous system (CNS). The cytoplasmic dynein light chain (LC8), which is involved in a variety of intracellular motile events, was shown to interact with RV phosphoprotein (P). In order to determine the functional significance of this interaction, P residues 143 to 149 or 139 to 149 encompassing a conserved LC8-interacting motif (K/RXTQT) were deleted from recombinant viruses SAD-L16 and SAD-D29. These viruses are identical except for a replacement of the arginine at position 333 (R333) of the RV glycoprotein by an aspartic acid in SAD-D29. SAD-L16 virus is fully pathogenic for mice, whereas SAD-D29 is nonpathogenic for adult mice but retained pathogenicity for suckling mice. The deletions introduced into the LC8 binding site abolished the P-LC8 interaction and blocked LC8 incorporation into virions. All the mutants propagated in cell culture as efficiently as the parent strains. The pathogenicity of the mutants was then compared with that of the parent viruses by inoculating suckling mice. SAD-L16 derivatives were as pathogenic as their parent virus after intramuscular inoculation, indicating that LC8 is dispensable for the spread of a pathogenic RV from a peripheral site to the CNS. In contrast, SAD-D29-derived deletion mutants were attenuated by as much as 30-fold after intramuscular inoculation but remained as pathogenic as the parent virus when inoculated directly into the brain. This remarkable attenuation after intramuscular but not after intracranial inoculation suggested that abolishing the P-LC8 interaction reduces the efficiency of peripheral spread of the more attenuated SAD-D29 strain. These results demonstrate that elimination of the LC8 ligand and simultaneous substitution of R333 considerably attenuate RV pathogenicity and may be helpful in designing and developing highly safe live-RV-based vaccines.

Molecular basis for the interaction between rabies virus phosphoprotein P and the dynein light chain LC8: dissociation of dynein-binding properties and transcriptional functionality of P.

The lyssavirus phosphoprotein P is a co-factor of the viral RNA polymerase and plays a central role in virus transcription and replication. It has been shown previously that P interacts with the dynein light chain LC8, which is involved in minus end-directed movement of organelles along microtubules. Co-immunoprecipitation experiments and the two-hybrid system were used to map the LC8-binding site to the sequence (139)RSSEDKSTQTTGR(151). Site-directed mutagenesis of residues D(143) and Q(147) to an A residue abolished binding to LC8. The P-LC8 association is not required for virus transcription, since the double mutant was not affected in its transcription ability in a minigenome assay. Based on the crystal structure of LC8 bound to a peptide from neuronal nitric oxide synthase, a model for the complex between the peptide spanning residues 140-150 of P and LC8 is proposed. This model suggests that P binds LC8 in a manner similar to other LC8 cellular partners.