Abstract: The intracellular retinoic acid-inducible gene I-like receptors (RLRs) sense viral ribonucleic acid and signal through the mitochondrial protein mitochondrial antiviral signalling (MAVS) to trigger the production of type I interferons and proinflammatory cytokines. In this study, we report that RLR activation promotes elongation of the mitochondrial network. Mimicking this elongation enhances signalling downstream from MAVS and favours the binding of MAVS to stimulator of interferon genes, an endoplasmic reticulum (ER) protein involved in the RLR pathway. By contrast, enforced mitochondrial fragmentation dampens signalling and reduces the association between both proteins. Our finding that MAVS is associated with a pool of mitofusin 1, a protein of the mitochondrial fusion machinery, suggests that MAVS is capable of regulating mitochondrial dynamics to facilitate the mitochondria-ER association required for signal transduction. Importantly, we observed that viral mitochondria-localized inhibitor of apoptosis, a cytomegalovirus (CMV) antiapoptotic protein that promotes mitochondrial fragmentation, inhibits signalling downstream from MAVS, suggesting a possible new immune modulation strategy of the CMV.
Abstract: Arenavirus and bunyavirus RNA genomes are unusual in that they are found in circular nucleocapsids, presumably due to the annealing of their complementary terminal sequences. Moreover, arenavirus genome synthesis initiates with GTP at position +2 of the template rather than at the precise 3' end (position +1). After formation of a dinucleotide, 5' pppGpC(OH) is then realigned on the template before this primer is extended. The net result of this "prime and realign" mechanism of genome initiation is that 5' pppG is found as an unpaired 5' nucleotide when the complementary genome ends anneal to form a double-stranded (dsRNA) panhandle. Using 5' pppRNA made in vitro and purified so that all dsRNA side products are absent, we have determined that both this 5' nucleotide overhang, as well as mismatches within the dsRNA (as found in some arenavirus genomes), clearly reduce the ability of these model dsRNAs to induce interferon upon transfection into cells. The presence of this unpaired 5' ppp-nucleotide is thus another way that some viruses appear to use to avoid detection by cytoplasmic pattern recognition receptors.
Abstract: Hepatitis C virus is a poor inducer of interferon (IFN), although its structured viral RNA can bind the RNA helicase RIG-I, and activate the IFN-induction pathway. Low IFN induction has been attributed to HCV NS3/4A protease-mediated cleavage of the mitochondria-adapter MAVS. Here, we have investigated the early events of IFN induction upon HCV infection, using the cell-cultured HCV JFH1 strain and the new HCV-permissive hepatoma-derived Huh7.25.CD81 cell subclone. These cells depend on ectopic expression of the RIG-I ubiquitinating enzyme TRIM25 to induce IFN through the RIG-I/MAVS pathway. We observed induction of IFN during the first 12 hrs of HCV infection, after which a decline occurred which was more abrupt at the protein than at the RNA level, revealing a novel HCV-mediated control of IFN induction at the level of translation. The cellular protein kinase PKR is an important regulator of translation, through the phosphorylation of its substrate the eIF2alpha initiation factor. A comparison of the expression of luciferase placed under the control of an eIF2alpha-dependent (IRES(EMCV)) or independent (IRES(HCV)) RNA showed a specific HCV-mediated inhibition of eIF2alpha-dependent translation. We demonstrated that HCV infection triggers the phosphorylation of both PKR and eIF2alpha at 12 and 15 hrs post-infection. PKR silencing, as well as treatment with PKR pharmacological inhibitors, restored IFN induction in JFH1-infected cells, at least until 18 hrs post-infection, at which time a decrease in IFN expression could be attributed to NS3/4A-mediated MAVS cleavage. Importantly, both PKR silencing and PKR inhibitors led to inhibition of HCV yields in cells that express functional RIG-I/MAVS. In conclusion, here we provide the first evidence that HCV uses PKR to restrain its ability to induce IFN through the RIG-I/MAVS pathway. This opens up new possibilities to assay PKR chemical inhibitors for their potential to boost innate immunity in HCV infection.
Abstract: The large RNA polymerase (L) protein of human parainfluenza virus type 2 (hPIV2) binds the nucleocapsid, phospho- and V proteins, as well as itself, and these interactions are essential for transcription and replication of the viral RNA genome. Although all of these interactions were found to be mediated through the domains within the N-terminus of L, the C-terminus of the L protein was also required for minigenome reporter gene expression. We have identified a highly conserved rubulavirus domain near the C-terminus of the L protein that is required for mRNA synthesis, but not for genome replication. Remarkably, this region of L shares homology with a conserved region of cellular capping enzymes that binds GTP and forms a lysyl-GMP enzyme intermediate; the first step in the cellular capping reaction. We propose that this conserved region of L also binds GTP (or GDP) to carry out the 2(nd) step of the unconventional nonsegmented negative strand virus capping reaction.
Abstract: Vaccinia virus, a large DNA virus that replicates in the cytoplasm, expresses its E3L protein to inhibit the cellular innate immune response and apoptosis. E3L is a bifunctional protein that contains an N-terminal DNA binding domain (BD) and a C-terminal double-stranded RNA (dsRNA)-BD (residues 100-190), both of which contribute to viral pathogenesis by blocking the activation of cellular genes that respond to the viral infection. We report that expression of the dsRNA-BD alone inhibits not only the dsRNA-induced activation of interferon beta (IFNbeta) but also that of 5'-triphosphate single-stranded RNA and DNA-induced IFNbeta activation even though E3L(100-190) does not bind the latter two pathogen-associated molecular patterns. This inhibition occurs in both human HeLa and A549 cells, where RIG-I appears to be required for dsDNA-induced IFNbeta activation. Unexpectedly, the two residues most important for dsRNA binding are also critical for this domain's ability to inhibit all three nucleic acid-induced cellular responses.
Abstract: Except for viruses that initiate RNA synthesis with a protein primer (e.g., picornaviruses), most RNA viruses initiate RNA synthesis with an NTP, and at least some of their viral (ppp)RNAs remain unblocked during the infection. Consistent with this, most viruses require RIG-I to mount an innate immune response, whereas picornaviruses require mda-5. We have examined a SeV infection whose ability to induce interferon depends on the generation of capped dsRNA (without free 5' tri-phosphate ends), and found that this infection as well requires RIG-I and not mda-5. We also provide evidence that RIG-I interacts with poly-I/C in vivo, and that heteropolymeric dsRNA and poly-I/C interact directly with RIG-I in vitro, but in different ways; i.e., poly-I/C has the unique ability to stimulate the helicase ATPase of RIG-I variants which lack the C-terminal regulatory domain.
Abstract: Trimethylguanosine synthase (Tgs1) is the enzyme that converts standard m(7)G caps to the 2,2,7-trimethylguanosine (TMG) caps characteristic of spliceosomal small nuclear RNAs. Fungi and mammalian somatic cells are able to grow in the absence of Tgs1 and TMG caps, suggesting that an essential function of the TMG cap might be obscured by functional redundancy. A systematic screen in budding yeast identified nonessential genes that, when deleted, caused synthetic growth defects with tgs1Delta. The Tgs1 interaction network embraced proteins implicated in small nuclear ribonucleoprotein function and spliceosome assembly, including Mud2, Nam8, Brr1, Lea1, Ist3, Isy1, Cwc21, and Bud13. Complementation of the synthetic lethality of mud2Delta tgs1Delta and nam8Delta tgs1Delta strains by wild-type TGS1, but not by catalytically defective mutants, indicated that the TMG cap is essential for mitotic growth when redundant splicing factors are missing. Our genetic analysis also highlighted synthetic interactions of Tgs1 with proteins implicated in RNA end processing and decay (Pat1, Lsm1, and Trf4) and regulation of polymerase II transcription (Rpn4, Spt3, Srb2, Soh1, Swr1, and Htz1). We find that the C-terminal domain of human Tgs1 can function in lieu of the yeast protein in vivo. We present a biochemical characterization of the human Tgs1 guanine-N2 methyltransferase reaction and identify individual amino acids required for methyltransferase activity in vitro and in vivo.
Abstract: Several cellular proteins are synthesized in the cytosol on free ribosomes and then associate with membranes due to the presence of short peptide sequences. These membrane-targeting sequences contain sites to which lipid chains are attached, which help direct the protein to a particular membrane domain and anchor it firmly in the bilayer. The intracellular concentration of these proteins in particular cellular compartments, where their interacting partners are also concentrated, is essential to their function. This paper reports that the apparently unmodified N-terminal sequence of the Sendai virus C protein (MPSFLKKILKLRGRR . . .; letters in italics represent hydrophobic residues; underlined letters represent basic residues, which has a strong propensity to form an amphipathic alpha-helix in a hydrophobic environment) also function as a membrane targeting signal and membrane anchor. Moreover, the intracellular localization of the C protein at the plasma membrane is essential for inducing the interferon-independent phosphorylation of Stat1 as part of the viral program to prevent the cellular antiviral response.
Abstract: Short RNA interference is more and more widely recognized as an effective method to specifically suppress viral functions in eukaryotic cells. Here, we used an experimental system that allows suppression of the Sendai virus (SeV) M protein by using a target sequence, derived from the green fluorescent protein gene, that was introduced in the 3' untranslated region of the M protein mRNA. Silencing of the M protein gene was eventually achieved by a small interfering RNA (siRNA) directed against this target sequence. This siRNA was constitutively expressed in a cell line constructed by transduction with an appropriate lentivirus vector. Suppression of the M protein was sufficient to diminish virus production by 50- to 100-fold. This level of suppression had no apparent effect on viral replication and transcription, supporting the lack of M involvement in SeV transcription or replication control.
Abstract: Mini-genomes expressing two reporter genes and a variable gene junction were used to study Sendai virus RNA polymerase (RdRp) scanning for the mRNA start signal of the downstream gene (gs2). We found that RdRp could scan the template efficiently as long as the initiating uridylate of gs2 (3' UCCCnnUUUC) was preceded by the conserved intergenic region (3' GAA) and the last 3 uridylates of the upstream gene end signal (ge1; 3' AUUCUUUUU). The end of the leader sequence (3' CUAAAA, which precedes gs1) could also be used for gene2 expression, but this sequence was considerably less efficient. Increasing the distance between ge1 and gs2 (up to 200 nt) led to the progressive loss of gene2 expression, in which half of gene2 expression was lost for each 70 nucleotides of intervening sequence. Beyond 200 nt, gene2 expression was lost more slowly. Our results suggest that there may be two populations of RdRp that scan at gene junctions, which can be distinguished by the efficiency with which they can scan the genome template for gs.
Abstract: As infection with wild-type (wt) Sendai virus (SeV) normally activates beta interferon (IFN-beta) very poorly, two unnatural SeV infections were used to study virus-induced IFN-beta activation in mouse embryonic fibroblasts: (i) SeV-DI-H4, which is composed mostly of small, copyback defective interfering (DI) genomes and whose infection overproduces short 5'-triphosphorylated trailer RNAs (pppRNAs) and underproduces viral V and C proteins, and (ii) SeV-GFP(+/-), a coinfection that produces wt amounts of viral gene products but that also produces both green fluorescent protein (GFP) mRNA and its complement, which can form double-stranded RNA (dsRNA) with capped 5' ends. We found that (i) virus-induced signaling to IFN-beta depended predominantly on RIG-I (as opposed to mda-5) for both SeV infections, i.e., that RIG-I senses both pppRNAs and dsRNA without 5'-triphosphorylated ends, and (ii) it is the viral C protein (as opposed to V) that is primarily responsible for countering RIG-I-dependent signaling to IFN-beta. Nondefective SeV that cannot specifically express C proteins not only cannot prevent the effects of transfected poly(I-C) or (ppp)RNAs on IFN-beta activation but also synergistically enhances these effects. SeV-V(minus) infection, in contrast, behaves mostly like wt SeV and counteracts the effects of transfected poly(I-C) or (ppp)RNAs.
Abstract: Closing the Sendai virus C protein open reading frames (rSeV-DeltaC virus) results in the production of virus particles with highly reduced infectivity. Besides, the Sendai virus C proteins interact with Alix/AIP1 and Alix suppression negatively affects Sendai virus like particle (VLP) budding. Similarly, the Sendai virus M protein has been shown to interact with Alix. On this basis, it has been suggested that Sendai virus budding involves recruitment of the multivesicular body formation machinery. We follow, here, the production of SeV particles upon regular virus infection. We find that neither Alix suppression nor dominant negative-VPS4A expression, applied separately or in combination, affects physical or infectious virion production. This contrasts with the observed decrease of SV5 virion production upon dominant negative-VPS4A expression. Finally, we show that suppression of more than 70% of a GFP/C protein in the background of a rSeV-DeltaC virus infection has no effect either on SeV particle production or on virus particle infectivity. Our results contrast with what has been published before. Possible explanations for this discrepancy are discussed.
Abstract: The ability of some Sendai virus stocks to strongly activate IFNbeta has long been known to be associated with defective-interfering (DI) genomes. We have compared SeV stocks containing various copyback and internal deletion DI genomes (and those containing only nondefective (ND) genomes) for their ability to activate reporter genes driven by the IFNbeta promoter. We found that this property was primarily due to the presence of copyback DI genomes and correlated with their ability to self-anneal and form dsRNA. The level of IFNbeta activation was found to be proportional to that of DI genome replication and to the ratio of DI to ND genomes during infection. Over-expression of the viral V and C proteins was as effective in blocking the copyback DI-induced activation of the IFNbeta promoter as it was in reducing poly-I/C-induced activation, providing evidence that these DI infections activate IFNbeta via dsRNA. Infection with an SeV stock that is highly contaminated with copyback DI genomes is thus a very particular way of potently activating IFNbeta, presumably by providing plentiful dsRNA under conditions of reduced expression of viral products which block the host antiviral response.
Abstract: The order Mononegavirales includes three virus families that replicate in the cytoplasm: the Paramyxoviridae, composed of two subfamilies, the Paramyxovirinae and Pneumovirinae, the Rhabdoviridae and the Filoviridae. These viruses, also called non-segmented negative-strand RNA viruses (NNV), contain five to ten tandemly linked genes, which are separated by conserved junctional sequences that act as mRNA start and poly(A)/stop sites. For the NNV, downstream mRNA synthesis depends on termination of the upstream mRNA, and all NNV RNA-dependent RNA polymerases reiteratively copy ("stutter" on) a short run of template uridylates during transcription to polyadenylate and terminate their mRNAs. The RNA-dependent RNA polymerase of a subset of the NNV, all members of the Paramyxovirinae, also stutter in a very controlled fashion to edit their phosphoprotein gene mRNA, and Ebola virus, a filovirus, carries out a related process on its glycoprotein mRNA. Remarkably, all viruses that edit their phosphoprotein mRNA are also governed by the "rule of six", i.e. their genomes must be of polyhexameric length (6n+0) to replicate efficiently. Why these two seemingly unrelated processes are so tightly linked in the Paramyxovirinae has been an enigma. This paper will review what is presently known about these two processes that are unique to viruses of this subfamily, and will discuss whether this enigmatic linkage could be due to the phenomenon of RNA virus error catastrophe.
Abstract: Some paramyxovirus V proteins induce STAT protein degradation, and the amino acids essential for this process in the human parainfluenza virus type 2 (hPIV2) V protein have been studied. Various recombinant hPIV2s and cell lines constitutively expressing various mutant V proteins were generated. We found that V proteins with replacement of Cys residues of the Cys cluster were still able to bind STATs but were unable to induce their degradation. The hPIV2 V protein binds STATs via a W-(X)3-W-(X)9-W Trp motif located just upstream of the Cys cluster. Replacements of two or more Trp residues in this motif resulted in a failure to form a V/STAT2 complex. We have also identified two Phe residues of the hPIV2 V protein that are essential for STAT degradation, namely, Phe207, lying within the Cys cluster, and Phe143, in the P/V common region of the protein. Interestingly, infection of BHK cells with hPIV2 led to the specific degradation of STAT1 and not STAT2. Other evidence for the cell species specificity of hPIV2-induced STAT degradation is presented. Finally, a V-minus hPIV2, which can express only the P protein from its P gene, was generated and partially characterized. In contrast to V-minus viruses of other paramyxovirus genera, this V-minus rubulavirus was highly debilitated, and its growth even in Vero cells was very limited. The structural rubulavirus V proteins, as expected, are thus clearly important in promoting virus growth, independent of their anti-interferon (IFN) activity. Interestingly, many of the residues that are essential for anti-IFN activity, e.g., the Cys of this cluster and Phe207 within this cluster, as well as the Trp of this motif, are also essential for promoting virus growth.
Abstract: The Sendai virus C protein acts to dismantle the interferon-induced cellular antiviral state in an MG132-sensitive manner, in part by inducing STAT1 instability. This activity of C maps to the first 23 amino acids (C(1-23)) of the 204-amino-acid (aa)-long protein (C(1-204)). C(1-23) was found to act as an independent viral element that induces STAT1 instability, since this peptide fused to green fluorescent protein (C(1-23)/GFP) is at least as active as C(1-204) in this respect. This peptide also induces the degradation of C(1-23)/GFP and other proteins to which it is fused. Most of C(1-204), and particularly its amino-terminal half, is predicted to be structurally disordered. C(1-23) as a peptide was found to be disordered by circular dichroism, and the first 11 aa have a strong potential to form an amphipathic alpha-helix in low concentrations of trifluoroethanol, which is thought to mimic protein-protein interaction. The critical degradation-determining sequence of C(1-23) was mapped by mutation to eight residues near its N terminus: (4)FLKKILKL(11). All the large hydrophobic residues of (4)FLKKILKL(11), plus its ability to form an amphipathic alpha-helix, were found to be critical for STAT1 degradation. In contrast, C(1-23)/GFP self-degradation did not require (8)ILKL(11), nor the ability to form an alpha-helix throughout this region. Remarkably, C(1-23)/GFP also stimulated C(1-204) degradation, and this degradation in trans required the same peptide determinants as for STAT1. Our results suggest that C(1-204) coordinates its dual activities of regulating viral RNA synthesis and counteracting the host innate antiviral response by sensing both its own intracellular concentration and that of STAT1.
Abstract: mRNA synthesis from nonsegmented negative-strand RNA virus (NNV) genomes is unique in tht the genome RNA is embedded in an N protein assembly (the nucleocapsid) and the viral RNA polymerase does not dissociate from the template after release of each mRNA, but rather scans the genome RNA for the next gene-start site. A revised model for NNV RNA synthesis is presented, in which RNA polymerase scanning plays a prominent role. Polymerase scanning of the template is known to occur as the viral transcriptase negotiates gene junctions without falling off the template.
Abstract: The Sendai virus (SeV) C proteins are known to interact with Stat1 to prevent interferon (IFN)-induced pY701-Stat1 formation and IFN signaling. Nevertheless, pY701-Stat1 levels paradoxically increase during SeV infection. The C proteins also induce bulk Stat1 instability in some cells, similar to rubulavirus V proteins. We have found that SeV infection increases pY701-Stat1 levels even in cells in which bulk Stat1 levels strongly decrease. Remarkably, both the decrease in bulk Stat1 levels and the increase in pY701-Stat1 levels were found to be independent of the IFN signaling system, i.e., these events occur in mutant cells in which various components of the IFN signaling system have been disabled. Consistent with this, the C-induced decrease in Stat1 levels does not require Y701 of Stat1. We present evidence that C interacts with Stat1 in two different ways, one that prevents IFN-induced pY701-Stat1 formation and IFN signaling that has already been documented, and another that induces pY701-Stat1 formation (while decreasing bulk Stat1 levels) in a manner that does not require IFN signaling. These two types of Stat1 interaction are also distinguishable by C gene mutations. In particular, the IFN signaling-independent Stat1 interactions specifically require the amino-terminal extensions of the longer C proteins. The actions of the SeV C proteins in counteracting the cellular antiviral response are clearly more extensive than previously appreciated.
Abstract: We have used cDNA arrays to compare the activation of various cellular genes in response to infection with Sendai viruses (SeV) that contain specific mutations. Three groups of cellular genes activated by mutant SeV infection, but not by wild-type SeV, were identified in this way. While some of these genes are well known interferon (IFN)-stimulated genes, others, such as those for interleukin-6 (IL-6) and IL-8, are not directly induced by IFN. The gene for beta IFN (IFN-beta), which is critical for initiating an antiviral response, was also specifically activated in mutant SeV infections. The SeV-induced activation of IFN-beta was found to depend on IFN regulatory factor 3, and the activation of all three cellular genes was independent of IFN signaling. Mutations that disrupt four distinct elements in the SeV genome (the leader RNA, two regions of the C protein, and the V protein) all lead to enhanced levels of IFN-beta mRNA, and at least three of these viral genes also appear to be involved in preventing activation of IL-8. Our results suggest that SeV targets the inflammatory and adaptive immune responses as well as the IFN-induced intracellular antiviral state by using a multifaceted approach.
Abstract: The genomic and antigenomic 3'-end replication promoters of Sendai virus are bipartite in nature and symmetrical, composed of le or tr sequences; a gene start or gene end site, respectively; and a simple hexameric repeat. The relative strengths of these 3'-end promoters determines the ratios of genomes and antigenomes formed during infection and whether model mini-genomes can be rescued from DNA by nondefective helper viruses. Using these tests of promoter strength, we have confirmed that tr is stronger than le in this respect. We have also found that the presence of a gene start site within either 3'-end promoter strongly reduces 3'-end promoter strength. The negative effects of the gene start site on the 3'-end promoter suggest that these closely spaced RNA start sites compete with each other for a common pool of viral RNA polymerase. The manner in which this competition could occur for polymerase off the template (in trans) and polymerase on the template (in cis) adds insight into how the viral RNA polymerase switches between its dual functions as transcriptase and replicase.
Abstract: Human parainfluenza virus type 1 (HPIV1), a major cause of croup in infants and young children, accounts for 6% of hospitalizations for pediatric respiratory tract disease. The antigenically related Sendai virus, referred to here as murine PIV1 (MPIV1), is being considered for use as a live-attenuated vaccine to protect against HPIV1 (J. L. Hurwitz, K. F. Soike, M. Y., Sangster, A. Portner, R. E. Sealy, D. H. Dawson, and C. Coleclough, 1997, Vaccine 15(5), 533-540) and also as a recombinant vaccine vector expressing antigens to protect against viral disease in humans. However, in the 1950s MPIV1 was reported to have been isolated from humans, suggesting that zoonotic transmission might have occurred. It is therefore important to examine the ability of MPIV1 to replicate in nonhuman primates, i.e., surrogate hosts for humans. In the present study the level of replication of MPIV1 and HPIV1 was compared in African green monkeys and chimpanzees. Surprisingly, MPIV1 replicated as efficiently as HPIV1 in the upper and lower respiratory tract of African green monkeys at doses of 10(4) and 10(6) and replicated only slightly less efficiently at both sites in chimpanzees. African green monkeys immunized with MPIV1 were highly resistant to subsequent challenge with HPIV1 even though MPIV1 did not induce a detectable HPIV1-neutralizing antibody response. The high level of replication of MPIV1 observed in the upper and lower respiratory tract of these primates suggests that MPIV1 likely would require significant attenuation before it could be given to humans as a vaccine against HPIV1 or as a vaccine vector. Its ability to efficiently replicate in nonhuman primates suggests that MPIV1 lacks a significant host range restriction in primates and could theoretically cause zoonotic disease in humans.
Abstract: Ambisense Sendai virus (SeV) was prepared in order to study the control of viral RNA synthesis. In these studies, we found that the relative ratios of genomes/antigenomes formed during infection are largely determined by the relative strengths of the replication promoters, independent of the presence of a functional mRNA start site. We also found that the ability of the viral polymerase (vRdRP) to respond to an mRNA editing site requires prior (re)initiation at an mRNA start site, similar to the acquisition of vRdRP processivity in the absence of nascent chain coassembly. During these studies, the inherent instability of ambisense SeV upon passage in embryonated chicken eggs was noted and was found to be associated with a point mutation in the ambisense mRNA (ambi-mRNA) start site that severely limited its expression. Since the interferon (IFN)-induced antiviral state is mediated in part via double-stranded RNA (dsRNA), the efficiency of the ambi-mRNA poly(A)/stop site was examined. This site was found to operate in a manner similar to that of other SeV mRNA poly(A)/stop sites, i.e., at approximately 95% efficiency. This modest level of vRdRP read-through is apparently tolerable for natural SeV because the potential to form dsRNA during infection remains limited. However, when mRNAs are expressed from ambisense SeV antigenomes, vRdRP read-through of the ambi-mRNA poly(A)/stop site creates a capped transcript that can potentially extend the entire length of the antigenome, since there are no further poly(A)/stop sites here. In support of this hypothesis, loss of ambi-mRNA expression during passage of ambisense SeV stocks in eggs is also characterized by conversion of virus that grows poorly in IFN-sensitive cultures and is relatively IFN sensitive to virus that grows well even in IFN-pretreated cells that restrict vesicular stomatitis virus replication, i.e., the wild-type SeV phenotype. The selection of mutants unable to express ambi-mRNA on passage in chicken eggs is presumably due to increased levels of dsRNA during infection. How natural ambisense viruses may deal with this dilemma is discussed.
Abstract: Sendai virus infection strongly induces interferon (IFN) production and has recently been shown to interdict the subsequent IFN signaling through the Jak/Stat pathway. This anti-IFN activity of SeV is due to its "C" proteins, a nested set of four proteins (C', C, Y1, Y2) that carry out a nested set of functions in countering the innate immune response. We previously reported that all four C proteins interact with Stat1 to prevent IFN signaling through the Jak/Stat pathway. Nevertheless, only the longer C proteins reduced Stat1 levels and prevented IFN from inducing an antiviral (VSV) state, or apoptosis, in IFN-competent murine cells. Here, we investigate the mechanism by which the various C proteins differentially affect the host antiviral defenses. All four C proteins were found to physically associate with Stat1 during cell culture infections, and in vitro in the absence of other viral gene products (as evidenced by co-immunoprecipitation). In addition, the inability of a null mutant (C(F170S)) to bind Stat1 suggests that this interaction is physiologically relevant. We have also shown that the proteasomal inhibitor MG132 can prevent the C protein-induced dismantling of the antiviral (VSV) state in murine cells; thus, the turnover of Stat1 correlates with the C protein-mediated counteraction of the antiviral (VSV) state. The C protein-induced instability of Stat1 was accompanied by a clear increase in the level of mono-ubiquinated Stat1, an unexpected hallmark of protein degradation. Finally, we show that a rSeV with mutant C proteins but wild-type Y proteins (CDelta10-15, that does not counteract the endogenous antiviral (VSV) state of MEFs even though their C proteins bind Stat1 and prevent its activity) is also unable to decrease bulk Stat1 levels or to increase the level of ubiquinated Stat1.
Abstract: Viruses of the Paramyxovirinae, similar to other viruses, have evolved specific proteins that interdict IFN action as part of a general strategy to counteract host innate immunity. In many (but not all) cases, this interdiction is accompanied by a lowering of the intracellular levels of the STAT proteins. Among rubulaviruses, there is a notable variation in how they interfere with IFN action. Whereas SV41, SV5, and MuV all act by lowering Stat1, hPIV2 acts by lowering Stat2. Here, we show that the mumps and hPIV2 V proteins both form a complex with several Stat proteins in a mixed-extract assay. This suggests that the specific degradation of these Stat proteins is not determined by complex formation, but presumably at some later stage of the degradation pathway. V/Stat complex formation requires a specific carboxyl segment of V. However, a previously unrecognized trp-rich motif, rather than the Zn(++)-binding cys-cluster of this segment, appears to be required for V/Stat interaction. The C protein of Sendai (respiro-) virus, another P gene encoded protein, also forms a complex with Stat1, and prebinding of MuV V to Stat1 prevents the subsequent binding of SeV C. Our results suggest that rubulavirus V proteins may be related to both the C and the V proteins of respiroviruses.
Abstract: The minus-strand genome of Sendai virus is an assembly of the nucleocapsid protein (N) and RNA, in which each N subunit is associated with precisely 6 nt. Only genomes that are a multiple of 6 nt long replicate efficiently or are found naturally, and their replication promoters contain sequence elements with hexamer repeats. Paramyxoviruses that are governed by this hexamer rule also edit their P gene mRNA during its synthesis, by G insertions, via a controlled form of viral RNA polymerase "stuttering" (pseudo-templated transcription). This stuttering is directed by a cis-acting sequence (3' UNN UUUUUU CCC), whose hexamer phase is conserved within each virus group. To determine whether the hexamer phase of a given nucleotide sequence within nucleocapsids affected its sensitivity to chemical modification, and whether hexamer phase of the mRNA editing site was important for the editing process, we prepared a matched set of viruses in which a model editing site was displaced 1 nt at a time relative to the genome ends. The relative abilities of these Sendai viruses to edit their mRNAs in cell culture infections were examined, and the ability of DMS to chemically modify the nucleotides of this cis-acting signal within resting viral nucleocapsids was also studied. Cytidines at hexamer phases 1 and 6 were the most accessible to chemical modification, whereas mRNA editing was most extensive when the stutter-site C was in positions 2 to 5. Apparently, the N subunit imprints the nucleotide sequence it is associated with, and affects both the initiation of viral RNA synthesis and mRNA editing. The N-subunit assembly thus appears to superimpose another code upon the genetic code.
Abstract: Sendai virus (SeV) leader (le) and trailer (tr) RNAs are short transcripts generated during abortive antigenome and genome synthesis, respectively. Recom binant SeV (rSeV) that express tr-like RNAs from the leader region are non-cytopathic and, moreover, prevent wild-type SeV from inducing apoptosis in mixed infections. These rSeV thus appear to have gained a function. Here we report that tr RNA binds to a cellular protein with many links to apoptosis (TIAR) via the AU-rich sequence 5' UUUUAAAUUUU. Duplication of this AU-rich sequence alone within the le RNA confers TIAR binding on this le* RNA and a non-cytopathic phenotype to these rSeV in cell culture. Transgenic overexpression of TIAR during SeV infection promotes apoptosis and reverses the anti-apoptotic effects of le* RNA expression. More over, TIAR overexpression and SeV infection act synergistically to induce apoptosis. These short viral RNAs may act by sequestering TIAR, a multivalent RNA recognition motif (RRM) family RNA-binding protein involved in SeV-induced apoptosis. In this view, tr RNA is not simply a by-product of abortive genome synthesis, but is also an antigenome transcript that modulates the cellular antiviral response.
Abstract: The Sendai virus (SeV) C gene codes for a nested set of four C proteins that carry out several functions, including the modulation of viral RNA synthesis and countering of the cellular antiviral response. Using mutant C genes (and in particular a C gene with a deletion of six amino acids present only in the larger pair of C proteins) and recombinant SeV carrying these mutant C genes, we find that the nested set of C proteins carry out a nested set of functions. All of the C proteins interdict interferon (IFN) signaling to IFN-stimulated genes (ISGs) and prevent pY701-Stat1 formation. However, only the larger C proteins can induce STAT1 instability, prevent IFN from inducing an antiviral state, or prevent programmed cell death. Remarkably, interdiction of IFN signaling to ISGs and the absence of pY701-Stat1 formation did not prevent IFN-alpha from inducing an anti-Vesicular stomatitis virus (VSV) state. It is possible that IFN-alpha signaling to induce an anti-VSV state can occur independently of the well-established Jak/Stat/ISGF3 pathway and that it is this parallel pathway that is targeted by the longer C proteins.
Abstract: Sendai virus (SeV) infection of interferon (IFN)-competent cells is one of the most efficient ways of inducing IFN production. Virus replication is nevertheless largely unaffected, since SeV infection also interfers with IFN action, a prerequisite for the establishment of an antiviral state. This property has been mapped by reverse genetics to the viral C gene, which is also known to act as a promoter-specific inhibitor of viral RNA synthesis. Using luciferase reporter plasmids containing IFN-responsive promoters, we have found that all four C proteins effectively interdict IFN signaling when expressed independently of SeV infection. The C proteins must therefore interact directly with cellular components to carry this out. The C gene in the context of an SeV infection was also found to induce STAT1 instability in some cells, whereas in other cells it apparently acts to prevent the synthesis of STAT1 in response to the virus infection or IFN treatment. The SeV C proteins appear to act in at least two ways to counteract the IFN induced by SeV infection.
Abstract: Editing of paramyxovirus P gene mRNAs occurs cotranscriptionally and functions to fuse an alternate downstream open reading frame to the N-terminal half of the P protein. G residues are inserted into a short G run contained within a larger purine run (AnGn) in this process, by a mechanism whereby the transcribing polymerase stutters (i.e., reads the same template cytosine more than once). Although Sendai virus (SeV) and bovine parainfluenza virus type 3 (bPIV3) are closely related, the G insertions in their P mRNAs are distributed differently. SeV predominantly inserts a single G residue within the G run of the sequence 5' AACAAAAAAGGG, whereas bPIV3 inserts one to six G's at roughly equal frequency within the sequence 5' AUUAAAAAAGGGG (differences are underlined). We have examined how the cis-acting editing sequence determines the number of G's inserted, both in a transfected cell system using minigenome analogues and by generating recombinant viruses. We found that the presence of four rather than three G's in the purine run did not affect the distribution of G insertions. However, when the underlined AC of the SeV sequence was replaced by the UU found in bPIV3, the editing phenotype from both the minigenome and the recombinant virus resembled that found in natural bPIV3 infections (i.e., a significant fraction of the mRNAs contained two to six G insertions). The two nucleotides located just upstream of the polypurine tract are thus key determinants of the editing phenotype of these viruses. Moreover, the minimum number of A residues that will promote SeV editing phenotype is six but can be reduced to five when the upstream AC is replaced by UU. A model for how the upstream dinucleotide controls the insertion phenotype is presented.
Abstract: We have studied the relationship between the Sendai virus (SeV) C proteins (a nested set of four proteins initiated at different start codons) and the interferon (IFN)-mediated antiviral response in IFN-competent cells in culture. SeV strains containing wild-type or various mutant C proteins were examined for their ability (i) to induce an antiviral state (i.e., to prevent the growth of vesicular stomatitis virus [VSV] following a period of SeV infection), (ii) to induce the elevation of Stat1 protein levels, and (iii) to prevent IFN added concomitant with the SeV infection from inducing an antiviral state. We find that expression of the wild-type C gene and, specifically, the AUG114-initiated C protein prevents the establishment of an antiviral state: i.e., cells infected with wild-type SeV exhibited little or no increase in Stat1 levels and were permissive for VSV replication, even in the presence of exogenous IFN. In contrast, in cells infected with SeV lacking the AUG114-initiated C protein or containing a single amino acid substitution in the C protein, the level of Stat1 increased and VSV replication was inhibited. The prevention of the cellular IFN-mediated antiviral response appears to be a key determinant of SeV pathogenicity.
Abstract: Paramyxoviruses cotranscriptionally edit their P gene mRNAs by expanding the number of Gs of a conserved AnGn run. Different viruses insert different distributions of guanylates, e.g., Sendai virus inserts a single G, whereas parainfluenza virus type 3 inserts one to six Gs. The sequences conserved at the editing site, as well as the experimental evidence, suggest that the insertions occur by a stuttering process, i.e., by pseudotemplated transcription. The number of times the polymerase "stutters" at the editing site before continuing strictly templated elongation is directed by a cis-acting sequence found upstream of the insertions. We have examined the stuttering process during natural virus infections by constructing recombinant Sendai viruses with mutations in their cis-acting sequences. We found that the template stutter site is precisely determined (C1052) and that a relatively short region (approximately 6 nucleotides) just upstream of the AnGn run can modulate the overall frequency of mRNA editing as well as the distribution of the nucleotide insertions. The positions more proximal to the 5' AnGn run are the most important in this respect. We also provide evidence that the stability of the mRNA/template hybrid plays a determining role in the overall frequency and range of mRNA editing. When the template U run is extended all the way to the stutter site, adenylates rather than guanylates are added at the editing site and their distribution begins to resemble the polyadenylation associated with mRNA 3' end formation by the viral polymerase. Our data suggest how paramyxovirus mRNA editing and polyadenylation are related mechanistically and how editing sites may have evolved from poly(A)-termination sites or vice versa.
Abstract: Wild-type Sendai virus expresses three proteins containing the N-terminal half of the P protein open reading frame due to mRNA editing; a full-length P protein (ca. 70% of the total), a V protein with the N-terminal half fused to a Cys-rich Zn(2+)-binding domain (ca. 25% of the total), and a W protein representing the N-terminal half alone (ca. 5% of the total). To examine the role of these proteins in the virus life cycle, we have prepared recombinant viruses in which the normal V mRNA expresses a W protein (V-stop; 70% P, 30% W), one which cannot edit its P gene mRNA (delta 6A; 100% P), and one which overedits its mRNA like parainfluenza virus type 3 (swap/8;20-40% P, 30% V, 30% W). All these viruses were readily recovered and grew to similar titers in eggs, and except for the P gene products, cell lines individually infected with these viruses accumulated similar amounts of viral macromolecules. The relative competitive advantage of each virus was determined by multiple cycle coinfections of eggs and found to be rSeV-Vstop = rSeV-wt >> rSeV-delta 6A > rSeV-swap/8. On the other hand, rSeV-swap/8 underwent multiple cycles of replication in C57BI/6 mouse lungs and was highly virulent for these animals, whereas rSeV-delta 6A was avirulent in mice and this infection was quickly cleared. Remarkably, rSeV-Vstop appeared to be more virulent for inbred C57BI/6 mice than rSeV-wt, but was partially attenuated in infections of outbred ICR mice. Thus, the expression of either the V or the W proteins is sufficient for multiple cycles of infection and pathogenesis in C57BI/6 mice, whereas W can only partially substitute for V for pathogenesis in ICR mice.
Abstract: The replication of nonsegmented minus-strand RNA genomes, like that of Sendai paramyxovirus (SeV), are controlled by the short leader regions present at each end of the linear genomes and antigenomes; the left and right promoters (PL and PR), respectively. Wild-type SeV is highly cytopathic in cell culture, because it induces programmed cell death (PCD). We have found that a recombinant SeV (rSeVGP42), in which the first 42 nt of le+ sequences at PL were replaced with the equivalent sequences of PR, and which produces infectious virus in amounts comparable to wild type, does not kill cells. Further, the increasing replacement of the terminal le+ sequences at PL with le- sequences led to a decreasing fraction of infected cells being apoptotic. This property (PCD-), moreover, is dominant in cells co-infected with SeVwt and rSeVGP42, and the mutant virus therefore appears to have gained a function which prevents PCD induced by SeVwt. Even though this virus has not been selected for naturally, it excludes SeVwt during co-infections of cultured cells or embryonated chicken eggs. The noncytopathic nature of cells infected or co-infected with rSeVGP42 leads automatically to stable, persistent infections. The mutation in rSeVGP42 is not in the protein coding regions of the viral genome, but in the 55-nt-long leader region which controls antigenome synthesis from genome templates. The SeV leader regions, which are expressed as short RNAs, thus appear to control the induction of PCD.
Abstract: The Sendai virus V protein is a nonstructural trans-frame protein in which a highly conserved cys-rich Zn2+-binding domain is fused to the N-terminal half of the P protein via mRNA editing. Using a recently developed system in which infectious virus is recovered from cDNA, we have engineered a virus in which a translation stop codon was placed at the beginning of the V ORF. Translation of the V(stop) mRNA yields a W-like protein, i.e., a protein composed of the N-terminal half of the P protein alone which is naturally expressed at low levels from the P gene. This V-minus but W-augmented virus was found to replicate normally in cell culture and embryonated chicken eggs. The Sendai virus V protein is thus an accessory protein, and the cys-rich Zn2+-binding domain is likely to function in a specialized role during virus propagation.
Abstract: A mutant Sendai virus (SevMVC), which grows much better than its progenitor virus (SeVM) in cell culture, but, in strong contrast to SeVM, is totally avirulent for mice, has been described. SeVMVC contains two amino acid substitutions relative to SeVM, namely, F170S in the C protein and E2050A in the L protein. We have examined which substitutions were responsible for the above phenotypes by exchanging the C gene of our reference strain Z with those of SeVH (another reference strain), SeVM, and SeVMVC, in turn. We have found that the F170S mutation in the CMVC protein is responsible both for enhanced replication in cell culture and for avirulence in mice. Avirulence appeared to be due to restricted viral replication primarily after day 1, implicating some aspect of innate immunity in this process. The SeV C proteins thus appear to be required for multiple cycles of replication in mice.
Abstract: Many paramyxoviruses express small basic C proteins, from an alternate, overlapping open reading frame of the P gene mRNA, which were previously found to inhibit mRNA synthesis. During recent experiments in which infectious Sendai virus (SeV) was recovered from cDNA via the initial expression of the viral N, P, and L genes from plasmids, the abrogation of C protein expression from the plasmid P gene was found to be necessary for virus recovery. We have investigated the effect of C coexpression on the amplification of an internally deleted defective interfering (DI) genome directly in the transfected cell, for which, in contrast to virus recovery experiments, genome amplification is independent of mRNA synthesis carried out by the SeV polymerase. We find that C protein coexpression also strongly inhibits the amplification of this DI genome but has little or no effect on that of a copy-back DI genome (DI-H4). We have also characterized the C protein from a mutant SeV and found that (i) it had lost most of its inhibitory activity on internally deleted DI genome amplification and (ii) its coexpression no longer prevented the recovery of SeV from DNA. However, consistent with the insensitivity of copy-back DI genomes to C protein inhibition, C coexpression did not prevent the recovery of copy-back nondefective viruses from DNA. The inhibitory effects of C coexpression thus appear to be promoter specific.
Abstract: We have used a cDNA copy of a natural, internally deleted, Sendai virus defective interfering genome to study the effect of insertions and deletions (which maintain the hexamer genome length) on the ability of viral genomes to be amplified in a transfected cell system. The insertion of 18 nt at nt72 (In the 5' untranslated region of the N gene, just downstream of the le+ region) was found to be lethal, whereas similar insertions further from the genome ends were well tolerated. Curiously, the insertion of 6 nt on either side of the le+/N junction (at nt47 and nt87) was well tolerated, but the insertion of 12 nt at either site, or of 6 nt at both sites, largely ablated genome amplification. These results suggest that an element of this replication promoter is located downstream of nt87, in the 5' untranslated region of the first gene. Remarkably, the addition of 6 nt by the insertion of 2, 3, or 4 nt at nt47 plus the insertion of 4, 3, or 2 nt, respectively, at nt87 was poorly tolerated, presumably because the hexamer phase of the intervening sequence was altered with respect to the N subunits of the template. These results suggest that the rule of six operates, at least in part, at the level of the initiation of antigenome synthesis.
Abstract: We examined the 5' ends of Hantaan virus (HTN) genomes and mRNAs to gain insight into the manner in which these chains were initiated. Like those of all members of the family Bunyaviridae described so far, the HTN mRNAs contained 5' terminal extensions that were heterogeneous in both length and sequence, presumably because HTN also "cap snatches" host mRNAs to initiate the viral mRNAs. Unexpectedly, however, almost all of the mRNAs contained a G residue at position -1, and a large fraction also lacked precisely one of the three UAG repeats at the termini. The genomes, on the other hand, commenced with a U residue at position +1, but only 5' monophosphates were found here, indicating that these chains may not have initiated with UTP at this position. Taken together, these unusual findings suggest a prime-and-realign mechanism of chain initiation in which mRNAs are initiated with a G-terminated host cell primer and genomes with GTP, not at the 3' end of the genome template but internally (opposite the template C at position +3), and after extension by one or a few nucleotides, the nascent chain realigns backwards by virtue of the terminal sequence repeats, before processive elongation takes place. For genome initiation, an endonuclease, perhaps that involved in cap snatching, is postulated to remove the 5' terminal extension of the genome, leaving the 5' pU at position +1.
Abstract: We have recovered infectious Sendai virus (SeV) from full-length cDNA (FL-3) by transfecting this cDNA and pGEM plasmids expressing the nucleocapsid protein (NP), phosphoprotein and large proteins into cells infected with a vaccinia virus which expresses T7 RNA polymerase. These cells were then injected into chicken eggs, in which SeV grows to very high titers. FL-3 was marked with a BglII site in the leader region and an NsiI site (ATGCAT) in the 5' nontranslated region of the NP gene, creating a new, out-of-frame, 5' proximal AUG. All the virus stocks generated eventually removed this impediment to NP expression, by either point mutation or recombination between FL-3 and pGEM-NP. The recovery system was found to be highly recombinogenic. Even in the absence of selective pressure, one in 20 of the recombinant SeV generated had exchanged the NP gene of FL-3 with that of pGEM-NP. When a fifth plasmid containing a new genomic 3' end without the presumably deleterious BglII site was included as another target for recombination, the new genomic 3' end was found in the recombinant SeV in 12 out of 12 recoveries. Using this approach, a novel copy-back nondefective virus was generated which interferes with wild-type virus replication.
Abstract: Subgenomic RNAs of both polarities corresponding to rice hoja blanca virus (RHBV) ambisense RNA4 were detected in RHBV-infected rice tissues. Total RNA extracted from RHBV-infected and noninfected rice tissues and RNA4 purified from RHBV ribonucleoprotein particles were used as templates for primer extension studies. The RNAs extracted from RHBV-infected tissues contain a population of RNA molecules with 10 to 17 nonviral nucleotides at their 5' end. The RNA-cDNA hybrids resulting from primer extension of such RNA molecules were specifically immunoselected with anti-cap antibodies, indicating that the subgenomic RNAs are capped and probably serve as mRNAs and that the additional nucleotides at their 5' end possibly derive from host mRNAs via a cap-snatching mechanism.
Abstract: In this report we have monitored viral gene expression, both at the RNA and protein level, after the establishment of a long-term persistent infection of Sendai virus. The persistent infection was initially established by infecting BHK cells with a viral stock containing a short (1.4 kb) copy-back DI (DIH4). After over 120 weeks in culture this short copy-back DI had been replaced by two large deletion DIs (approximately 7 and 12 kb) from which was expressed an N-terminally truncated form of the P protein. The mRNA for this protein was detected in cells and the deletion within the P gene was mapped by PCR cloning and sequencing of intracellular nucleocapsid RNA. This truncated P protein (derived by deleting the N-terminal half of the cloned Pwt gene) has already been shown to function as a dominant negative for DI replication when driven by cloned viral genes. Cloning and expression of the truncated P from the long-term persistent infection revealed that this protein had retained the dominant negative phenotype. The presence of such a protein would severely depress viral gene expression and may therefore play an important role in the maintenance of persistence.
Abstract: An antiserum to a peptide of the Tacaribe virus Z protein was used to determine whether this small Zn(2+)-binding protein was required for viral RNA synthesis in infected cell extracts. Specific immunodepletion of the extracts invariably reduced genome synthesis to near background levels, but strong effects on mRNA synthesis occurred only early in the infection or when mRNA synthesis was relatively weak. Our results suggest that the Z protein is required for both mRNA and genome synthesis, but in somewhat different manners.
Abstract: A Tacaribe virus in vitro system for RNA synthesis was established and found in large part to faithfully reproduce RNA synthesis in vivo. Similar to influenza virus and bunyavirus in vitro systems, this system was also highly dependent on added oligonucleotides. Of the eight tested, only three were active, in the order GpC greater than CpG greater than ApApC. Determination of the 5' ends of the transcripts suggested that the oligonucleotides were acting as primers. In particular, whereas stimulation with CpG (complementary to positions +1 and +2 of the template) led to RNAs whose 5' ends were at position +1 as expected, GpC stimulation led to transcripts whose 5' ends were at position -1 rather than at position +2, as GpC is complementary to positions +2 and +3 of the template. This finding suggests a model for the initiation of genome replication in which pppGpC is first made on the template at positions +2 and +3 but slips backwards on the template so that the 5' end is at position -1 before elongation can continue.
Abstract: Infection of human epidermoid carcinoma-2 (HEp-2) cells by Herpes simplex virus type 1 (HSV-1) leads to significant activation of inositol phospholipid turnover after 15 min. The effect of neomycin, an inhibitor of inositol phospholipid turnover, has been investigated for its effect on HSV-1 multiplication in HEp-2 cells. HSV-1 multiplication is inhibited by neomycin. This inhibition is not due to a block of virus adsorption or penetration. Neomycin inhibits the expression of virus immediate-early genes, as well as expression of early genes and viral DNA synthesis. In neomycin-treated cells, the usual virion-associated shut off of host protein synthesis does not occur. These results indicate that the inositol phospholipid pathway is involved in immediate-early gene expression and shut off of host protein synthesis in HEp-2 cells.
Abstract: In order to examine cellular gene involvement in HSV-1 expression, we constructed different rat embryo fibroblast cell lines immortalized by adenovirus E1A or c-myc, with or without the human EJ bladder carcinoma transforming oncogene EJ-ras. HSV-1 multiplication was strongly inhibited in cells expressing EJ-ras genes compared to immortalized control cells. Virus adsorption and penetration were not quantitatively modified, but HSV-1 DNA replication was inhibited. The expression of viral thymidine kinase (TK) activity after infection by recombinant virus with the TK coding sequence under immediate-early (IE) promoter control showed that IE gene expression is inhibited in cells expressing EJ-ras. Analysis of IE gene transcription by Northern-blot hybridization and by nuclear run-off transcription assay indicates that this inhibition takes place at the transcriptional level.
Abstract: Neomycin, an inhibitor of inositol phospholipid turnover, prevents Herpes-simplex-virus-type-1 (HSV-1)-induced stimulation of ribosomal protein S6 phosphorylation, but does not impair the S6 phosphorylation induced by serum. Long-term treatment with phorbol 12-myristate 13-acetate, which down-regulates protein kinase C activity, does not inhibit virus-induced S6 phosphorylation. In ras-transformed cells, S6 phosphorylation is not stimulated after HSV-1 infection. These results suggest that activation of the inositol phospholipid pathway is involved in the HSV-1-induced stimulation of S6 phosphorylation. However, protein kinase C activation does not appear to be necessary for HSV-1-induced S6 phosphorylation.
Abstract: The ends of arenavirus genome and antigenome RNAs are highly conserved and where determined directly, always contain a 3' G (referred to as position +1). However, primers extended to the 5' ends of Tacaribe virus genomes and antigenomes extend to position -1. When genomes and antigenomes are annealed either inter or intramolecularly and treated with RNase A or T1, there appears to be a single unpaired G at the 5' ends of the hybrids. A single extra G is also found by cloning the 5' ends of S antigenomes, and studies with capping enzyme detect (p)ppG at the 5' ends of genome and antigenome chains. A model is proposed in which genome replication initiates with pppGpC to create the nontemplated extra G. In contrast, the nontemplated bases at the 5' ends of the N mRNAs, which extend to positions -1 to -5, were found to be capped and also heterogeneous in sequence.
Abstract: Centrifugation of Tacaribe arenavirus-infected cell extracts on CsCl density gradients was used to separate genomes and antigenomes, which band at 1.31 g/ml as nucleocapsids, from mRNAs which pellet. Primer extensions on the banded RNAs showed that the 5' ends of the genomes and antigenomes were unique, whereas primer extensions on the mRNAs showed that their 5' ends were heterogenous in length, extending 0-4 bases beyond the 3' ends of the templates for their synthesis. This suggests that arenavirus mRNAs may initiate by a cap-snatching mechanism, somewhat similar to influenza viruses and bunyaviruses. We also found an extra G residue at the 5' end of the genome RNA, which was not predicted according to current models. This is now the third time that the unexpected G residue has been found at the 5' end of arenavirus genomes.
Abstract: Modifications of ribosomes have been investigated in human epidermoid carcinoma-2 cells at different stages of herpes simplex virus type 1 infection. Very early in infection, there is an increase in ribosomal protein S6 phosphorylation even in the absence of serum. The same result is obtained in the presence of actinomycin D. At early infection time, ribosomal proteins S2, S3a and Sa are newly phosphorylated. At early and early-late times, three phosphorylated non-ribosomal proteins (v1, v2 and v3) are differently associated temporally to ribosomes. Analyses of proteins extracted from 40S subunits, 80S ribosomes and polysomes show that v1 and v2 are distributed differently among the different ribosomal populations. S6 phosphopeptides were found to be identical after serum stimulation and after viral infection. In every case phosphoserine and phosphothreonine were identified in S6. Only phosphoserine was found in other phosphorylated proteins. Our results indicate that herpes simplex virus type 1 is able to modify pre-existing ribosomes: (i) by stimulating a pre-existing kinase for S6 phosphorylation even in the absence of serum and of viral genome expression; (ii) by inducing new specific kinase activity(ies); and (iii) by association of new, phosphorylated proteins to ribosomes. These ribosomal modifications are correlated with changes in protein synthesis, as shown by two-dimensional electrophoretic analyses of newly synthesized 35S-labelled proteins.
