Heterogneeous 5'-terminal structures occur on vesicular stomatitis virus mRNAs.

Four alternative structures occur at the 5' ends of vesicular stomatitis virus mRNAs synthesized in infected cells and are separated conveniently by a technique described here. Sixty-five to seventy per cent of the mRNA molecules have the 5' end structure m7G5'ppp5'(m)AmpAp and about 20% have a more highly modified structure m7G5'ppp5'(m)AmpmAmpCp. The base of the first adenosine in each sequence is methylated in about one-half of the ends of each type and kinetic experiments suggest that the latter sequence is derived from the former by further methylations. The remaining 10 to 15% of the 5' ends are pppAp and pppGp in approximately equimolar yields. This heterogeneity with respect to 5' end structure is found within each of the vesicular stomatitis virus mRNA species examined. The mRNA molecules with 5'-triphosphate ends accumulate throughout the infection but are not found on ribosomes, suggesting that they lack a structure(s) required for ribosome recognition. In contrast to mRNA, virion RNA has a single 5' end structure, pppAp.     

The 5' terminal structure of the methylated mRNA synthesized in vitro by vesicular stomatitis virus.

The 5' terminal structure of the mRNA synthesized in vitro by the virion-associated RNA polymerase of vesicular stomatitis virus in the presence of S-adenosyl-L-methione consists of 7-methyl guanosine linked to 2'-O-methyl adenosine through a 5'-5' pyrophosphate bond as m7G(5')ppp(5')A-m-p ... The alpha and beta phosphated of GTP and alpha phosphate of ATP are incorporated into the blocked 5' terminal structure.     

Two methyltransferase activities in the purified virions of vesicular stomatitis virus.

In addition to an RNA-dependent RNA polymerase, purified vesicular stomatitis virus contains a methyltransferase activity which transfers the methyl group from the methyl donor, S-adenosyl-L-methionine, to two positions in the 5'-terminal capped structure of the nascent mRNA's synthesized in vitro as 7mG-(5)'ppp(5')Apm... In the present study it is shown that two distinct methyltransferase activities are discernible in the purified virus. The in vitro concentrations of the methyl donor specify the number and location of the methyl groups transferred to the capped 5'-termini of VSV mRNA's. Limited concentrations of the methyl donor result in a single methylation of the penultimate base in the 2'-hydroxyl position, that is, G(5')ppp(5')Apm..., whereas saturating concentrations of the methyl donor methylate the blocking guanosine residue at the 7-position, resulting in the dimethylated cap, 7mG(5')ppp(5')Apm... Pulse-chase experiments demonstrate that the monomethylated cap structure is the precursor substrate for the dimethylated cap. In this respect, vesicular stomatitis virus system is quite distinct from the vaccinia and reovirus systems. Virus purified from different host cells including hamster, mouse, and human contain both methyltransferase activities. The mRNA's containing monomethylated capped structures are poor templates for protein synthesis in vitro.     

Effect of viral infection on host protein synthesis and mRNA association with the cytoplasmic cytoskeletal structure

We studied the association of several eucaryotic viral and cellular mRNAs with cytoskeletal fractions derived from normal and virus-infected cells. We found that all mRNAs appear to associate with the cytoskeletal structure during protein synthesis, irrespective of their 5' and 3' terminal structures: e.g., poliovirus that lacks a 5' cap structure or reovirus and histone mRNAs that lack a 3' poly A tail associated with the cytoskeletal framework to the same extent as capped, polyadenylated actin mRNA. Cellular (actin) and viral (vesicular stomatitis virus and reovirus) mRNAs were released from the cytoskeletal framework and their translation was inhibited when cells were infected with poliovirus. In contrast, actin mRNA was not released from the cytoskeleton during vesicular stomatitis virus infection although actin synthesis was inhibited. In addition, several other conditions under which protein synthesis is inhibited did not result in the release of mRNAs from the cytoskeletal framework. We conclude that the association of mRNA with the cytoskeletal framework is required but is not sufficient for protein synthesis in eucaryotes. Furthermore, the shut-off of host protein synthesis during poliovirus infection and not vesicular stomatitis virus infection occurs by a unique mechanism that leads to the release of host mRNAs from the cytoskeleton.     

Defective interfering particle generated by internal deletion of the vesicular stomatitis virus genome.

The genome structure of the long, truncated defective interfering particle derived from the heat-resistant strain of vesicular stomatitis virus has been examined. Stocks of this defective interfering particle are shown to contain several different species having information primarily from the 3' half of the vesicular stomatitis virus genome; the proportions of these components vary depending on the passage history of the stock. The two most abundant types have been identified and characterized. One has complementary 5' and 3' termini and consequently appears as a circular molecule when examined by electron microscopy. The other cannot circularize and remains linear. The circular forms are consistently 8 to 10% longer than the linear molecules. Rapid sequencing analyses reveal that both forms retain the 5' parental viral terminal sequence, but only the linear form retains the parental 3'-terminal sequence which is the complement of the 5' end. Hybridization experiments and electron microscopic analyses indicate that the linear form has retained 320 to 350 nucleotides of the 5' parental sequence and was probably generated by an internal deletion of the vesicular stomatitis virus genome.     

Regulation of MuIFN-alpha/beta and MuIFN-gamma-induced antiviral states: differential effects with different challenge viruses and lack of correlation with 2'5' oligoadenylate synthetase levels

The MuIFN-alpha/beta and MuIFN-gamma induced antiviral states which are directed against mengovirus have been shown previously to be differentially regulated. Following interferon removal, the MuIFN-alpha/beta-induced antiviral state decays rapidly, while the MuIFN-gamma-induced antiviral state increases dramatically. To determine whether these observations with mengovirus represent part of a general phenomenon, these studies have been extended using vesicular stomatitis virus and vaccinia virus, which represent two distinctly different groups of viruses. The antiviral states induced by MuIFN-gamma against all three viruses increased dramatically following interferon removal. The antiviral state induced by MuIFN-alpha/beta against vesicular stomatitis virus was stable following interferon removal, while the antiviral states induced by MuIFN-alpha/beta against mengovirus and vaccinia virus decayed rapidly. Also, levels of 2'5' oligoadenylate synthetase were determined at various times following interferon removal. MuIFN-alpha/beta was found to be a relatively strong inducer of 2'5' oligoadenylate synthetase, while MuIFN-gamma was a relatively weak inducer. Further, while the changes in 2'5' oligoadenylate synthetase levels paralleled the changes in the levels of the antiviral states induced by MuIFN-alpha/beta and MuIFN-gamma against mengovirus and vaccinia virus, the changes in 2'5' oligoadenylate synthetase levels did not parallel the changes in the antiviral state induced by MuIFN-alpha/beta against vesicular stomatitis virus. The results suggested that the 2'5' oligoadenylate synthetase levels did not correlate with the level of antiviral state.     

Vesicular stomatitis virus-infected cells fuse when the intracellular pool of functional M protein is reduced in the presence of G protein.

Five highly cytolytic strains of both Indiana and New Jersey serotypes of vesicular stomatitis virus were shown to induce cell fusion in BHK-21 and R(B77) cells. Inhibition of protein synthesis after the eclipse period of viral replication is a prerequisite for vesicular stomatitis virus-induced cell fusion. Pulse-chase experiments showed that inhibition of protein synthesis would lead to a drastic reduction in the intracellular pool of M protein as compared with other proteins. A temperature-sensitive mutant defective in M protein function (G31) was the only mutant of the five complementation groups to spontaneously induce polykaryocytes at the nonpermissive temperature. Previously, G protein has been shown to play a role in vesicular stomatitis virus-induced cell fusion. These results suggest that the combination of the presence of G protein on the virus-infected cell surface and the absence of functional M protein or a reduced level of intracellular M protein promotes cell fusion. On the basis of this study, we propose that vesicular stomatitis virus infection can induce cell fusion when the functional M protein pool declines to a critical level while G protein remains on the cell surface.     

Injection of mice with antibody to mouse interferon alpha/beta decreases the level of 2''-5'' oligoadenylate synthetase in peritoneal macrophages.

Injection of conventional or axenic weanling mice with potent sheep or goat antibody to mouse interferon alpha/beta resulted in a decrease in the basal level of 2-5A synthetase in resting peritoneal macrophages and rendered these cells permissive for vesicular stomatitis virus. There was a good inverse correlation between the level of 2-5A synthetase in peritoneal macrophages and the permissivity of these cells for vesicular stomatitis virus. The peritoneal macrophages of 1- and 2-week-old mice had low levels of 2-5A synthetase and were permissive for vesicular stomatitis virus, whereas at 3 weeks (and after) there was a marked increase in the level of 2-5A synthetase in peritoneal macrophages, and these cells were no longer permissive for vesicular stomatitis virus. We suggest that low levels of interferon alpha or beta or both are produced in normal mice, and that this interferon contributes to host defense by inducing and maintaining an antiviral state in some cells.     

In vitro RNA transcription by the New Jersey serotype of vesicular stomatitis virus. I. Characterization of the mRNA species.

The in vitro RNA synthesis by the virion-associated RNA polymerase of vesicular stomatitis virus (VSV), New Jersey serotype, was compared with that of the serologically distinct Indiana serotype of VSV. The New Jersey serotype of VSV synthesized five distinct mRNA species in vitro, three of which were smaller than the corresponding species synthesized by the Indiana serotype of VSV. These included the mRNA's coding for the G, M, and NS proteins. By hybridization experiments, virtually no sequence homology was detected between the mRNA's of the two serotypes. Despite this lack of overall homology, the 12 to 18S mRNA species of both serotype contained a common 5'-terminal hexanucleotide sequence, G(5')ppp(5')A-A-C-A-G. The signicance of this finding in light of specific interactions between the two serotypes of VSV in vivo is discussed.     

Translation of capped and uncapped vesicular stomatitis virus and reovirus mRNA'S. Sensitivity to m7GpppAm and ionic conditions.

In an attempt to elucidate the role of the 5'-terminal 7-methylguanosine residue in translation of mammalian mRNAs, vesicular stomatitis virus (VS virus), and reovirus mRNAs containing and lacking this residue, and also Qbeta RNA, were translated in cell-free extracts from reticulocytes and wheat germ under a variety of ionic conditions. Optimal translation of mRNAs lacking a 5' m7G occurred at concentrations of KOAc or KCl which were lower than those optimal for normal "capped" mRNAs. However, this salt dependence was much less marked in the mammalian reticulocyte extract and, at salt concentrations optimal for translation of normal capped mRNAs, reticulocyte lysates translated uncapped with mRNAs at 30 to 60% the normal efficiency. At low K+ concentrations, wheat germ ribosomes bound and translated appreciable amounts of uncapped VS virus mRNA; controls showed that no m7G residue is added to the 5' end of the bound RNA. Analogues of the 5' end, such as m7GpppAm, inhibited translation of both normal and uncapped VS virus RNAs in wheat germ extracts to about the same extent, but the efficiency of its action was reduced at low K+ concentrations. We conclude that there is a reduced importance of the 5' m7G residue in ribosome-mRNA recognition at low K+ concentrations, and that translation of mRNAs in reticulocyte extract is, under any reaction conditions, less dependent on the presence of a 5' "cap" than in wheat germ extracts.     

Nucleotide sequences from the 3''-ends of vesicular stomatitis virus mRNA''s as determined from cloned DNA.

Molecular clones of vesicular stomatitis virus mRNA's were used to determine the 3'-terminal sequences of mRNA's encoding the N and NS proteins. This new approach to VSV mRNA sequencing allowed the first comparison of 3'-terminal sequences. The sequences showed a tetranucleotide homology, UAUG, immediately preceding the polyadenylic acid. In addition, both mRNA's had an AU-rich region including the tetranucleotide AUAU at positions 16 to 19 nucleotides from the polyadenylic acid. A possible secondary structure between the 3' end of N mRNA and the 5' end of the adjacent NS mRNA is noted. These structural features may serve as signals for termination (or cleavage) and polyadenylation of vesicular stomatitis virus mRNA's. Neither mRNA had the polyadenylic acidproximal hexanucleotide, AAUAAA, found in eucaryotic cellular and viral mRNA's transcribed from nuclear DNA. The probable location of the translation termination codon for the NS protein is only six nucleotides from polyadenylic acid in NS mRNA.     

Relative importance of 7-methylguanosine in ribosome binding and translation of vesicular stomatitis virus mRNA in wheat germ and reticulocyte cell-free systems.

Vesicular stomatitis virus mRNAs with these four types of 5'-termini, (a) m7G5'ppp5'(m)Am, (b) ppp5'(m)Am, (c) m7G5'-ppp5' Am, and (d) G5'ppp5'A, were prepared and their translation and ribosome binding analyzed in wheat germ and reticulocyte cell-free protein synthesis systems. The relative efficiencies of translation of individual vesicular stomatitis virus (VSV) mRNAs having type 2 termini ranged from 23 to 29% of the control (type 1) RNA in the reticulocyte system and 6 to 7% of control RNA in the wheat germ system. A similar difference between the two systems was seen in ribosome-binding experiments in which type 2 RNA formed an 80 S initiation complex with high efficiency (70% of control type 1 RNA) in the reticulocyte system, but with low efficiency (17% of control RNA) in the wheat germ system. Similar differences in the importance of m7G in translation in the two systems were seen when VSV mRNAs synthesized in vitro with type 3 and type 4 termini were analyzed. However, the analysis of type 4 RNA (which was synthesized in vitro in the presence of S-adenosylhomocysteine) was complicated by the presence of abnormally large poly(A) at its 3'-end. Another series of experiments showed that compounds such as 5'pm7G and m7G5'ppp5'Np are potent and specific inhibitors of translation of all types of VSV mRNAs in the wheat germ system (greater than 98% inhibition) but cause less than 20% inhibition of translation in the reticulocyte system. Taken together, all of the results indicate that a 5'-terminal m7G is far more important in translation of VSV mRNAs in the heterologous plant cell-free system than in the reticulocyte lysate system.     

Fluorescence anisotropy of a fatty acid covalently linked in vivo to the glycoprotein of vesicular stomatitis virus

The covalently-attached fatty acid of the membrane glycoprotein (G) of vesicular stomatitis virus was fluorescently labeled biologically by isolating vesicular stomatitis virus from infected baby hamster kidney clone 21 cells that had been grown in the presence of 16(9-anthroyloxy)palmitate. The fluorescent labeling was specific for the G protein; the other viral membrane protein, the matrix (M) protein, was not labeled. Steady state fluorescence anisotropy of the 16(9-anthroyloxy)palmitate-labeled G protein reconstituted into dipalmitoylphosphatidylcholine vesicles indicated that the fatty acid attached to G protein is located in a dipalmitoylphosphatidylcholine domain that does not undergo the gel to liquid-crystalline phase transition.     

Characterization of conserved viral leader RNA sequences that stimulate innate immunity through TLRs

Viruses of the order Mononegavirales encompass life-threatening pathogens with single-stranded segmented or nonsegmented negative-strand RNA genomes. The RNA genomes are characterized by highly conserved sequences at the extreme untranslated 3' and 5' termini that are most important for virus infection and viral RNA synthetic processes. The 3' terminal genome regions of negative-strand viruses such as vesicular stomatitis virus, Sendai virus, or influenza virus contain a high number of conserved U and G nucleotides, and synthetic oligoribonucleotides encoding such sequences stimulate sequence-dependent cytokine responses via TLR7 and TLR8. Immune cells responding to such sequences include NK cells, NK/T cells, plasmacytoid, and myeloid dendritic cells, as well as monocytes and B cells. Strong Th1 and pro-inflammatory cytokine responses are also induced upon in vivo application of oligoribonucleotides. It appears possible that the presence of highly conserved untranslated terminal regions in the viral genome fulfilling fundamental functions for the viral replication may enable the host to induce directed innate immune defense mechanisms, by allowing pathogen detection through essential RNA regions that the virus cannot readily mutate.     

Role of the vesicular stomatitis virus matrix protein in maintaining the viral nucleocapsid in the condensed form found in native virions.

Vesicular stomatitis virus was extracted with 60 mM octylglucoside in the absence of salts and in the presence of 0.5 M NaCl. The resulting extracted virus particles were examined by electron microscopy, and the proteins present were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Extraction in the absence of salts yielded subviral structures which we cell "skeletons" as originally suggested by Cartwright et al. (J. Gen. Virol. 7:19-32, 1970). The skeletons contained the viral N, M, and L proteins, but they lacked the glycoprotein (G) entirely. Morphologically, the skeletons resembled intact vesicular stomatitis virus but they were slightly longer and smaller in diameter. Like native vesicular stomatitis virus, skeletons were found to have lateral striations spaced 5.0 to 6.0 nm apart along the length of the structure. In contrast to extraction in the absence of NaCl, extraction of vesicular stomatitis virus with 60 mM octylglucoside in the presence of 0.5 M NaCl yielded highly extended viral nucleocapsids in which N was the predominant protein; no M or G proteins could be detected. These results support the view that the M protein is involved in maintaining the nucleocapsid in the compact form found in native virions.