The large (L) proteins of non-segmented, negative-strand RNA viruses, a group that includes Ebola and rabies viruses, catalyze RNA-dependent RNA polymerization with viral ribonucleoprotein as template, a noncanonical sequence of capping and methylation reactions, and polyadenylation of viral messages. We have determined by electron cryomicroscopy the structure of the vesicular stomatitis virus (VSV) L protein. The density map, at a resolution of 3.8 Å, has led to an atomic model for nearly all of the 2109-residue polypeptide chain, which comprises three enzymatic domains [RNA-dependent RNA polymerase (RdRp), polyribonucleotidyl transferase (PRNTase), and methyl transferase] and two structural domains. The RdRp resembles the corresponding enzymatic regions of dsRNA virus polymerases and influenza virus polymerase. A loop from the PRNTase (capping) domain projects into the catalytic site of the RdRp, where it appears to have the role of a priming loop and to couple product elongation to large-scale conformational changes in L.

Nonsegmented negative-sense (NNS) RNA viruses cap their mRNA by an unconventional mechanism. Specifically, 5 monophosphate mRNA is transferred to GDP derived from GTP through a reaction that involves a covalent intermediate between the large polymerase protein L and mRNA. This polyribonucleotidyltransferase activity contrasts with all other capping reactions, which are catalyzed by an RNA triphosphatase and guanylyltransferase. In these reactions, a 5 diphosphate mRNA is capped by transfer of GMP via a covalent enzyme-GMP intermediate. RNA guanylyltransferases typically have a KxDG motif in which the lysine forms this covalent intermediate. Consistent with the distinct mechanism of capping employed by NNS RNA viruses, such a motif is absent from L. To determine the residues of L protein required for capping, we reconstituted the capping reaction of the prototype NNS RNA virus, vesicular stomatitis virus, from highly purified components. Using a panel of L proteins with single-amino-acid substitutions to residues universally conserved among NNS RNA virus L proteins, we define a new motif, GxxT[n]HR, present within conserved region V of L protein that is essential for this unconventional mechanism of mRNA cap formation.The 5Ј terminus of eukaryotic mRNA is modified by the addition of a 7 m GpppN cap structure. The cap structure is essential for mRNA stability, mRNA transportation, splicing of pre-mRNAs, and translation (13, 28). Formation of this structure requires a series of enzymatic reactions. An RNA triphosphatase (RTPase) hydrolyzes the 5Ј triphosphate (pppN) end of mRNA to yield a 5Ј diphosphate (ppN). This is capped by an RNA guanylyltransferase (GTase) which transfers Gp derived from GTP to form the cap structure. The cap is subsequently methylated by guanine-N-7 (G-N-7) methyltransferase (MTase) to yield 7 m GpppN, which can be further methylated by ribose-2Ј-O (2Ј-O) MTase to yield 7 m GpppN m (14, 36). Cap formation in nonsegmented negative-strand (NNS) RNA viruses involves a different reaction mechanism. For vesicular stomatitis virus (VSV) (2), spring viremia of carp virus (16), and respiratory syncytial virus (RSV) (5), the underlined phosphates of the 5Ј GpppN triphosphate bridge were shown to be derived from GDP rather than GMP. Recent studies with VSV demonstrated that this reaction does not involve transfer of guanylate onto the mRNA, but rather involves a polyribonucleotidyltransferase activity (29). Here, the mRNA capping reaction proceeds via a covalent intermediate between the 241-kDa viral polymerase protein L and the 5Ј monophosphate mRNA. This monophosphate mRNA is transferred onto GDP derived from GTP to yield the GpppA mRNA cap. Consequently, this mechanism of cap formation is in marked contrast with those catalyzed by conventional GTases. The crystal structures of representative GTases have been solved and their reaction mechanisms studied in biochemical detail (17). GTases typically contain a KxDG motif, in which the lysine forms the covalent intermediate with GMP, and consistent with...

Nonsegmented negative-strand (NNS) RNA viruses initiate infection by delivering into the host cell a highly specialized RNA synthesis machine comprising the genomic RNA completely encapsidated by the viral nucleocapsid protein and associated with the viral polymerase. The catalytic core of this protein-RNA complex is a 250-kDa multifunctional large (L) polymerase protein that contains enzymatic activities for nucleotide polymerization as well as for each step of mRNA cap formation. Working with vesicular stomatitis virus (VSV), a prototype of NNS RNA viruses, we used negative stain electron microscopy (EM) to obtain a molecular view of L, alone and in complex with the viral phosphoprotein (P) cofactor. EM analysis, combined with proteolytic digestion and deletion mapping, revealed the organization of L into a ring domain containing the RNA polymerase and an appendage of three globular domains containing the cap-forming activities. The capping enzyme maps to a globular domain, which is juxtaposed to the ring, and the cap methyltransferase maps to a more distal and flexibly connected globule. Upon P binding, L undergoes a significant rearrangement that may reflect an optimal positioning of its functional domains for transcription. The structural map of L provides new insights into the interrelationship of its various domains, and their rearrangement on P binding that is likely important for RNA synthesis. Because the arrangement of conserved regions involved in catalysis is homologous, the structural insights obtained for VSV L likely extend to all NNS RNA viruses.L protein | structure and function | viral replication N onsegmented negative-strand (NNS) RNA viruses initiate infection by delivering into the host cell a highly specialized RNA synthesis machine. This machine consists of a ribonucleoprotein complex (RNP) comprising the genomic RNA completely coated by the viral nucleocapsid (N) protein and associated with the RNA-dependent RNA polymerase (RdRP) complex (1). The catalytic core of the RNP is a single large (L) 250-kDa protein that contains enzymatic activities for nucleotide polymerization, mRNA cap addition, cap methylation, and polyadenylation (2-6). The location of all of the enzymatic activities necessary for transcription within a single polypeptide chain contrasts with the arrangement exhibited by the host cell and many other viruses, in which the different activities reside within separate proteins that assemble into a larger transcription complex (7).Our understanding of the different activities of NNS RNA virus L proteins has been largely shaped by studies of vesicular stomatitis virus (VSV) because it is the only member of this order of viruses for which robust transcription can be reconstituted in vitro. The enzymatic activities of VSV L have been mapped at the single amino acid level. Within the primary sequence of L are six conserved regions (CRs I-VI) shared among all NNS RNA virus L proteins (8). The RdRP activity maps to CRIII (3), and it is also required for polyadenylation, which occur...

Positive-strand and double-strand RNA viruses typically compartmentalize their replication machinery in infected cells. This is thought to shield viral RNA from detection by innate immune sensors and favor RNA synthesis. The picture for the non-segmented negative-strand (NNS) RNA viruses, however, is less clear. Working with vesicular stomatitis virus (VSV), a prototype of the NNS RNA viruses, we examined the location of the viral replication machinery and RNA synthesis in cells. By short-term labeling of viral RNA with 5′-bromouridine 5′-triphosphate (BrUTP), we demonstrate that primary mRNA synthesis occurs throughout the host cell cytoplasm. Protein synthesis results in the formation of inclusions that contain the viral RNA synthesis machinery and become the predominant sites of mRNA synthesis in the cell. Disruption of the microtubule network by treatment of cells with nocodazole leads to the accumulation of viral mRNA in discrete structures that decorate the surface of the inclusions. By pulse-chase analysis of the mRNA, we find that viral transcripts synthesized at the inclusions are transported away from the inclusions in a microtubule-dependent manner. Metabolic labeling of viral proteins revealed that inhibiting this transport step diminished the rate of translation. Collectively those data suggest that microtubule-dependent transport of viral mRNAs from inclusions facilitates their translation. Our experiments also show that during a VSV infection, protein synthesis is required to redirect viral RNA synthesis to intracytoplasmic inclusions. As viral RNA synthesis is initially unrestricted, we speculate that its subsequent confinement to inclusions might reflect a cellular response to infection.

Mammalian DNA polymerase ␦ (pol ␦), a key enzyme of chromosomal DNA replication, consists of four subunits as follows: the catalytic subunit; p125, which is tightly associated with the p50 subunit; p68, a proliferating cell nuclear antigen (PCNA)-binding protein; and a fourth subunit, p12. In this study, the functional roles of the p12 subunit of pol ␦ were studied. The inter-subunit interactions of the p12 subunit were determined by yeast two-hybrid assays and by pulldown assays. These assays revealed that p12 interacts with p125 as well as p50. This dual interaction of p12 suggests that it may serve to stabilize the p125-p50 interaction. p12 was shown to be a novel PCNA-binding protein. This was confirmed by identification of a PCNA-binding motif at its N terminus by binding assays and by site-directed mutagenesis. The activities and reaction products of recombinant pol ␦ containing a p12 mutant defective in PCNA binding, as well as purified recombinant pol ␦ and its subassemblies, were analyzed. Our results indicate that p12 contributes to PCNA-dependent pol ␦ activity, i.e. the p12-PCNA interaction is functional. Our data indicate that both p12 and p68 are required for optimal pol ␦ activity. This supports the hypothesis that the interaction between pol ␦ and PCNA is a divalent one that involves p12 and p68. We propose a model in which pol ␦ interacts with PCNA via at least two of its subunits, and one in which p12 could play a role in stabilizing the overall pol ␦-PCNA complex as well as pol ␦ itself.Chromosomal DNA replication in eukaryotic cells requires the following three distinct DNA polymerases: polymerase ␣, polymerase ␦ (pol ␦), 3 and polymerase ⑀. DNA pol ␦ is the key enzyme that is thought to play a central role in the elongation of both the leading and the lagging strands of DNA and the maturation of Okazaki fragments (1-3). DNA pol ␦ was originally identified as a new type of DNA polymerase possessing an intrinsic 3Ј-5Ј-exonuclease activity (4). Mammalian pol ␦ holoenzyme consists of the p125 catalytic subunit (which harbors both 5Ј-3Ј DNA polymerase and 3Ј-5Ј-exonuclease activities) and a tightly associated second subunit p50; this core is associated with two other subunits, p68 and p12, that are also referred to as the third and fourth subunits (5-9). The function of pol ␦ as a chromosomal DNA polymerase is dependent on its association with PCNA, which functions as a molecular sliding clamp (10, 11). The third subunits of pol ␦ in both mammalian (p68/p66) and in yeast cells (Cdc27 in Schizosaccharomyces pombe and pol 32 in Saccharomyces cerevisiae) harbor a PCNA-binding motif, and it has been shown that this provides a PCNA interaction site for pol ␦ (12-16). However, the exact nature of the subunit contacts of mammalian pol ␦ with PCNA has yet to be clarified; we (17-20) and others (8) have reported that human pol ␦ p125 binds to PCNA, although other reports have come to the opposite conclusion (14, 21). There is also a report that the p50 subunit of mammalian pol ␦ binds to PCNA (21).The fou...