By immunoaffinity column chromatography, we have purified two RNA polymerase complexes, the transcriptase and replicase, from vesicular stomatitis virus-infected baby hamster kidney cells. The transcriptase is a multiprotein complex, containing the virus-encoded RNA polymerase L and P proteins, and two cellular proteins, translation elongation factor-1␣ and heat-shock protein 60. In addition, the complex contains a submolar amount of cellular mRNA cap guanylyltransferase. The replicase, on the other hand, is a complex containing the viral proteins, L, P, and the nucleocapsid (N), but lacking elongation factor-1␣, heat-shock protein 60, and guanylyltransferase. The transcriptase complex synthesizes capped mRNAs and initiates transcription at the first gene (N) start site, whereas the replicase complex initiates RNA synthesis at the precise 3 end of the genome RNA and synthesizes encapsidated replication products in the presence of the N-P complex. We propose that two RNA polymerase complexes that differ in their content of virally and host-encoded proteins are separately responsible for transcription and replication of vesicular stomatitis virus genome RNA. Ahallmark of all nonsegmented negative-strand (ns)RNA viruses (mononegavirales order) such as rabies, measles, Sendai, parainfluenza, Ebola, and many others is that mature virions contain a virally encoded RNA-dependent RNA polymerase (referred to as transcriptase), which transcribes the negative-sense genome RNA into discrete mRNAs on entry into the cell to initiate infection (1, 2). To replicate the genome RNA, the transcriptase is hypothesized to be modified by an unknown mechanism to form a replicase that synthesizes the full-length positive-strand genome RNA (3, 4); the replicase then synthesizes multiple copies of nsRNA using the positive RNA as template. During each step of the replication reaction, both plusand minus-strand RNAs are concomitantly enwrapped by the newly synthesized nucleocapsid (N) protein (5, 6) to form the ribonucleoprotein (RNP) complex. The composition of the replicase and the process of replication remain an enigma.We have been studying vesicular stomatitis virus (VSV) as a prototypic nonsegmented negative-strand RNA virus to probe the structure and function of transcriptase and the putative replicase to delineate the mechanism of transcription and replication of this class of viruses. VSV, like rabies virus, belongs to the rhabdovirus family and contains a single-strand genome RNA of negative polarity (Ϸ11.2 kb long) tightly associated with the N protein and helically packed within a bullet-shaped shell that is surrounded by the host-cell plasma membrane (3, 4). Two membrane proteins, spike glycoprotein (G) and matrix (M) protein, are located outside and inside of the membrane, respectively. Two proteins are associated with the helical RNP, the RNA polymerase large (L) (241 kDa) and phosphoprotein (P) (29 kDa) (7), which together constitute the active transcriptase holoenzyme complex (3, 4). It is generally believed that the L pro...

Several host proteins have been shown to play key roles in the life-cycle of vesicular stomatitis virus (VSV). We have identified an additional host protein, cyclophilin A (CypA), a chaperone protein possessing peptidyl cis-trans prolyl-isomerase activity, as one of the cellular factors required for VSV replication. Inhibition of the enzymatic activity of cellular CypA by cyclosporin A (CsA) or SDZ-211-811 resulted in a drastic inhibition of gene expression by VSV New Jersey (VSV-NJ) serotype, while these drugs had a significantly reduced effect on the genome expression of VSV Indiana (VSV-IND) serotype. Overexpression of a catalytically inactive mutant of CypA resulted in the reduction of VSV-NJ replication, suggesting a requirement for functional CypA for VSV-NJ infection. It was also shown that CypA interacted with the nucleocapsid (N) protein of VSV-NJ and VSV-IND in infected cells and was incorporated into the released virions of both serotypes. VSV-NJ utilized CypA for post-entry intracellular primary transcription, since inhibition of CypA with CsA reduced primary transcription of VSV-NJ by 85-90 %, whereas reduction for VSV-IND was only 10 %. Thus, it seems that cellular CypA binds to the N protein of both serotypes of VSV. However, it performs an obligatory function on the N protein activity of VSV-NJ, while its requirement is significantly less critical for VSV-IND N protein function. The different requirements for CypA by two serologically different viruses belonging to the same family has highlighted the utilization of specific host factors during their evolutionary lineages.

An RNA-dependent RNA polymerase is packaged within the virions of purified vesicular stomatitis virus, a nonsegmented negative-strand RNA virus, which carries out transcription of the genome RNA into mRNAs both in vitro and in vivo. The RNA polymerase is composed of two virally encoded polypeptides: a large protein L (240 kDa) and a phosphoprotein P (29 kDa). Recently, we obtained biologically active L protein from insect cells following infection by a recombinant baculovirus expressing L gene. During purification of the L protein from Sf21 cells, we obtained in addition to an active L fraction an inactive fraction that required uninfected insect cell extract to restore its activity. The cellular factors have now been purified, characterized, and shown to be ␤ and ␥ subunits of the protein synthesis elongation factor EF-1. We also demonstrate that the ␣ subunit of EF-1 remains tightly bound to the L protein in the inactive fraction and ␤␥ subunits associate with the L(␣) complex. Further purification of L(␣) from the inactive fraction revealed that the complex is partially active and is significantly stimulated by the addition of ␤␥ subunits purified from Sf21 cells. A putative inhibitor(s) appears to co-elute in the inactive fraction that blocked the L(␣) activity. The purified virions also package all three subunits of EF-1. These findings have a striking similarity with Q␤ RNA phage, which also associates with the bacterial homologue of EF-1 for its replicase function, implicating a possible evolutionary relationship between these host proteins and the RNA-dependent RNA polymerase of RNA viruses.Vesicular stomatitis virus (VSV), a prototype of nonsegmented negative-strand RNA viruses, has long been a paradigm for studying gene expression of this class of RNA viruses that infect vertebrates, invertebrates, and plants (1). Some of the most common human pathogens that belong to this category are rabies, measles, mumps, and human parainfluenza. A hallmark of all negative strand RNA viruses is the obligate packaging of an RNA-dependent RNA polymerase within the mature virions (2) that transcribes the genome RNA into mRNAs both in vitro and in vivo (3). For VSV, the virion-associated RNA polymerase is generally thought to consist of two virally encoded protein subunits, L (240 kDa) and P (29 kDa), which remain tightly complexed within the virion (3). Studies on the structure and function of VSV RNA polymerase have been greatly aided by the ability to isolate the polymerase subunits from the virions in a relatively pure form (4, 5). Active reconstitution of transcription is achieved by mixing the genome RNA enwrapped with the nucleocapsid protein (N) (referred to as N-RNA template) and purified L and P proteins (4, 5). From a large body of evidence, it appears that the L protein possesses the catalytic activity for RNA synthesis and the P protein is a transcription factor essential for L function (3); no cellular protein(s) has so far been shown to be required for the RNA polymerase activity. Only recently, ...

Phosphorylation by casein kinase II at three specific residues (S-60, T-62, and S-64) within the acidic domain I of the P protein of Indiana serotype vesicular stomatitis virus has been shown to be critical for in vitro transcription activity of the viral RNA polymerase (P-L) complex. To examine the role of phosphorylation of P protein in transcription as well as replication in vivo, we used a panel of mutant P proteins in which the phosphate acceptor sites in domain I were substituted with alanines or other amino acids. Analyses of the alanine-substituted mutant P proteins for the ability to support defective interfering RNA replication in vivo suggest that phosphorylation of these residues does not play a significant role in the replicative function of the P protein since these mutant P proteins supported replication at levels >70% of the wild-type P-protein level. However, the transcription function of most of the mutant proteins in vivo was severely impaired (2 to 10% of the wild-type P-protein level). The level of transcription supported by the mutant P protein (P 60/62/64 ) in which all phosphate acceptor sites have been mutated to alanines was at best 2 to 3% of that of the wild-type P protein. 81% of the wild-type level), substitution with phenylalanine (P FFF ) rendered the protein much less active in transcription (<5%). Substitution with arginine residues led to significantly reduced activity in replication (6%), whereas glutamic acid substituted P protein (P EEE ) supported replication (42%) and transcription (86%) well. In addition, the mutant P proteins that were defective in replication (P RRR ) or transcription (P 60/62/64 ) did not behave as transdominant repressors of replication or transcription when coexpressed with wild-type P protein. From these results, we conclude that phosphorylation of domain I residues plays a major role in in vivo transcription activity of the P protein, whereas in vivo replicative function of the protein does not require phosphorylation. These findings support the contention that different phosphorylated states of the P protein regulate the transcriptase and replicase functions of the polymerase protein, L. Increasing the amount of P 60/62/64 expression in transfected cells did not rescue significant levels of transcription. Substitution with other amino acids at these sites had various effects on replication and transcription. While substitution with threonine residues (P TTT ) had no apparent effect on transcription (113% of the wild-type level) or replication (