The vesicular stomatitis virus (VSV) RNA polymerase synthesizes viral mRNAs with 5-cap structures methylated at the guanine-N7 and 2-O-adenosine positions (7mGpppA m ). Previously, our laboratory showed that a VSV host range (hr) and temperature-sensitive (ts) mutant, hr1, had a complete defect in mRNA cap methylation and that the wild-type L protein could complement the hr1 defect in vitro. Here, we sequenced the L, P, and N genes of mutant hr1 and found only two amino acid substitutions, both residing in the Lpolymerase protein, which differentiate hr1 from its wild-type parent. These mutations (N505D and D1671V) were introduced separately and together into the L gene, and their effects on VSV in vitro transcription and in vivo chloramphenicol acetyltransferase minigenome replication were studied under conditions that are permissive and nonpermissive for hr1. Neither L mutation significantly affected viral RNA synthesis at 34°C in permissive (BHK) and nonpermissive (HEp-2) cells, but D1671V reduced in vitro transcription and genome replication by about 50% at 40°C in both cell lines. Recombinant VSV bearing each mutation were isolated, and the hr and ts phenotypes in infected cells were the result of a single D1671V substitution in the L protein. While the mutations did not significantly affect mRNA synthesis by purified viruses, 5-cap analyses of product mRNAs clearly demonstrated that the D1671V mutation abrogated all methyltransferase activity. Sequence analysis suggests that an aspartic acid at amino acid 1671 is a critical residue within a putative conserved S-adenosyl-L-methionine-binding domain of the L protein.Vesicular stomatitis virus (VSV, a rhabdovirus) is a prototypic nonsegmented negative-strand (NNS) RNA virus belonging to the order Mononegavirales, whose members share a similar genome organization and common mechanisms of genome replication and gene expression. This order includes many medically important pathogens, including the lethal rabies, Ebola, Marburg, Nipah, and Hendra viruses. The RNA-dependent RNA polymerase (RdRp) of NNS RNA viruses is packaged into mature virions and consists of two viral subunits, the phosphoprotein (P) and the large (L) protein. The RNA genome of NNS viruses is tightly encapsidated by the nucleocapsid (N) protein, and the resulting nucleocapsid serves as the template for the sequential transcription of monocistronic mRNAs and for genome replication. Recent studies on VSV suggest that two separate RdRp complexes, which differ in their protein content, are involved in genome replication versus mRNA transcription (14, 47).The VSV RdRp produces mRNA transcripts modified at their 5Ј end by capping and cap methylation (71). The mechanism of mRNA 5Ј capping in VSV and other NNS RNA viruses is unusual, where, in contrast to cellular cap structures, both the ␣ and ␤ phosphates in the GpppA triphosphate bridge are derived from a GDP donor (2, 5, 23). The cytoplasmic localization of virus transcription and the unusual mechanism of capping suggest that the guanylyltransferas...

NF-κB and inflammasomes both play central roles in orchestrating anti-pathogen responses by rapidly inducing a variety of early-response cytokines and chemokines following infection. Myxoma virus (MYXV), a pathogenic poxvirus of rabbits, encodes a member of the cellular pyrin domain (PYD) superfamily, called M013. The viral M013 protein was previously shown to bind host ASC-1 protein and inhibit the cellular inflammasome complex that regulates the activation and secretion of caspase 1-regulated cytokines such as IL-1β and IL-18. Here, we report that human THP-1 monocytic cells infected with a MYXV construct deleted for the M013L gene (vMyxM013-KO), in stark contrast to the parental MYXV, rapidly induce high levels of secreted pro-inflammatory cytokines like TNF, IL-6, and MCP-1, all of which are regulated by NF-κB. The induction of these NF-κB regulated cytokines following infection with vMyxM013-KO was also confirmed in vivo using THP-1 derived xenografts in NOD-SCID mice. vMyxM013-KO virus infection specifically induced the rapid phosphorylation of IKK and degradation of IκBα, which was followed by nuclear translocation of NF-κB/p65. Even in the absence of virus infection, transiently expressed M013 protein alone inhibited cellular NF-κB-mediated reporter gene expression and nuclear translocation of NF-κB/p65. Using protein/protein interaction analysis, we show that M013 protein also binds directly with cellular NF-κB1, suggesting a direct physical and functional linkage between NF-κB1 and ASC-1. We further demonstrate that inhibition of the inflammasome with a caspase-1 inhibitor did not prevent the induction of NF-κB regulated cytokines following infection with vMyxM013-KO virus, but did block the activation of IL-1β. Thus, the poxviral M013 inhibitor exerts a dual immuno-subversive role in the simultaneous co-regulation of both the cellular inflammasome complex and NF-κB-mediated pro-inflammatory responses.

No abstract

Our study suggests that MYXV in combination with rapamycin infects and kills both the BTICs and the differentiated compartments of GBM and may be an effective treatment even in TMZ-resistant patients.

No abstract

The Sendai virus nested set of C proteins which are expressed in an alternative open reading frame from the P mRNA has been shown to downregulate viral RNA synthesis. Utilizing a glutathione S-transferase (gst) C fusion protein (gstC), we have shown that C protein forms a complex with the L, but not the P, subunit of the viral RNA polymerase. When P, L, and gstC are coexpressed, an oligomer of P, through its interaction with L, is also bound to beads. Since binding of C to L in the P-L complex does not disrupt P binding, the C and P binding sites appear to be different. GstC binding to L occurs only when the proteins are coexpressed in the same cell. The gstC, but not gst, protein inhibits viral transcription in vitro, showing that the fusion protein retains biological function. Pulse-chase experiments of the various complexes show that L protein synthesized alone has a half-life of 1. 2 hr, which is increased 12.5-fold by binding P, but is not significantly increased by binding gstC. Analyses of complex formation with truncations of L protein show that the C-terminal 1333 amino acids of L are not required for binding C. The dose-response curves show that replication of the genomic DI-H RNA is more sensitive to inhibition by C protein than is the synthesis of DI leader RNA, suggesting that the downregulation of RNA synthesis may be more complex than just the inhibition of the initiation of RNA synthesis.

The interactions of Sendai virus proteins required for viral RNA synthesis have been characterized both by the yeast two-hybrid system and through the use of glutathione S-transferase (gst)-viral fusion proteins synthesized in mammalian cells. Using the two-hybrid system we have confirmed the previously identified P-L (RNA polymerase), NPo-P (encapsidation substrate), and P-P complexes and now demonstrate NP-NP and NPo-V protein interactions. Expression of gstP and P proteins and binding to glutathione-Sepharose beads as a measure of complex formation confirmed the P-P interaction. The P-gstP binding occurred only on expression of the proteins in the same cell and was mapped to amino acids 345-411. We also show that full-length and deletion gstV and gstW proteins bound NPo protein when these sets of proteins were coexpressed and have identified one required region from amino acids 78-316. Neither gstV nor gstW bound NP assembled into nucleocapsids. Furthermore, both V and W proteins lacking the N-terminal 77 amino acids inhibited DI-H genome replication in vitro, showing the biological relevance of the remaining region. We propose that the specific inhibition of genome replication by V and W proteins occurs through interference with either the formation or the use of the NPo-P encapsidation substrate.

To begin to map functional domains of the Sendai P-L RNA polymerase complex we wanted to characterize the P binding site on the Sendai L protein. Analysis of in vitro and in vivo P-L polymerase complex formation with carboxyl-truncations of the L protein showed that the N-terminal half of the protein was required. Site-directed mutagenesis of the Sendai virus L gene was employed to change amino acids within a highly conserved region of the N-terminal domain I from amino acids (aa) 348-379 singly or in pairs from the Sendai to the corresponding measles L sequence or to alanine. The mutant L proteins coexpressed with the viral P and NP proteins in mammalian cells were assayed for their ability to form the P-L complex and to synthesize RNA in vitro and showed a variety of defective phenotypes. While most of the mutant L proteins still formed the P-L polymerase complex, a change from serine to arginine at aa 368 and a three-amino-acid insertion at aa 379 virtually abolished both complex formation and RNA synthesis. Changes of aas 370 and 376-377 in the L protein gave only small decreases in viral RNA synthesis. Substitutions at either aas 349-350 or aas 354-355 and a three-amino-acid insertion at aa 348 in the L protein yielded enzymes that catalyzed significant transcription, but were defective in DI RNA replication, thus differentially affecting the two processes. Since DI leader RNA, but not genome RNA, was still synthesized by this class of mutants, the defect in replication appears to be in the ability of the mutant enzyme to package newly synthesized nascent RNA. Single changes at aas 362, 363, and 366 in the L protein gave enzymes with severely decreased overall RNA synthesis, although some leader RNA was synthesized, suggesting that they cannot transcribe or replicate past the leader gene. These studies have identified a region in conserved domain I critical for multiple functions of the Sendai virus L protein.