A Methyltransferase-Defective Vesicular Stomatitis Virus-Based SARS-CoV-2 Vaccine Candidate Provides Complete Protection against SARS-CoV-2 Infection in Hamsters.

The current pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to dramatic economic and health burdens. Although the worldwide SARS-CoV-2 vaccination campaign has begun, exploration of other vaccine candidates is needed due to uncertainties with the current approved vaccines, such as durability of protection, cross-protection against variant strains, and costs of long-term production and storage. In this study, we developed a methyltransferase-defective recombinant vesicular stomatitis virus (mtdVSV)-based SARS-CoV-2 vaccine candidate. We generated mtdVSVs expressing SARS-CoV-2 full-length spike (S) protein, S1, or its receptor-binding domain (RBD). All of these recombinant viruses grew to high titers in mammalian cells despite high attenuation in cell culture. The SARS-CoV-2 S protein and its truncations were highly expressed by the mtdVSV vector. These mtdVSV-based vaccine candidates were completely attenuated in both immunocompetent and immunocompromised mice. Among these constructs, mtdVSV-S induced high levels of SARS-CoV-2-specific neutralizing antibodies (NAbs) and Th1-biased T-cell immune responses in mice. In Syrian golden hamsters, the serum levels of SARS-CoV-2-specific NAbs triggered by mtdVSV-S were higher than the levels of NAbs in convalescent plasma from recovered COVID-19 patients. In addition, hamsters immunized with mtdVSV-S were completely protected against SARS-CoV-2 replication in lung and nasal turbinate tissues, cytokine storm, and lung pathology. Collectively, our data demonstrate that mtdVSV expressing SARS-CoV-2 S protein is a safe and highly efficacious vaccine candidate against SARS-CoV-2 infection. IMPORTANCE Viral mRNA cap methyltransferase (MTase) is essential for mRNA stability, protein translation, and innate immune evasion. Thus, viral mRNA cap MTase activity is an excellent target for development of live attenuated or live vectored vaccine candidates. Here, we developed a panel of MTase-defective recombinant vesicular stomatitis virus (mtdVSV)-based SARS-CoV-2 vaccine candidates expressing full-length S, S1, or several versions of the RBD. These mtdVSV-based vaccine candidates grew to high titers in cell culture and were completely attenuated in both immunocompetent and immunocompromised mice. Among these vaccine candidates, mtdVSV-S induces high levels of SARS-CoV-2-specific neutralizing antibodies (Nabs) and Th1-biased immune responses in mice. Syrian golden hamsters immunized with mtdVSV-S triggered SARS-CoV-2-specific NAbs at higher levels than those in convalescent plasma from recovered COVID-19 patients. Furthermore, hamsters immunized with mtdVSV-S were completely protected against SARS-CoV-2 challenge. Thus, mtdVSV is a safe and highly effective vector to deliver SARS-CoV-2 vaccine.

Structures of the Mononegavirales Polymerases.

Mononegavirales, known as nonsegmented negative-sense (NNS) RNA viruses, are a class of pathogenic and sometimes deadly viruses that include rabies virus (RABV), human respiratory syncytial virus (HRSV), and Ebola virus (EBOV). Unfortunately, no effective vaccines and antiviral therapeutics against many Mononegavirales are currently available. Viral polymerases have been attractive and major antiviral therapeutic targets. Therefore, Mononegavirales polymerases have been extensively investigated for their structures and functions. Mononegavirales mimic RNA synthesis of their eukaryotic counterparts by utilizing multifunctional RNA polymerases to replicate entire viral genomes and transcribe viral mRNAs from individual viral genes as well as synthesize 5' methylated cap and 3' poly(A) tail of the transcribed viral mRNAs. The catalytic subunit large protein (L) and cofactor phosphoprotein (P) constitute the Mononegavirales polymerases. In this review, we discuss the shared and unique features of RNA synthesis, the monomeric multifunctional enzyme L, and the oligomeric multimodular adapter P of Mononegavirales We outline the structural analyses of the Mononegavirales polymerases since the first structure of the vesicular stomatitis virus (VSV) L protein determined in 2015 and highlight multiple high-resolution cryo-electron microscopy (cryo-EM) structures of the polymerases of Mononegavirales, namely, VSV, RABV, HRSV, human metapneumovirus (HMPV), and human parainfluenza virus (HPIV), that have been reported in recent months (2019 to 2020). We compare the structures of those polymerases grouped by virus family, illustrate the similarities and differences among those polymerases, and reveal the potential RNA synthesis mechanisms and models of highly conserved Mononegavirales We conclude by the discussion of remaining questions, evolutionary perspectives, and future directions.

The VSV polymerase can initiate at mRNA start sites located either up or downstream of a transcription termination signal but size of the intervening intergenic region affects efficiency of initiation.

Transcription by the vesicular stomatitis virus (VSV) polymerase has been characterized as obligatorily sequential with transcription of each downstream gene dependent on termination of the gene immediately upstream. In studies described here we investigated the ability of the VSV RNA-dependent RNA polymerase (RdRp) to access mRNA initiation sites located at increasing distances either downstream or upstream of a transcription termination signal. Bi-cistronic subgenomic replicons were constructed containing progressively extended intergenic regions preceding the initiation site of a downstream gene. The ability of the RdRp to access the downstream sites was progressively reduced as the length of the intergenic region increased. Alternatively, bi-cistronic replicons were constructed containing an mRNA start signal located at increasing distances upstream of a termination site. Analysis of transcription of these "overlapped" genes showed that for an upstream mRNA start site to be recognized it had to contain not only the canonical 3'-UUGUCnnUAG-5' gene start signal, but that signal needed also to be preceded by a U7 tract. Access of these upstream mRNA initiation sites by the VSV RdRp was proportionately reduced with increasing distance between the termination site and the overlapped initiation signal. Possible mechanisms for how the RdRp accesses these upstream start sites are discussed.

Unconventional mechanism of mRNA capping by the RNA-dependent RNA polymerase of vesicular stomatitis virus.

All known eukaryotic and some viral mRNA capping enzymes (CEs) transfer a GMP moiety of GTP to the 5'-diphosphate end of the acceptor RNA via a covalent enzyme-GMP intermediate to generate the cap structure. In striking contrast, the putative CE of vesicular stomatitis virus (VSV), a prototype of nonsegmented negative-strand (NNS) RNA viruses including rabies, measles, and Ebola, incorporates the GDP moiety of GTP into the cap structure of transcribing mRNAs. Here, we report that the RNA-dependent RNA polymerase L protein of VSV catalyzes the capping reaction by an RNA:GDP polyribonucleotidyltransferase activity, in which a 5'-monophosphorylated viral mRNA-start sequence is transferred to GDP generated from GTP via a covalent enzyme-RNA intermediate. Thus, the L proteins of VSV and, by extension, other NNS RNA viruses represent a new class of viral CEs, which have evolved independently from known eukaryotic CEs.

Cell-type-specific growth restriction of vesicular stomatitis virus polR mutants is linked to defective viral polymerase function.

Vesicular stomatitis virus polR mutants synthesize defective RNA replication products in vitro and display growth restriction in some cultured cells (J. L. Chuang, R. L. Jackson, and J. Perrault, Virology 229:57-67, 1997). We show here that a recombinant virus carrying the polR N protein mutation (R179H) yielded approximately 100-fold- and approximately 40-fold-lower amounts of infectious virus than the wild type in mouse L-929 and rat 3Y1 cells, respectively, but only approximately 3-fold less in hamster BHK cells. Virus genome accumulation was inhibited 6- to 10-fold in restricting cells, but transcription was not affected. No defect in encapsidation of replication products was detected, but virus protein accumulation was reduced two- to threefold in both restricting and nonrestricting cells. polR virus particles released from the latter were 5- to 10-fold less infectious than the wild type but showed no difference in protein composition. Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2alpha) was enhanced approximately 3-fold in polR versus wild-type virus-infected L-929 cells, but neither inhibition of host gene transcription nor inhibition of double-stranded RNA (dsRNA)-activated protein kinase showed significant effects on restriction. Conditioned medium studies revealed no evidence for secretion of antiviral factors from restricting cells. We conclude that the block in polR growth is due to the combined effect of reduced genome replication and lower infectivity of released virus particles and may be due to overproduction of dsRNA. An accompanying paper (D. Ostertag, T. M. Hoblitzell-Ostertag, and J. Perrault, J. Virol. 81:503-513, 2007) provides compelling evidence for the role of dsRNA in this unique restriction phenomenon.

Identification of a new region in the vesicular stomatitis virus L polymerase protein which is essential for mRNA cap methylation.

The vesicular stomatitis virus (VSV) L polymerase protein possesses two methyltransferase (MTase) activities, which catalyze the methylation of viral mRNA cap structures at the guanine-N7 and 2'-O-adenosine positions. To identify L sequences required for the MTase activities, we analyzed a host range (hr) and temperature-sensitive (ts) mutant of VSV, hr8, which was defective in mRNA cap methylation. Sequencing hr8 identified five amino acid substitutions, all residing in the L protein. Recombinant VSV were generated with each of the identified L mutations, and the presence of a single G1481R substitution in L, located between conserved domains V and VI, was sufficient to produce a dramatic reduction (about 90%) in overall mRNA methylation. Cap analysis showed residual guanine-N7 methylation and reduced 2'-O-adenosine methylation, identical to that of the original hr8 virus. When recombinant viruses were tested for virus growth under conditions that were permissive and nonpermissive for the hr8 mutant, the same single L mutation, G1481R, was solely responsible for both the hr and ts phenotypes. A spontaneous suppressor mutant of the rG1481R virus that restored both growth on nonpermissive cells and cap methylation was identified and mapped to a single change, L1450I, in L. Site-directed mutagenesis of the region between domains V and VI, amino acids 1419-1672 of L, followed by the rescue of recombinant viruses identified five additional virus mutants, K1468A, R1478A/D1479A, G1481A, G1481N, and G1672A, that were all hr and defective in mRNA cap methylation. Thus, in addition to the previously characterized domain VI [Grdzelishvili, V.Z., Smallwood, S., Tower, D., Hall, R.L., Hunt, D.M., Moyer, S.A., 2005. A single amino acid change in the L-polymerase protein of vesicular stomatitis virus completely abolishes viral mRNA cap methylation. J. Virol. 79, 7327-7337; Li, J., Fontaine-Rodriguez, E.C., Whelan, S.P., 2005. Amino acid residues within conserved domain VI of the vesicular stomatitis virus large polymerase protein essential for mRNA cap methyltransferase activity. J. Virol. 79, 13373-13384], a new region between L amino acids 1450-1481 was identified which is critical for mRNA cap methylation.

Two RNA polymerase complexes from vesicular stomatitis virus-infected cells that carry out transcription and replication of genome RNA.

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-1alpha 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-1alpha, 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.

The oncolytic effect of recombinant vesicular stomatitis virus is enhanced by expression of the fusion cytosine deaminase/uracil phosphoribosyltransferase suicide gene.

Vesicular stomatitis virus (VSV) has recently been demonstrated to exhibit significant oncolytic capabilities against a wide variety of tumor models in vitro and in vivo. To potentially enhance the oncolytic effect, we generated a novel recombinant VSV (rVSV) that expressed the fusion suicide gene Escherichia coli cytosine deaminase (CD)/uracil phosphoribosyltransferase (UPRT). rVSV encoding the CD/UPRT fusion gene (VSV-C:U) exhibited normal growth properties and generated high levels of biologically active CD/UPRT that could catalyze the modification of 5-fluorocytosine into chemotherapeutic 5-fluorouracil (5-FU), which exhibited considerable bystander effect. Intratumoral inoculation of VSV-C:U in the presence of the systemically administered prodrug 5-fluorocytosine produced statistically significant reductions in the malignant growth of syngeneic lymphoma (A20) or mammary carcinoma (TSA) in BALB/c mice compared with rVSV treatments or with control 5-FU alone. Aside from detecting prolonged therapeutic levels of 5-FU in VSV-C:U-treated animals harboring TSA tumors and enhancing bystander killing of tumor cells, we demonstrated marked activation of IFN-gamma-secreting cytotoxic T cells by enzyme-linked immunospot analysis that may have also facilitated tumor killing. In conclusion, the insertion of the fusion CD/UPRT suicide gene potentiates the oncolytic efficiency of VSV by generating a strong bystander effect and by contributing to the activation of the immune system against the tumor without detrimentally altering the kinetics of virus-mediated oncolysis and may be useful in the treatment of malignant disease.

Transcriptional control of the RNA-dependent RNA polymerase of vesicular stomatitis virus.

The nonsegmented negative strand (NNS) RNA viruses include some of the mosr problematic human, animal and plant pathogens extant: for example, rabies virus, Ebola virus, respiratory syncytial virus, the parainfluenza viruses, measles and infectious hemapoietic necrosis virus. The key feature of transcriptional control in the NNS RNA viruses is polymerase entry at a single 3' proximal site followed by obligatory sequential transcription of the linear array of genes. The levels of gene expression are primarily regulated by their position on the genome. The promoter proximal gene is transcribed in greatest abundance and each successive downstream gene is synthesized in progressively lower amounts due to attenuation of transcription at each successive gene junction. In addition, NNS RNA virus gene expression is regulated by cis-acting sequences that reside at the beginning and end of each gene and the intergenic junctions. Using vesicular stomatitis virus (VSV), the prototypic NNS, many of these control elements have been identified.The signals for transcription initiation and 5' end modification and for 3' end polyadenylation and termination have been elucidated. The sequences that determine the ability of the polymerase to slip on the template to generate polyadenylate have been identified and polyadenylation has been shown to be template dependent and integral to the termination process. Transcriptional termination is a key element in control of gene expression of the negative strand RNA viruses and a means by which expression of individual genes may be silenced or regulated within the framework of a single transcriptional promoter. In addition, the fundamental question of the site of entry of the polymerase during transcription has been reexamined and our understanding of the process altered and updated. The ability to engineer changes into infectious viruses has confirmed the action of these elements and as a consequence, it has been shown that transcriptional control is key to controlling the outcome of a viral infection. Finally, the principles of transcriptional regulation have been utilized to develop a new paradigm for systematic attenuation of virulence to develop live attenuated viral vaccines.

Unique capping activity of the recombinant RNA polymerase (L) of vesicular stomatitis virus: association of cellular capping enzyme with the L protein.

Vesicular stomatitis virus (VSV), a prototype of non-segmented negative strand RNA viruses, packages an RNA-dependent RNA polymerase (L) which, together with an associated phosphoprotein (P), transcribes the genome RNA, in vitro and in vivo, into mRNAs that are capped at the 5'-ends. However, unlike cellular guanlylyltransferase (GT), the RNA polymerase incorporates GDP in the capped structure, as Gp(alpha)p(beta)-p(alpha)A. In an effort to characterize the capping activity of the RNA polymerase, we have purified recombinant L (rL) protein expressed in insect cells. The rL, like the virion L polymerase, also caps transcribed mRNAs with identical unique cap structure. Interestingly, the purified rL is found to be tightly bound to the GT of the insect cell during all stages of purification. VSV grown in baby hamster kidney cells also packages cellular GT of the murine cell, suggesting that VSV L protein or its associated proteins may have a strong affinity for the cellular GT. The GT bound to rL, however, formed E-GMP complex, whereas no such complex was detected with the rL protein. It appears that the L protein may contain the putative active site for the unique capping reaction or the tightly bound cellular GT may by some unknown mechanism participate in the unique capping reaction.

Polymerase slippage at vesicular stomatitis virus gene junctions to generate poly(A) is regulated by the upstream 3'-AUAC-5' tetranucleotide: implications for the mechanism of transcription termination.

Termination of mRNA synthesis in vesicular stomatitis virus (VSV), the prototypic rhabdovirus, is controlled by a 13-nucleotide gene end sequence which comprises the conserved tetranucleotide 3'-AUAC-5', the U(7) tract and the intergenic dinucleotide. mRNAs terminated at this sequence possess 100- to 300-nucleotide-long 3' poly(A) tails which are thought to result from polymerase slippage (reiterative transcription) by the VSV polymerase on the U(7) tract. Previously we determined that in addition to the AUAC tetranucleotide, the U(7) tract was an essential signal in the termination process. Shortening or interrupting the U(7) tract abolished termination. These altered U tracts also prevented the polymerase from performing reiterative transcription necessary for generation of the mRNA poly(A) tail and thus established seven residues as the minimum length of U tract that allowed reiterative transcription to occur. In this study we investigated whether sequences other than the essential U(7) tract are involved in controlling polymerase slippage. We investigated whether the AUAC tetranucleotide affected the process of reiterative transcription by analyzing the nucleotide sequence of RNAs transcribed from altered subgenomic templates and infectious VSV variants. The tetranucleotide was found to regulate reiterative transcription on the U(7) tract. The extent of polymerase slippage was governed not by specific tetranucleotide sequences but rather by nucleotide composition such that slippage occurred when the tetranucleotide was composed of A or U residues but not when it was composed of G or C residues. This suggested that polymerase slippage was controlled, at least in part, by the strength of base pairing between the template and nascent strands. Further data presented here indicate that the tetranucleotide contains both a signal that directs the VSV polymerase to slip on the downstream U(7) tract and also a signal that directs a slipping polymerase to terminate mRNA synthesis.

Host cell protein kinases in nonsegmented negative-strand virus (mononegavirales) infection.

Phosphorylation of one or more viral proteins is probably an essential step in the life cycle of every member of the nonsegmented negative-strand RNA virus (mononegavirales [MNV]) group. Since no virally encoded protein kinases have been discovered in this group, phosphorylation is effected entirely by host cell kinases. The virally encoded P proteins of the MNV are the only ones consistently phosphorylated with a stoichiometry > or =1. The P protein of vesicular stomatitis virus (VSV), and perhaps also of respiratory syncytial virus, are the only ones for which a function of phosphorylation has been established. Phosphorylation by casein kinase 2 at one or more identified sites in the VSV P protein activates transcriptional activity by promoting formation of a homotrimer, which is then capable of binding the RNA polymerase and attaching it to the N protein-RNA template. A second phosphorylation of VSV P protein by a different kinase also occurs, dependent upon prior modification by casein kinase 2, but its function is not definitely known. Phosphorylation of the other MNV P proteins may serve a different purpose. No evidence has been obtained yet for any function for phosphorylation of any other MNV protein.

RNA polymerase of vesicular stomatitis virus specifically associates with translation elongation factor-1 alphabetagamma for its activity.

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 beta and gamma subunits of the protein synthesis elongation factor EF-1. We also demonstrate that the alpha subunit of EF-1 remains tightly bound to the L protein in the inactive fraction and betagamma subunits associate with the L(alpha) complex. Further purification of L(alpha) from the inactive fraction revealed that the complex is partially active and is significantly stimulated by the addition of betagamma subunits purified from Sf21 cells. A putative inhibitor(s) appears to co-elute in the inactive fraction that blocked the L(alpha) activity. The purified virions also package all three subunits of EF-1. These findings have a striking similarity with Qbeta 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.

Phosphorylation within the amino-terminal acidic domain I of the phosphoprotein of vesicular stomatitis virus is required for transcription but not for replication.

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 > or = 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. 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 (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.

cis-Acting signals involved in termination of vesicular stomatitis virus mRNA synthesis include the conserved AUAC and the U7 signal for polyadenylation.

We investigated the cis-acting sequences involved in termination of vesicular stomatis virus mRNA synthesis by using bicistronic genomic analogs. All of the cis-acting signals necessary for termination reside within the first 13 nucleotides of the 23-nucleotide conserved gene junction. This 13-nucleotide termination sequence at the end of the upstream gene comprises the tetranucleotide AUAC, the tract containing seven uridines (U7 tract), and the intergenic dinucleotide (GA), but it does not include the downstream gene start sequence. Data presented here show that upstream mRNA termination is independent of downstream mRNA initiation. Alteration of any nucleotide in the 13-nucleotide sequence decreased the termination activity of the gene junction and resulted in increased synthesis of a bicistronic readthrough RNA. This finding indicated that the wild-type gene junction has evolved to achieve the maximum termination efficiency. The most critical position of the AUAC sequence was the C, which could not be altered without complete loss of mRNA termination. Reducing the length of the wild-type U7 tract to zero, five, or six U residues also totally abolished mRNA termination, resulting in exclusive synthesis of the bicistronic readthrough mRNA. Shortening the wild-type U7 tract to either five or six U residues abolished VSV polymerase slippage during readthrough RNA synthesis. Since neither the U5 nor U6 template was able to direct mRNA termination, these data imply that polymerase slippage is a prerequisite for termination. Evidence is also presented to show that in addition to causing polymerase slippage, the U7 tract itself or its poly(A) product constitutes an essential signal for mRNA termination.

Inhibition of vesicular stomatitis virus in cells constitutively expressing an antisense RNA targeted against the virus RNA polymerase gene.

To study the effect of virus-specific antisense RNA expression on vesicular stomatitis virus (VSV) infectivity in cultured cells, a HeLaS3 cell line constitutively expressing antisense RNA complimentary to a portion of the VSV large RNA-dependent RNA polymerase gene (L) was established (HeAntiL). At an m.o.i. of 0.01 or 0.1, the HeAntiL cell line was able to reduce virus titre and delay virus-induced cell death by 9 or 5 h, respectively, when compared to a HeLa cell line stably transfected with the expression vector devoid of antisense sequence. Ribonuclease protection experiments showed a 10-20-fold reduction of hybridizable virus L mRNA in infected HeAntiL cells compared to infected control cells at various times before cell death. These results indicate that the antisense RNA approach can significantly reduce VSV mRNA transcription and virus production for a reasonable period of time. The robust growth rate of VSV eventually overwhelms the available antisense RNA and leads to delayed cell death.

Role of cellular casein kinase II in the function of the phosphoprotein (P) subunit of RNA polymerase of vesicular stomatitis virus.

The phosphorylation of the P protein of vesicular stomatitis virus by cellular casein kinase II (CKII) is essential for its activity in viral transcription. Recent in vitro studies have demonstrated that CKII converts the inactive unphosphorylated form of P (P0) to an active phosphorylated form P1, after phosphorylation at two serine residues, Ser-59 and Ser-61. To gain insight into the role of CKII-mediated phosphorylation in the structure and function of the P protein, we have carried out circular dichroism (CD) and biochemical analyses of both P0 and P1. The results of CD analyses reveal that phosphorylation of P0 to P1 significantly increases the predicted alpha-helical structure of the P1 protein from 27 to 48%. The phosphorylation defective double serine mutant (P59/61), which is transcriptionally inactive, possesses a secondary structure similar to that of P0. P1, at a protein concentration of 50 micrograms/ml, elutes from a gel filtration column apparently as a dimer, whereas both P0 and the double serine mutant elute as a monomer at the same concentration. Interestingly, unlike wild-type P1 protein, the P mutants in which either Ser-59 or Ser-61 is altered to alanine required a high concentration of CKII for optimal phosphorylation. We demonstrate here that phosphorylation of either Ser-59 or Ser-61 is necessary and sufficient to transactivate L polymerase although alteration of one serine residue significantly decreases its affinity for CKII. We have also shown that P1 binds to the N-RNA template more efficiently than P0 and the formation of P1 is a prerequisite for the subsequent phosphorylation by L protein-associated kinase. In addition, mutant P59/61 acts as a transdominant negative mutant when used in a transcription reconstitution assay in the presence of wild-type P protein.

Casein kinase II is the P protein phosphorylating cellular kinase associated with the ribonucleoprotein complex of purified vesicular stomatitis virus.

Protein kinase activities associated with a highly purified transcriptionally active ribonucleoprotein complex from the virions of vesicular stomatitis virus (VSV) were isolated and characterized. Based upon several biochemical and immunological criteria, the protein kinase activity, which phosphorylated the bacterially expressed unphosphorylated (Po) protein, was shown to be cellular casein kinase II (CKII). These studies included inhibition of the protein kinase by specific inhibitors, phosphorylation of mutant phosphoproteins (P), immunoprecipitation by CKII antibody and Western blot analyses, and finally its ability to activate Po to synthesize RNA in a transcription-reconstitution reaction. The P protein is phosphorylated intracellularly by cellular CKII. The present study demonstrates that VSV specifically packages CKII which remains strongly associated with the ribonucleoprotein complex during morphogenesis.