ReviewStructural insights into the rhabdovirus transcription/replication complex
Section snippets
The rhabdoviruses
The Rhabdoviridae is a family of enveloped viruses that have a non-segmented genome ((−)RNA) made of a single stranded negative-sense RNA molecule. The Rhabdoviridae family is grouped in the order Mononegavirales (MNV) (the name is a composition of three elements: Mono – single; nega – negative sense; virales – virus) with the Filoviridae (Ebola and Marburg viruses), the Paramyxoviridae (measles, mumps, respiratory syncytial viruses) and the Bornaviridae (Borna disease virus). All these
The viral transcription/replication machinery
The genome of the Rhabdoviridae is of 9–18 kb in length and comprises up to ten genes flanked by untranslated leader (le) and trailer (tr) RNA regions. RAV and VSV genomes contain only five genes that are common to all members of the MNV and encode successively from the 3′ terminus, the nucleoprotein (N), the phosphoprotein (P), the matrix protein (M), the glycoprotein (G) and the large subunit of the RNA-dependent RNA polymerase (L) (Fig. 1A).
The replication cycle of both RAV and VSV occurs
The nucleoprotein and the N–RNA complex
In rhabdovirus NCs, each N molecule binds nine nucleotides (Iseni et al., 1998, Thomas et al., 1985). The number of N molecules present in a virion of VSV (1250 N) (Ge et al., 2010, Thomas et al., 1985) corresponds closely to the theoretical number required for covering the entire genome (VSV: 11161 nt → 1240 N), although the length of the genomic RNA molecule is not an entire multiple of nine nucleotides. By contrast, in Paramyxoviridae each N molecule binds six nucleotides and the length of
The phosphoprotein
Early studies showed that P contains independent functional regions (Das et al., 1997, Takacs et al., 1993) and its hydrodynamic properties suggested that it is a non-globular molecule (Gérard et al., 2007). These characteristic features are explained by the recent findings that P is made of concatenated disordered regions and structured domains. The structure and functions of rhabdovirus phosphoproteins have been reviewed recently (Leyrat et al., 2010, Leyrat et al., 2011b), and therefore,
Complexes between the nucleoprotein and the phosphoprotein
During the replication cycle, P forms two different complexes with N, the N0–P complex and the N–RNA–P complex. These interactions are independent of each other, and the recent structural characterization of these two complexes reveals the molecular origin of the dual behavior of P.
The L subunit of the viral polymerase
The L protein is a 250 kDa multi-enzymatic protein, which catalyzes RNA synthesis as well as mRNA capping, methylation and polyadenylation. Its sequence is well conserved among all non-segmented (−)RNA viruses, and sequence alignments revealed the existence of six conserved regions, numbered I–VI (Poch et al., 1990). The conserved region III contains different motives involved in the RNA-dependent RNA polymerase activity, while the conserved regions V and VI carry out mRNA capping. The capping
The viral particle
Animal rhabdoviruses have an overall bullet shape with one conical end and one flat end, while plant rhabdoviruses have a bacillus shape with two conical ends. Fig. 6A and B shows negative-staining electron micrographs of RAV and VSV particles. Recently, the cryo-electron microscopy (EM) reconstruction of the virion of VSV, revealed the molecular organization of the viral proteins in three concentric layers (Ge et al., 2010). The outer shell is a host cell-derived lipid membrane decorated with
Future work
The recent progress made in the preparation of highly purified components of the rhabdovirus transcription/replication complex and in the characterization of their stoichiometry, size, shape and structure opens new avenues for deciphering the molecular mechanisms of this machinery and the subtleties of their regulations. In the near future, it should be possible to reconstitute a functional system from purified components and to test hypotheses concerning the motion of the polymerase on its
Acknowledgments
Research activities in the authors’ laboratory were supported by grants from the French ANR (ANR-07–001-01 (ANRAGE)), the FINOVI foundation and Lyonbiopôle. Ivan Ivanov was supported by a PhD fellowship from the ILL and Filip Yabukarski was supported by a MENRT fellowship from the French government. We thank Guy Schoehn and Leandro Estrozi for their help in preparing Fig. 2, Fig. 6, our colleagues for extensive discussions and the Partnership for Structural Biology for the excellent structural
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