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...
Rabies virus P protein is a co-factor of the viral RNA polymerase. It has been shown previously that P mRNA directs the synthesis of four N-terminally truncated P products P2, P3, P4, and P5 due to translational initiation by a leaky scanning mechanism at internal Met codons. Whereas P and P2 are located in the cytoplasm, P3, P4, and P5 are found in the nucleus. Here, we have analyzed the molecular basis of the subcellular localization of these proteins. Using deletion mutants fused to GFP protein, we show the presence of a nuclear localization signal (NLS) in the C-terminal part of P (172-297). This domain contains a short lysine-rich stretch ((211)KKYK(214)) located in close proximity with arginine 260 as revealed by the crystal structure of P. We demonstrate the critical role of lysine 214 and arginine 260 in NLS activity. In the presence of Leptomycin B, P is retained in the nucleus indicating that it contains a CRM1-dependent nuclear export signal (NES). The subcellular distribution of P deletion mutants indicates that the domain responsible for export is the amino-terminal part of the protein. The use of fusion proteins that have amino terminal fragments of P fused to beta-galactosidase containing the NLS of SV40 T antigen allows us to identify a NES between residues 49 and 58. The localization of NLS and NES determines the cellular distribution of the P gene products.
Vesicular stomatitis virus (VSV) is an oncolytic rhabdovirus and its glycoprotein G is widely used to pseudotype other viruses for gene therapy. Low-density lipoprotein receptor (LDL-R) serves as a major entry receptor for VSV. Here we report two crystal structures of VSV G in complex with two distinct cysteine-rich domains (CR2 and CR3) of LDL-R, showing that their binding sites on G are identical. We identify two basic residues on G, which are essential for its interaction with CR2 and CR3. Mutating these residues abolishes VSV infectivity even though VSV can use alternative receptors, indicating that all VSV receptors are members of the LDL-R family. Collectively, our data suggest that VSV G has specifically evolved to interact with receptor CR domains. These structural insights into the interaction between VSV G and host cell receptors provide a basis for the design of recombinant viruses with an altered tropism.
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