Different host-cell shutoff strategies related to the matrix protein lead to persistence of vesicular stomatitis virus mutants on fibroblast cells.

Acute infection of fibroblastic cell lines by the Indiana strain of vesicular stomatitis virus (VSV) usually induces dramatic cytopathic effects and shutoff of cellular gene expression. We have compared a series of independent mutants with differences in shutoff induction and found that M was mutated either in the N-terminus (M(51)R) or C-terminus (V(221)F and S(226)R). Furthermore, only double mutants (M mutation and a ts mutation related or not to M) were able to persist on fibroblast cell lines at 39 degrees C. A more detailed investigation of the infection was performed for the mutants T1026, TP3 and G31, differing in their host shutoff effects related to M protein. Viral activity in persistently infected mouse L-929 and monkey Vero cell lines was followed by viral proteins detection, RNA synthesis throughout infection and finally detection of infectious particles. All three mutants cause extensive CPE followed by emergence of persistently infected cells on Vero cells. The same thing is seen on L-929 cells except for T1026 which causes little CPE. Taken together, the results form a basis of further studies to clarify how various viral and cellular factors interact in the establishment of a persistent infection by VSV mutants.

Expression and DNA rearrangement of proto-oncogenes in Cas-Br-E-induced non-T-, non-B-cell leukemias.

The Cas-Br-E murine leukemia virus is a non-defective retrovirus that induces non-T-, non-B-cell leukemias in susceptible NIH/Swiss mice. A collection of tumors was examined for genomic DNA structure and RNA expression of known or putative proto-oncogenes and one tumor-suppressor gene, with the aim of identifying genes involved in Cas-Br-E-induced non-T-, non-B-cell leukemogenesis. Fli-1, p53, and Evi-1 were found to be rearranged in 72%, 23%, and 18% of the tumors, respectively, whereas no DNA alteration were detected for c-myc, c-myb, Pim-1, Evi-2, and EpoR genes. Evi-1 rearrangements are rarely associated with p53 or Fli-1 alterations. However, rearrangements of these last two genes are very often associated within the same tumor. Moreover, patterns of coordinated expression of critical cell growth-regulating genes are consistently associated with specific tumor types. These data suggest that Cas-Br-E can induce two types of hematopoietic neoplasias by different mechanisms.

Analysis of proviruses integrated in Fli-1 and Evi-1 regions in Cas-Br-E MuLV-induced non-T-, non-B-cell leukemias.

The DNAs of the Cas-Br-E MuLV-induced leukemias always contain somatically acquired mink cell focus-forming (MCF) recombinant proviruses. MCF recombinants could be involved during leukemogenesis at both preleukemic times and in late-stage tumors. Among the Cas-Br-E-induced non-T-, non-B-cell leukemias, viral integrations were found in the Fli-1 and Evi-1 region in 71% (36 out of 51) and 22% (16 out of 72) of the tumors analyzed, respectively. As an approach to evaluate the contribution of Cas-Br-E MCF recombinant formation in cis-activation of proto-oncogenes, we analyzed the structure of the Fli-1- and Evi-1-associated proviruses by Southern blot hybridization. In Fli-1, we found that the proviruses, ecotropic as well as MCF, are all integrated within a very short DNA region immediately upstream of the initiator ATG, toward the 3' end of a 5' exon (Ben-David, Giddens, Letwin, and Bernstein, 1991, Genes Dev. 5, 908-918). All proviruses are oriented the same way, in the 5' to 3' transcriptional sense. Both provirus types are able to direct the Fli-1 expression to the same extent presumably via a promoter insertion mechanism. Most of the proviruses had no detectable deletion and contained both 5' and 3' LTR sequences with similar U3 sequences. MCF recombinants did not show any selective advantage over ecotropic proviruses for the Fli-1 locus since the frequency of ecotropic to MCF-recombinant virus at the Fli-1 locus was identical to that observed at any other locus. This suggests that the formation of these MCF recombinants is not essential for activation of Fli-1 and that ecotropic Cas-Br-E already possesses the required sequences for full cis-activation of Fli-1. On the other hand, in Evi-1, there is a strict selection for ecotropic proviruses. Presumably, viral genetic elements outside of the U3 region could be critical for the Evi-1 cis-activation.

Determinants of thymotropism in Kaplan radiation leukemia virus and nucleotide sequence of its envelope region.

Radiation leukemia viruses (RadLVs) are a group of murine leukemia viruses which are induced by radiation and cause T-cell leukemia. Viral clones isolated from the BL/VL3 lymphoid cell line derived from a thymoma show variable tropism and leukemogenic potential. We have constructed chimeric viruses by in vitro recombination between two viruses, a RadLV that is thymotropic and an endogenous ecotropic virus that is nonthymotropic. We show here that, in contrast to thymotropism determinants identified previously, which lie in the long terminal repeat (LTR), it is the envelope region that is responsible for the thymotropism of BL/VL3 RadLV. The nonthymotropic virus which we have rendered thymotropic by transfer of the env region of RadLV in the present study has been shown previously to become thymotropic when the LTR of another thymotropic virus is inserted in its genome. Thus, the LTR and envelope gene may be involved in complementary action to lead to thymotropism.

The human homolog of the mouse common viral integration region, FLI1, maps to 11q23-q24.

FLI1 is a common mouse viral integration region in virus-induced leukemias and lymphomas. Using an evolutionarily conserved mouse probe and Southern hybridization to (rodent x human) somatic cell hybrid DNAs, the human homolog of FLI1 has been shown to lie on a fragment of chromosome 11 flanked on the centromeric side by the acute lymphoblastic leukemia-associated t(4;11)(q21;q23) translocation breakpoint and on the telomeric side by the Ewing- and neuroepithelioma-associated t(11;22) (q24;q12) breakpoint.

Identification of a common viral integration region in Cas-Br-E murine leukemia virus-induced non-T-, non-B-cell lymphomas.

The Cas-Br-E murine leukemia virus is a nondefective retrovirus that induces non-T-, non-B-cell lymphomas in susceptible NIH/Swiss mice. By using a DNA probe derived from Cas-Br-E provirus-flanking sequences, we identified a DNA region, originally called Sic-1, rearranged in 16 of 24 tumors analyzed (67%). All proviruses were integrated in a DNA segment smaller than 100 bp and were in the same 5'-to-3' orientation. Ecotropic as well as mink cell focus-forming virus types were found integrated in that specific DNA region. On the basis of Southern blot analysis of somatic cell hybrids and progeny of an interspecies backcross, the Sic-1 region was localized on mouse chromosome 9 near the previously described proto-oncogenes or common viral integration sites: Ets-1, Cbl-2, Tpl-1, and Fli-1. Restriction map analysis shows that this region is identical to the Fli-1 locus identified in Friend murine leukemia virus-induced erythroleukemia cell lines and thus may contain sequences also responsible for the development of mouse non-T-, non-B-cell lymphomas.

The isolation of interferon-inducing mutants of vesicular stomatitis virus with altered viral P function for the inhibition of total protein synthesis.

We have previously reported that T1026, a temperature-sensitive (ts) noncytocidal mutant of VSV, and its ts revertant, T1026-R1, are nonconditional mutants in the VSV function "P" for the inhibition of total protein synthesis (viral plus cellular) in infected cells (C. P. Stanners, A. M. Francoeur, and T. Lam, 1977, Cell 11, 273-281; C. P. Stanners, S. Kennedy, and L. Poliquin, 1987, Virology 160, 255-258). We have also shown that P- mutants such as these are superior interferon inducers relative to their parental P+ wild-type virus, HR, and that P- mutants may be distinguished from P+ virus using the plaque interferon production of PIF assay. (A. M. Francoeur, T. Lam, and C. P. Stanners, 1980, Virology 105, 526-536). In order to carry the analysis of VSV P function further, a number of independent mutants in the VSV P function are required. We show here that the PIF assay may be used to isolate spontaneously occurring interferon-inducing mutants (PIF+ mutants) from wild-type VSV (PIF- virus) populations. About one-half of the PIF+ mutants isolated with the PIF assay were found to have alterations in the VSV P function. As well as mutants that were defective for the inhibition of total protein synthesis, the assay yielded a new class of VSV P function mutants which appear to inhibit protein synthesis more severely than does P+ virus. The majority of newly isolated PIF+ mutants was also found to be temperature sensitive for growth. The ts phenotype, however, could be reverted for most PIF+ mutants with little effect on the PIF or P phenotype. These findings show that interferon induction and P function are related functions of VSV; this fact has allowed the isolation of a repertoire of mutants with widely varying P function.

Vesicular stomatitis virus P function depends on cellular growth cycle.

The P function of vesicular stomatitis virus (VSV) is defined as the viral function which results in a reduced rate of total protein synthesis (viral plus cellular) arising from a nonspecific reduction in the efficiency of the translational machinery in infected cells. The existence of P function has been challenged by Lodish and Porter who were unable to detect it in L-strain mouse cells infected with wild-type VSV (HR) or, as expected, with the P- mutant, T1026-R1. Although other groups have subsequently confirmed the existence of P function and the difference between HR and T1026-R1, we have sought an explanation for the difference between Lodish and Porter's results and those of other laboratories. We show that the VSV P function depends on the phase of the growth cycle of infected L-cell cultures. In very early exponential phase, as used by Lodish and Porter, HR has very little demonstrable P function; as the growth cycle proceeds toward stationary phase, P function becomes more and more manifest. Under the same conditions, T1026-R1 shows no P function throughout the growth cycle. Furthermore we show that the VSV M protein mutant tsG31 has a P++ phenotype reducing total protein synthesis below that seen with wild-type HR. P function can be observed in cells infected with tsG31, even early in the exponential phase of the cellular growth cycle.

Properties of a GTP sensitive microdomain in rough microsomes.

Stripped rough microsomes (SRM) fuse when incubated with physiological concentrations of GTP and MgCl2. In order to examine further to what extent such fusions are associated with other membrane functions of rough endoplasmic reticulum, we have evaluated the role of cytosolically exposed peptide constituents of SRM in fusion, and the possible relationship of GTP/MgCl2-induced fusion in protein transport across endoplasmic reticulum (ER) membranes, and in ER-Golgi interactions. Controlled proteolytic digestion of SRM led to the loss of fusion capability at 15 micrograms/ml trypsin--a concentration which maintained the latency of intraluminal mannose-6-phosphatase. Hence, a cytosolically exposed protein(s) regulated fusion. Based on ribonuclease-induced ribosome capping experiments, it was further concluded that the cytosolic oriented protein(s) was sequestered beneath the ribosome. As co-translational cell free translocation of placental lactogen across SRM was similar in control membranes compared to those rendered incapable of fusing, it was concluded that the fusion phenomenon may not be related to translocation. Under conditions promoting homologous fusion of SRM or Golgi membranes, mixtures of the two membranes showed no heterologous membrane fusion as assessed morphologically or by the transport of newly synthesized membrane glycoprotein. These experiments attest to the specificity of cytosolically exposed protein(s) in regulating nucleotide/divalent cation-induced membrane fusion.

Involvement of Golgi apparatus and a restructured nuclear envelope during biogenesis and transport of herpes simplex virus glycoproteins.

Following infection of BHK-21 cells with Herpes simplex virus type 1 (HSV-1), progeny nucleocapsids in the nucleus acquire a glycoprotein-rich envelope by budding through host-cell nuclear membranes. To investigate the nature of the glycoprotein products assembled in the virion at the nuclear envelope, infected cells were pulse-labeled with [3H]-mannose, an oligosaccharidal core sugar, or [3H]-fucose, a terminal sugar. After various chase periods, the incorporation of these sugars was monitored by electron microscope radioautography. The results show that HSV glycoproteins accumulate very rapidly in nuclear membranes, where they exist only as core-glycosylated precursors, i.e., containing [3H]-mannose but not [3H]-fucose. [3H]-fucose grains are seen mainly over Golgi membranes and over virions located in the Golgi and in other cytoplasmic vesicular structures. Our data support a model where addition of terminal sugars (e.g., fucose) to HSV-1 glycoprotein precursors can occur at the surface of newly enveloped viral particles as the virions themselves egress from the cell via the Golgi apparatus.

[Isolation and characterization of E. coli B mutants sensitive or resistant to ozone]. / Isolement et charactérisation de mutants sensibles ou résistants à l'ozone chez Escherichia coli B.

Escherichia coli B strains, either more sensitive or resistant to ozone than wild-type (OZs or OZr), were obtained after mutagenesis. OZs strains carrying a mutation in ozrA or orzB, two genes located on both sides of malB, appeared as phenotypically different with regards to radiosensitivity and cellular filamentation. OZr strains, on the other hand, probably carry an allele of ozrA but were radioresistant and divided normally even with induction. A single gene (ozrA) thus seems to determine three levels of sensitivity to ozone, sensitivity to radiation and cellular filamentation in this organism. The possible involvement of a specific endonuclease in the repair of ozone-induced DNA lesions is considered.