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The Homologies of Spontaneous and Induced Temperature-sensitive Mutants of Vesicular Stomatitis Virus Isolated in Chick Embryo and BHK 21 Cells

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Abstract

A series of 71 spontaneous temperature-sensitive mutants of vesicular stomatitis virus (type indiana) have been isolated in chick embryo cells (Flamand, 1970). In addition, 175 induced mutants have been isolated from a different wild-type strain propagated in BHK 21 cells (Pringle 1970b). These mutants have been classified into complementation groups independently. Reciprocal complementation experiments are now described which establish the genetic homologies of these mutants. The results obtained in the two systems are in good agreement, despite differences in experimental procedure, restrictive temperature, wild-type strain and host cell. It can be concluded that complementation groups I–IV in the Glasgow classification correspond to the groups represented by ts 4, ts 52, ts 23 and ts 100 in the Orsay system. Mutant ts 45 (Orsay) belongs to a fifth group not represented among the Glasgow mutants.

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... The rVSVgag5 vector used in this work as an nonattenuated reference standard during immunogenicity studies was generated by cloning the gag p55 open reading frame into an XhoI/NheI expression cassette located between the virus G and L genes (34). To construct vectors encoding temperature-sensitive (ts) mutations in the N and L genes (rVSVtsNϩLgag1), the N and L genes in vector genomic cDNA were replaced with the corresponding genes from mutant viruses, tsG11(N) and tsG41(L) (63,64). Briefly, viral RNA was isolated from Vero cells infected with mutant virus at a permissive temperature. ...
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Unlabelled: In previous work, a prototypic recombinant vesicular stomatitis virus Indiana serotype (rVSIV) vector expressing simian immunodeficiency virus (SIV) gag and human immunodeficiency virus type 1 (HIV-1) env antigens protected nonhuman primates (NHPs) from disease following challenge with an HIV-1/SIV recombinant (SHIV). However, when tested in a stringent NHP neurovirulence (NV) model, this vector was not adequately attenuated for clinical evaluation. For the work described here, the prototypic rVSIV vector was attenuated by combining specific G protein truncations with either N gene translocations or mutations (M33A and M51A) that ablate expression of subgenic M polypeptides, by incorporation of temperature-sensitive mutations in the N and L genes, and by deletion of the VSIV G gene to generate a replicon that is dependent on trans expression of G protein for in vitro propagation. When evaluated in a series of NHP NV studies, these attenuated rVSIV variants caused no clinical disease and demonstrated a very significant reduction in neuropathology compared to wild-type VSIV and the prototypic rVSIV vaccine vector. In spite of greatly increased in vivo attenuation, some of the rVSIV vectors elicited cell-mediated immune responses that were similar in magnitude to those induced by the much more virulent prototypic vector. These data demonstrate novel approaches to the rational attenuation of VSIV NV while retaining vector immunogenicity and have led to identification of an rVSIV N4CT1gag1 vaccine vector that has now successfully completed phase I clinical evaluation. Importance: The work described in this article demonstrates a rational approach to the attenuation of vesicular stomatitis virus neurovirulence. The major attenuation strategy described here will be most likely applicable to other members of the Rhabdoviridae and possibly other families of nonsegmented negative-strand RNA viruses. These studies have also enabled the identification of an attenuated, replication-competent rVSIV vector that has successfully undergone its first clinical evaluation in humans. Therefore, these studies represent a major milestone in the development of attenuated rVSIV, and likely other vesiculoviruses, as a new vaccine platform(s) for use in humans.
... Different steps of glycosylation of G protein start at the two glycosylation sites (residues 179 and 335) when G protein was either still bound to the ribosome as a nascent polypeptide chain or shortly after the biosynthesis is over (Robertson, Etchison & Summers, 1976). The signal peptide of 16 amino acids is cleaved cotranslationally after translocation from ER lumen (Gallione & Rose, 1983;Gallione & Rose, 1985;Flamand & Pringle, 1971). ...
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Full-text available
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Cells infected with temperature-sensitive (ts) mutants of complementation group V of vesicular stomatitis virus (VSV) give an enhanced yield at nonpermissive temperature when co-infected or superinfected with UV-irradiated virus. Virions produced in these mixed infections are temperature sensitive and do not complement ts V45. Rescue of group V mutants is multiplicity dependent. It can occur in the presence of cycloheximide; kinetics of rescue are similar in the absence or in the presence of the drug. Rescue is due to nongenetic complementation and is interpreted as a trigger effect on maturation of a small quantity of biologically active protein V molecules provided by UV-irradiated virus. These results are comfirmed by rescue of ts V45 by UV-irradiated, defective, interferring T particles.
Chapter
There are five families of RNA viruses in which the negative strand is sequestered in the extracellular virion. Viruses of two of these families, the Rhabdoviridae and the Paramyxoviridae, have unitary linear genomes, whereas viruses of the other three families, Arenaviridae, Bunyaviridae, and Orthomyxoviridae, have segmented genomes comprising, respectively, two, three, and seven or eight subunits. The informational macromolecules that comprise the genomes of rhabdoviruses and paramyxoviruses are among the largest functional RNA molecules and are exceeded in size only by those of the plus-strand coronaviruses. Reanney (1982, 1984) has calculated that the upper size limit for any RNA virus genome cannot be much in excess of 17,600 nucleotides (mol. wt. ≈5.7 × 106) as a consequence of the low copying fidelity of RNA polymerases. The segmentation of the genomes of the other negative-strand viruses may be a consequence of such constraints on molecular size or a device for decoupling the transcription of individual genes. Whatever the reason, the genetic properties of the segmented-genome viruses differ substantially from those of the unsegmented-genome viruses, because variation in the former is generated by reassortment of genome subunits as well as by mutation. Mutation is the sole mechanism of variation in unsegmented-genome viruses, since the intramolecular recombination observed with positivestrand RNA viruses does not seem to be permissible for any negativestrand RNA virus.
Chapter
The rhabdoviruses are enveloped bullet-shaped viruses infecting a wide range of organisms (for reviews, see Howatson, 1970; Knudsen, 1973; Wagner, 1975). The host may be a vertebrate (bird, fish, or mammal), an invertebrate (insect or arachnid), or a plant. Some rhabdoviruses, like Chandipura virus or vesicular stomatitis virus (VSV), have a wide host range and may be vector transmitted in nature. Indeed, although VSV is known because of the disease it produces in man and domestic animals, it is capable of multiplying in both its invertebrate vector and the vertebrate host, and it has been suggested that VSV is not primarily a virus of vertebrates (Tech et al., 1970). The plant rhabdovirus, lettuce necrotic yellows virus, can also multiply in its insect vector, and one rhabdovirus, sigma virus, is known only as a hereditary infection of the insect Drosophila. However, other rhabdoviruses, such as rabies virus, are restricted in their host range and no insect vector appears to be involved in their spread.
Article
Following the discovery of the androgens in the early nineteen thirties and the first demonstration of their anabolic effects in 1935, evidence rapidly accumulated to show that practically no tissue in the body was immune from possible androgen stimulation, whether assessed on histological or on biochemical criteria. Considering the diversity of the biochemical effects attributable to the androgens, it is not surprising that an equal diversity is to be seen in the experiments designed to study these effects. Arbitrarily, although with some justification on historical and biochemical grounds, these studies can be said to fall into two main groups.
Eighty-four temperature-sensitive (ts) mutants were isolated from Lumbo virus, an African strain of Tahyna virus (Bunyaviridae) after chemical mutagenesis by either 5-fluorouracil or N-methyl-N-nitroso-N'nitroguanidine.These mutants were analysed by complementation-recombination test.Genetic analysis showed that the mutants are distributed in three complementation-recombination groups.RésuméQuatre-vingt quatre mutants thermosensibles ont été isolés à partir du virus Lumbo (Bunyavirus du groupe California) par mutagenèse à l'aide de 5-fluoro-uracile ou de N-methyl-N-nitroso-N'nitroguanidine.L'analyse génétique a montré que ces mutants se répartissent en trois groupes de complémentation-recombinaison.
Article
Summary Temperature-sensitive mutants of vesicular stomatitis virus were examined for their ability to synthesize intracellular virus-specific RNA species at the nonpermissive temperature. Four mutants from complementation group I (ts 11, ts 12, ts 13, ts 114) were compared to two mutants from complementation group IV (ts 41, ts 44). Mutants within one group did not resemble each other in their phenotypes; nevertheless, two distinct pathways of RNA synthesis were detected: one for transcription and the other for replication.Copyright © 1974 S. Karger AG, Basel
Temperature shift experiments done on infectious centres reveal the existence of two classes of thermosenstive mutants. Early mutants are characterized by a defect in a necessary function during the first 24 h after inoculation at 20° C. On the contrary, late mutants are characterized by a defect in a function which is necessary after the first 24 h of infection. Two types of early mutants are defined and allow to distinguish two early steps of sigma virus cycle at 20°C. The temperature-sensitive period of the haP7 mutant characterizes the first step lasting from 0 to 9 h; the ts4 mutant has a temperature-sensitive period extending from 4 to 15 h and defines the second step. It is proposed that the viral genome replication takes place between 4 and 15 h. Analogy between the two types of early ts mutants of sigma virus and some ts mutants of vesicular stomatitis virus will be discussed in this paper.
Article
To isolate new types of vesicular stomatitis virus (VSV) mutants, a four-stage screen was developed which identifies and characterizes mutants capable of complementing the defect in the VSV temperature-sensitive mutant tsG11. Two types of mutants of VSV, Indiana serotype, have been found by using the screen; they are new temperature-sensitive mutants which are, of necessity, not in complementation group I and mutants which do not produce plaques under conditions of single infection at 31 C (the normal permissive temperature) and are, therefore, called complementation-dependent mutants. The newly isolated, temperature-sensitive mutants fall into three complementation groups, two of which are congruent with known complementation groups; the newly identified group extends to six the number of complementation groups of VSV Indiana. The nature of the complementation-dependent mutants has not been established, but one was shown to not contain a significant deletion in its nucleic acid.
Article
The complementation properties of the virus progeny released from cells mixedly infected with mutants of vesicular stomatitis virus belonging to four different complementation groups have been examined. The group IV mutant, tsW16B, was tested in combinations with three group I mutants (tsW4, tsW28, and tsG11), one group II mutant (tsG22), and one group III mutant (tsW29). Virus stocks were grown from isolated plaques appearing on the cell monolayers used to assay the mixed infection yields and tested, in a second series of mixed infections, for their ability to complement each of the two parents. It was found that the virus harvested from each one of the first series of mixed infections contained mutants of both parental types.
Article
Noninfectious spikeless particles have been obtained from vesicular stomatitis virus (VSV, Indiana serotype) by bromelain or Pronase treatment. They lack the viral glycoprotein (G) but contain all the other viral components (RNA, lipid, and other structural proteins). Triton-solubilized VSV-Indiana glycoprotein preparations, containing the viral G protein as well as lipids (including phospholipids), have been extracted from whole virus preparations, freed from the majority of the detergent, and used to restore infectivity to spikeless VSV. The infectivity of such particles has been found to be enhanced by poly-L-ornithine but inhibited by Trition or homologous antiserum pretreatment. Heat-denatured glycoprotein preparations were not effective in restoring the infectivity to spikeless VSV. Heterologous glycoprotein preparations from the serologically distinct VSV-New Jersey serotype were equally capable of making infectious entities with VSV-Indiana spikeless particles, and the infectivity of these structures was inhibited by VSV-New Jersey antiserum but not by VSV-Indiana antiserum. Purified, detergent-free glycoprotein selectively solubilized from VSV-Indiana by the dialyzable detergent, octylglucoside, also restored infectivity of spikeless virions of VSV-Indiana and VSV-New Jersey.
Chapter
Early work on the genetics of animal viruses was reviewed, with extensive references, by Fenner and Sambrook (1964), and work done between 1963 and 1969 by Fenner (1970a). A general account of the biology of animal viruses is available in Fenner (1968) and has been brought up to date in the second edition of that book (Fenner et al., 1974). Names used for viral groups (families and genera) in general follow Wildy (1971).
Article
Rescue virions obtained after superinfection of ts O45(V) Indiana serotype-infected cells by uv-irradiated vesicular stomatitis virus (VSV) at a nonpermissive temperature do not incorporate G protein synthesized at this temperature. They apparently contain G protein in their envelope since they are neutralized by homotypic antivirion and antispike sera. Rescue of ts O45(V) mutant Indiana serotype by uv-irradiated VSV New Jersey serotype is demonstrated. Ultraviolet-inactivated VSV of New Jersey serotype has therefore been used to determine the origin of the incorporated G protein molecules. The progeny-rescue virions belong genetically to VSV Indiana complementation group V. They are however neutralized by anti-New Jersey serum and not by anti-Indiana serum. Rescue ts O45(V) virions have thus probably reincorporated G protein molecules supplied by the uv-irradiated New Jersey virus. This provides further evidence suggesting that protein G is encoded for by gene V.
Article
The dose response of cell killing particle (CKP) activity of vesicular stomatitis virus (VSV) in GMK Vero cells treated with interferon or mouse L cells treated with poly(rI)·poly(rC) was determined from single-cell survival curves, and compared with dose-response curves for infectivity and viral transcript accumulation. “Pulse-infection” with temperature-sensitive (ts) mutants of VSV was used to confine formation of putative cell killing factor or its precursors to the time equivalent of a single cycle of growth. Cells which through interferon or poly(rI)·poly(rC) action survived a pulse-infection with ts-mutant virus at permissive temperature produced colonies normal in both appearance and growth rate. Cells treated with interferon or poly(rI)·poly(rC) and challenged with VSV generated a family of survival curves for CKP activity whose slopes decreased as the dose of the interferon-interference inducer increased. These survival curves revealed that the capacity to express CKP activity or accumulate transcripts in interferon-treated Vero cells were both lost initially at an exponential rate about four to five times slower than that observed for the loss of plaque or yield reducing capacity, demonstrating that it required four to five times more interferon to prevent cell killing or transcript accumulation by VSV than was needed to reduce infectivity by an equivalent amount. A marked tailing of the survival curves was observed as the interferon dose was increased, resulting in surviving activity for CKP and transcript accumulation far in excess of that expected by extrapolation from low-dose regions of the curve. These results are discussed in light of our previous hypothesis which stated that viral transcription was a requisite, though insufficient reaction to produce cell killing by VSV and that integrity of about one-fifth of the viral genome sufficed to provide these transcripts (Marcus, P. I., and Sekellick, M. J. (1974). Virology57, 321–338; (1975). Virology63, 176–190). We extend this hypothesis and propose that the lethal action of VSV on cells requires: (i) functional virion transcriptase, (ii) transcription of a special one-fifth of the genome, most likely that coding for proteins N and NS, (iii) translation of these transcripts into minimally functional polypeptides which lead to (iv) formation and subsequent action by a putative cell killing factor.
Article
Fv-1 gene-mediated host restriction of Friend leukemia virus replication was investigated in terms of coat protein synthesis. By using the assay of pseudotype formation with vesicular stomatitis virus. it was shown that under restricting growth conditions the availablity of leukemia virus coat protein for pseudotype formation was decreased. These studies appear to eliminate a pure assembly defect as the mechanism of Fv-1 host restriction.
Article
In vitro transcriptase activity of three group I temperature-sensitive (ts) mutants of vesicular stomatitis virus restricted at 39 C was restored by L-protein fractions derived from wild-type (wt) vesicular stomatitis virion nucleo-capsids. Soluble NS protein from wt nucleocapsids did not reconstitute restricted transcriptions of the group I RNA-ts mutants. NS protein activity, but not L protein activity, was purified from the group I ts mutants; this NS fraction always displayed the wt phenotype in reconstitution assays. Neither the L nor the NS protein was capable of restoring the defective transcriptive activity of the group IV vesicular stomatitis virus mutant ts W16B.
Article
Infectious B particles of vesicular stomatitis virus (VSV) are capable of inhibiting the replication of pseudorabies virus (PSR) in a variety of cell lines. Even under conditions of an abortive infection in a continuous line of rabbit cornea cells (RC-6O), B particles interfere with the replication of PSR with high efficiency. Particle per cell dose-response analysis of B particle populations revealed that the number of VSV particles capable of inhibiting PSR replication exceeds the number of PFU by a factor of 32 to 64. When B particles are treated with UV irradiation, a drastic increase in the multiplicity of infection is required to inhibit PSR replication. Whereas one infective B particles per cell is sufficient to prevent replication of PSR, 800 to 1,000 VSV particles rendered noninfective by UV irradiation are required to compensate for the loss of VSV synthetic activity that results from irradiation. Temperature-sensitive mutants representing five complementation groups of VSV were tested at low multiplicities of infection for their effect on PSR replication at the nonpermissive temperature. Generally, the ability of the different complementation groups to amplify virion products at the nonpermissive temperature is associated with their ability to inhibit PSR replication. These results imply that at low multiplicities of infection, amplification of infecting VSV components is necessary for inhibition of PSR replication., but at high multiplicities of infection with VSV, a virion component can prevent PSR replication in the absence of de novo VSV RNA or protein synthesis.
Article
The RNA polymerase in cells infected with three group I mutants of vesicular stomatitis virus has been examined. Mouse L cells were incubated at the permissive temperature (30 degrees C) for a few hours after infection to allow the development of secondary transcription. The temperature dependence of the secondary transcription system was determined from the incorporation of labelled uridine, in the presence of cycloheximide, at 30 and at 38 degrees C, the later temperature being non-permissive for viral replication. In cells infected with mutants W14, W28, and G11 at a low multiplicity (20 PFU/cells) secondary transcriptase activity was markedly temperature-sensitive after 3 and 5 h of infection at 30 degrees C. At a high multiplicity of infection (1000 PFU/cell) cells infected with W28 showed considerable RNA synthesis at 38 degrees C after 3 h at 30 degrees C. RNA synthesis was also observed in W28-infected cells in which protein synthesis was allowed to continue after the shift from 30 to 38 degrees C. In the latter two cases the RNA synthesized contained 12-18S species but little or no 30S mRNA.
Article
Rescue at the nonpermissive temperature of temperature-sensitive mutants of vesicular stomatitis virus (VSV) by ultraviolet-irradiated VSV has been investigated by means of its wavelength dependence. Targets of uv irradiation that are responsible for the rescue were determined. Rescue of ts O45(V) by a structural protein supplied by the irradiated parent is confirmed. The action spectra suggest that, in the dose range explored, the target acting in the rescue of ts O53(I) is exclusively protein in nature while the rescue of ts O111(IV) is mainly due to the functional survival of gene IV. The rescue of ts O52(II) could be related partly to the functional survival of gene II and partly to the structural protein II molecules provided by the infecting irradiated helper virus. The proposed explanation for the latter case is discussed taking into account the hypothesis that the site of the mutational lesion of group II is the gene encoding for structural protein N.
Article
In cultures of Chinese hamster ovary cells infected by vesicular stomatitis virus (VSV), viral polypeptides can be found in cellular, virion-associated, and soluble fractions. In order to determine whether or not the soluble polypeptides resulted from degradation of virions, progeny formation was inhibited and the distribution of polypeptides in these fractions was examined. Synthesis of infectious progeny was prevented in three ways: (1) addition of defective interfering (DI) particles; (2) utilization of a replication-minus mutant, tsG41; and (3) utilization of a glycoprotein mutant, ts045. In experiments involving either the coinfection of standard infectious particles and DI particles or the infection of cells at the nonpermissive condition for tsG41, overall virus-specific RNA synthesis was reduced by 80%, whereas the appearance of extracellular soluble viral polypeptides was not greatly inhibited. Cell-associated polypeptides were also present in normal amounts. By using tsO45, it was shown that there was no virion maturation but, surprisingly, the appearance of soluble viral polypeptides was enhanced. Therefore, the distribution of viral proteins in the cell-associated and extracellular soluble fractions was not dependent on the production of infectious progeny. The major soluble viral polypeptide was a glycoprotein which migrated more rapidly than the virion-associated glycoprotein; these two glycoproteins had about the same amount of carbohydrate, even when the soluble glycoprotein was synthesized by tsO45 under nonpermissive conditions. These results lead to the hypothesis that G protein interaction with the plasma membrane exists in three different states: (1) an initial very unstable association; (2) possible stabilization at the cell surface via interactions with another membrane protein called matrix protein; and (3) aggregation of G protein at the cell surface initiated by nucleocapsids interacting through the matrix proteins. The first two stages of this interaction probably result in the shedding of G protein from the cell surface.
Article
The replication of vesicular stomatitis virus (VSV) is inhibited by tunicamycin (TM), an antibiotic that blocks the formation of N-acetylglucosaminelipid intermediates. We had shown previously that the viral glycoprotein (G) synthesized in cells treated with TM is not glycosylated and is not found on the outer surface of the cell plasma membrane. In this report, we shown that cells exposed to TM produce a low yield of infectious particles. The yield is increased when the temperature during infection is lowered from 37 to 30 degrees C. At 30 degrees C in the presence of TM, both wild-type VSV and the temperature-sensitive mutant ts045 produce particles that do not bind to concanavalin A Sepharose and contain only the nonglycosylated form of G. These particles have a specific infectivity (pfu/cpm) comparable to that of VSV containing glycosylated G.
Article
Temperature-sensitive (ts) mutants of vesicular stomatitis virus belonging to the RNA(-) complementation group IV were investigated under various conditions to study both their RNA and protein syntheses. In infected cells maintained at 39.2 C, viral RNA species were recovered only in the 13 to 15S region of the gradient in an amount depending on the ts mutant used. In the presence of cycloheximide at 39.2 C, the primary transcription was deficient, especially for 28S mRNA production. When mutant-infected cells were shifted to nonpermissive temperature, a shutoff of 28S mRNA synthesis occurred as a general feature. On the contrary under this condition, the two mutants chosen, ts IV100 and ts IV111, behaved very differently in their 13 to 15S and 38S RNA production. However, treatment with cycloheximide at the time of the transfer to 39.2 C resulted in a similar recovery of 13 to 15S RNA in both mutants, whereas the 28S remained very depressed. The viral proteins synthesized by cells infected with the same two mutants also showed a distinct pattern, especially regarding the N protein; a correlation between 38S RNA and protein N syntheses was tentatively drawn. The whole set of data suggested that the lesion in group IV mutants concerned a viral structural protein required for the process of in vivo transcription and which probably intervened in the replication mechanism.
Etude des mutants thermosensibles du Virus de la Stomatite V6siculaire
  • A Rlamand
rLAMAND, A. (~969)-Etude des mutants thermosensibles du Virus de la Stomatite V6siculaire. Mise au point d'un test de compl6mentation. Comptes rendus hebdomadaires des sdances de l'Acaddmie des Sciences" 268, 2305.