Biological properties of a type C virus isolated from a human X mouse hybrid cell line.

The biological properties of the HMV-1 virus, spontaneously released from a human X C57BL/6 mouse hybrid cell line, were similar to those of RadLV, the prototype B-tropic virus of C57BL/6 mice. Both viruses replicated on B-type mouse cells and in the wild mouse cell line SC-1. The plaque-forming abilities of the two viruses were relatively low, but gradually increased after passage in new host cells. Both viruses were neutralized by AKR antisera but not by FMR antisera. HMV-1 virus could rescue the defective sarcoma genome from S+H- mouse cells. The pseudotype sarcoma virus so produced was deficient in "helper virus" activity. Newborn mice inoculated with HMV-1 virus remained tumor-free over a 1-yr observation period.     

Murine Sarcoma and Leukemia Viruses: Assay Using Clonal Lines of Contact-Inhibited Mouse Cells

A continuous cell line of highly contact-inhibited cells (NIH/3T3) has been developed from NIH Swiss mouse embryo cultures. Its growth properties are similar to those of 3T3 and BALB/3T3. Although 3T3 is relatively insensitive to focus formation by murine sarcoma viruses, cloned lines of both NIH/3T3 and BALB/3T3 have been isolated that are highly sensitive to sarcoma virus focus formation and leukemia virus growth. The sensitivity and specificity are comparable to those found with primary embryo cells. MSV-transformed lines of NIH/3T3 have been obtained.     

Factors Affecting the Sensitivity of Different Viruses to Interferon

When the sensitivities to interferon of Newcastle disease virus (NDV) and vesicular stomatitis virus (VSV) were compared by the plaque reduction method in chick embryo cell cultures, NDV was found to be 45-fold more resistant than VSV. This difference was exaggerated when a multiple-cycle yield inhibition method was employed. In marked contrast, when the same viruses were tested by a single-cycle yield inhibition method, the difference in sensitivity to interferon of the two viruses was virtually eliminated. Further investigation showed that, in chick embryo cells exposed to interferon, the resistance to NDV decayed more rapidly than resistance to VSV. This finding explained the divergent results obtained with the two viruses when single- or multiple-cycle replication techniques were employed. Experiments carried out with L cells showed that cellular antiviral resistance decayed much more slowly in these cells than in chick embryo cells. Consequently, when measured by the plaque reduction method in L cells, no difference was observed in the sensitivity to interferon of VSV and NDV(pi), a mutant of NDV which replicates efficiently in L cells. A procedure is suggested for determining the relative sensitivities to interferon of different viruses under conditions which minimize the role of decay of antiviral resistance in the host cells.     

UV-Irradiation of related mouse hybrid cells: Similar increase in capacity to replicate intact minute-virus-of-mice but differential enhancement of survival of UV-irradiated virus

3 hybrid cell lines between mouse fibroblasts (A9) and mouse lymphoma cells (L5178YS) were compared with regard to the ability of UV-pre-irradiated cells to replicated intact (unirradiated) Minute-Virus-of-Mice (MVM) and to reactivate UV-irradiated MVM. UV irradiation of cells before virus infection enhanced their ability to plaque intact virus (Enhanced Capacity) to a similar extent in the 3 hybrid cell lines. However, pretreatment of cells with UV radiation enhanced the survival of UV-damaged virus (Enhanced Reactivation) in only 1 of these hybrids. The lack of detectable Enhanced Reactivation in the other hybrids without concomitant change in their Enhanced Capacity, suggets that these processes are at least partly independent. Virus survival in unirradiated cells was similar for all 3 hybrid cell lines, indicating that the absence of detectable Enhanced Reactivation in 2 of the hybrids was not due to the constitutive expression of this process, but might rather result from its loss or extinction. The expression of both Enhanced Capacity and Enhanced Reactivation requires synthesis of protein de novo shortly after cell irradiation.     

Differential expression of murine leukemia virus loci in chemically induced hybrid cells.

The time course of murine leukemia virus production after chemical induction was determined in hamster-mouse somatic cell hybrids containing the xenotropic murine leukemia virus induction locus Bxv-1 or the ecotropic locus Akv-2. By using these hybrids, induction could be studied in the absence of secondary virus spread because xenotropic viruses cannot infect hybrid cells and ecotropic viruses cannot infect hybrids which have lost mouse chromosome 5. After induction, hybrids with Bxv-1 produced only a transient burst of virus, whereas those with Akv-2 continued to produce virus for periods in excess of 3 months. The presence or absence of other mouse chromosomes in the hybrid lines did not alter these induction patterns. Thus, endogenous murine leukemia virus loci differ in their response to induction, and both inducibility and the kinetics of virus expression are controlled at or near these proviral loci.     

Retroviruses have differing requirements for proteasome function in the budding process

Proteasome inhibitors reduce the budding of human immunodeficiency virus types 1 (HIV-1) and 2, simian immunodeficiency virus, and Rous sarcoma virus. To investigate this effect further, we examined the budding of other retroviruses from proteasome inhibitor-treated cells. The viruses tested differed in their Gag organization, late (L) domain usage, or assembly site from those previously examined. We found that proteasome inhibition decreased the budding of murine leukemia virus (plasma membrane assembly, PPPY L domain) and Mason-Pfizer monkey virus (cytoplasmic assembly, PPPY L domain), similar to the reduction observed for HIV-1. Thus, proteasome inhibitors can affect the budding of a virus that assembles within the cytoplasm. However, the budding of mouse mammary tumor virus (MMTV; cytoplasmic assembly, unknown L domain) was unaffected by proteasome inhibitors, similar to the proteasome-independent budding previously observed for equine infectious anemia virus (plasma membrane assembly, YPDL L domain). Examination of MMTV particles detected Gag-ubiquitin conjugates, demonstrating that an interaction with the ubiquitination system occurs during assembly, as previously found for other retroviruses. For all of the cell lines tested, the inhibitor treatment effectively inactivated proteasomes, as measured by the accumulation of polyubiquitinated proteins. The ubiquitination system was also inhibited, as evidenced by the loss of monoubiquitinated histones from treated cells. These results and those from other viruses show that proteasome inhibitors reduce the budding of viruses that utilize either a PPPY- or PTAP-based L domain and that this effect does not depend on the assembly site or the presence of monoubiquitinated Gag in the virion.     

Growth of Murine Cytomegalovirus in Various Cell Lines

Murine cytomegalovirus (MCMV) was capable of infecting and replicating in both primary and continuous cell lines obtained from various species. In African green monkey kidney (BSC-1) cells, primary rabbit kidney cells, and baby hamster kidney (BHK-21) cells, there were cytopathic effects (CPE) and virus replication upon initial exposure of cells to virus. In primary fetal sheep brain (FSB) cells, L cells, and rabbit kidney (RK-13) cells, it was necessary to subculture the infected cells one or more times before appearance of CPE and replication of virus. In the case of the infected FSB cultures, it was found that the virus effect could be induced if subculturing were accomplished by trypsinization but did not occur if cells were subcultured by scraping. FSB-grown virus replicated better in FSB than in mouse embryo fibroblast (MEF) cells. The CPE produced in all of the above cell lines was similar to that observed in MEF infected with MCMV. The virus grown in different cell lines was completely neutralized when mixed with several reference sera prepared in rabbits or mice. The populations of virions released from infected MEF and FSB cells were compared by isopycnic centrifugation in potassium tartrate, and no differences were revealed in the buoyant densities of the populations. Human embryonic brain cells, human embryonic kidney cells, a human lung fibroblast cell strain (WI-38), HeLa, and Hep-2 were not susceptible to MCMV.     

Expression of nervous system antigen-3 by lines of mouse fibroblasts and kidney cells and continued expression in hybrid cells

In a survey of the expression on cultured mouse cells of the cell surface antigen known as nervous system antigen-3 (NS-3), it was found that RAG, a renal adenocarcinoma line, expressed that antigen. It was also observed that 3T3, a fibroblast line of unknown tissue origin, expressed NS-3. Cells of these two lines were hybridized with cells of two mouse L cell lines that did not express NS-3. Four hybrid clones were tested for both the 3T3 × L cell cross and the RAG × L cell cross, and all the hybrids were found to be NS-3 positive. All the hybrids had at least 40% as much activity as the NS-3 positive parent. Of the four parental mouse cell lines used, only 3T3 expressed Thy-1.2 antigen on the cell surface. In contrast to the continued expression of NS-3 on hybrid cells, Thy-1.2 antigen was not detectable on two clones of 3T3 × L cell hybrids that were tested.     

Component of Strain MC29 Avian Leukosis Virus with the Property of Defectiveness

Three clones of morphologically altered cells (L(-)MC29) of singular properties were isolated from MC29 (subgroup A) leukosis virus-infected chick embryo cells. Supernatant fluids from cultures of the cloned cells produced no transforming or interfering activity on chick embryo cells susceptible to known avian leukosis-sarcoma viruses. No virus associated with the cells was demonstrable by fluorescent-antibody staining or by electron microscopy. All L(-)MC29 clone cells were activated, however, by four strains of Rous-associated viruses (RAV) representative of A, B, C, and D subgroup avian leukosis viruses and by two strains of MC29 virus. Virus L(-)MC29 cells activated by superinfection with RAV-1 and RAV-2 was characterized by helper-dependent and helper-independent properties. These findings suggest that the strain MC29 leukosis virus, or a component thereof, possesses properties of defectiveness similar to those of the Bryan high-titer Rous sarcoma virus.     

Genetic evidence for a product of the Fv-1 locus that transfers resistance to mouse leukemia viruses.

Extracts of mouse cells have been shown to transfer to N- or B-trophic host range types of mouse leukemia viruses. The genetic specificity of the inhibition was tested in two ways: (i) by correlating the Fv-1 genotype of a number of mouse strains with the restriction-transferring activity of extracts of the respective embryo cell cultures, and (ii) by correlating the Fv-1 genotype of BLC3F2 (C57BL/6 female [Fv-1bb] by C3H male [Fv-1nn] parental strains) mouse embryos, which segregate the Fv-1 alleles in a 12:1 ratio, with the inhibitor activity of extracts of the cells from each embryo. Five independent matings, totaling 45 individual embryos, were tested. Each embryo was cultured, and the Fv-1 genotype was determined independently by titration of N- and B-tropic viruses; the extracts of replicate secondary cultures were tested for their effect on infection of permissive cells by N- and B-tropic viruses. The specific-restriction-transferring activity of the embryos was found to segregate with the appropriate Fv-1 genotype. These res-lts confirm the suggestion that the inhibitor of the leukemia virus host range types in the cellular extracts is a product of the Fv-1 locus.     

Host restriction of friend leukemia virus; fate of input virion RNA

Host restriction of oncogenesis by RNA tumor viruses may be studied in vitro by measuring the replication of the lymphatic leukemia component of the Friend Virus Complex (LLV-F) in either NIH-Swiss or Balb/C mouse embryo cells. These cells derive from mice differing at the Fv-1 locus, which controls the replication of all murine RNA leukemia viruses. Studies of early events in the replication of LLV-F were carried out by following the infection of permissive and restrictive mouse embryo cells by ³²P labeled LLV-F. ³²P labeled viral genome RNA rapidly becomes associated with cell nuclei and may be found integrated to the same extent with high molecular weight host DNA of either permissive or restrictive cells. These results suggest that Fv-1 mediated host restriction of LLV-F occurs at a step following integration of viral genome RNA into host DNA.Two other conclusions are suggested by these data. The nucleus appears to be the site of activation and synthesis of DNA of the infecting virus; and the “provirus”, at least transiently, is represented as an RNA-DNA hybrid molecule covalently integrated with host cell DNA.     

The kinetics of VP5 mRNA expression is not critical for viral replication in cultured cells

We generated recombinant viruses in which the kinetics of expression of the leaky-late VP5 mRNA was altered. We then analyzed the effect of such alterations on viral replication in cultured cells. The VP5 promoter and leader sequences from positions -36 to +20, containing the TATA box and an initiator element, were deleted and replaced with a strong early (dUTPase), an equal-strength leaky-late (VP16), or a strict-late (U(L)38) promoter. We found that recombinant viruses containing the dUTPase promoter inserted in the VP5 locus expressed VP5-encoding mRNA with early kinetics, while virus with the U(L)38 promoter inserted expressed such mRNA with strict-late kinetics. Further, in spite of differences in its functional architecture, the VP16 promoter fully substituted for the VP5 promoter. Western blot analysis demonstrated that the amounts of VP5 capsid protein produced by the recombinant viruses differed somewhat; however, on complementing C32 and noncomplementing Vero cells, such viruses replicated to titers equivalent to those of the rescued wild-type virus controls. Multistep virus growth in mouse embryo fibroblasts, rabbit skin cells, and Vero cells also demonstrated equivalent replication efficiencies for both recombinant and wild-type viruses. Further, recombinant viruses did not show any impairment in their ability to replicate on serum-starved or quiescent human lung fibroblasts. We conclude that the kinetics of the essential VP5 mRNA expression is not critical for viral replication in cultured cells.     

Propagation of MM Virus in Continuous Cell Lines

Baby hamster kidney (BHK), McCoy, and L cell lines were found to be suitable for isolation of MM virus from infected mouse brain tissue. The virus was recovered in high titer in the first passage in BHK and McCoy cells, with concomitant cytopathic effect (CPE). In L cells, virus yield was lower than in the other two cell lines and CPE was incomplete. Adaptation of the virus to BHK and McCoy cells by serial passages was evidenced by accelerated development of the CPE and increase in the virus titer. Plaques were obtained in all three cell lines when inoculated with infected mouse brain or with the tissue culture-propagated virus. In the BHK cells, the virus release preceded the appearance of CPE and maximal yield of virus was obtained after 1 to 3 days of incubation, depending on the size of inoculum. The BHK-propagated virus had the same lethality for mice as did the mouse brain-propagated stock, and there was no difference in the course of the disease caused by the two preparations.     

Manitoba virus, a new rhabdovirus isolated from Culex tarsalis mosquitoes collected in Manitoba, Canada

A rhabdovirus, Mn 936-77, was isolated from a pool of two Culex tarsalis collected on August 16, 1977, from Morris, Manitoba. Isolate Mn 936-77 was not pathogenic for suckling Swiss white mice inoculated by the intracerebral route. The virus propagated in three vertebrate cell lines (Vero, primary chick embryo, mouse neuroblastoma), but apparently not in Aedes albopictus C6/36 cells. Isolate Mn 936-77 did not react by amplified enzyme-linked immunosorbant assay with 230 viruses of proven or possible arbovirus etiology or by immunofluorescence with 88 members of the family Rhabdoviridae. Isolate Mn 936-77 appears to be a newly discovered virus for which the name Manitoba virus is proposed.     

Identification of a 30S RNA with properties of a defective type C virus in murine cells.

A novel species of 30S RNA has been detected in a variety of mouse cell lines. The 30S RNA is specifically packaged by helper-independent type C viruses propagated in such cells. Nucleic acid hybridization detects no homology between the 30SRNA and the genomic RNA of helper-independent mouse type C viruses. The properties of the 30S RNA suggest that it is a defective endogenous mouse type C virus and that it is analogous to a previously described class of defective endogenous rat type C virus, which has been shown previously to be the progenitor of Kirsten and Harvey murine sarcoma viruses.     

Radioimmunoassay for tubulin: a quantitative comparison of the tubulin content of different established tissue culture cells and tissues

A quantitative estimate of the cellular tubulin concentration can be obtained by the use of a radioimmunoassay based upon the competition between tubulin in cell extracts and a known amount of radioactively labeled homogeneous tubulin during binding to a limited amount of antitubulin antibodies. This assay shows that a variety of widely used tissue culture cells (mouse L cells, mouse 3T3 cells, chick embryo fibroblasts) have a tubulin content which corresponds to approximately 2.5-3.3% of their total protein. Transformation of mouse 3T3 cells by the DNA virus SV40, and of chick embryo cells by the RNA Rous sarcoma virus, does not change the intracellular tubulin concentration. Transformed cells of brain origin, such as some glia tumor cell lines and some neuroblastoma cell lines, have a much lower tubulin content than does normal brain tissue.The intracellular concentration of tubulin in mouse 3T3 cells is discussed in relation to the number of microtubules detected during interphase by immunofluorescence microscopy. These results are also discussed in view of a mechanism of microtubule elongation in vivo driven by self-assembly.     

Role of Mitochondria in the Production of RNA-Containing Tumor Viruses

The role of mitochondria in the reproduction of RNA-containing tumor viruses was examined by using ethidium bromide (EB) to induce degenerative effects in mitochondria. The effects of EB in murine and avian cells were monitored by electron microscopy. Chronically infected mouse (JLS-V5) cells, in which extensive mitochondrial changes were induced, continued to produce murine leukemia virus. Also, complete reproductive cycles of Rous sarcoma virus (RSV) occurred in newly infected chicken embryo cells exposed to EB. Morphological transformation characteristic of infection of chicken embryo cells by RSV occurred in cells which contained induced aberrant mitochondria. The results demonstrate that mitochondria play a relatively minor role, if any, in the reproduction of RNA-containing tumor viruses.     

Fv-1 determinants in xenotropic murine leukemia viruses studied with biological assay systems: Isolation of xenotropic virus with N-tropic Fv-1 activity in the cryptic form.

By a biological assay system using phenotypically mixed ecotropic and xenotropic murine leukemia viruses, we investigated whether in the virions of a xenotropic virus there is N- or B-tropic Fv-1 determinant in active form. The existence of N-tropic Fv-1 determinant was demonstrated in SL-XT-1 xenotropic virus isolated from the spleen of a 3-month-old SL mouse, and the N-tropic Fv-1 tropism was confirmed by analysis of the phenotypically mixed viruses harvested from clonal SC-1 cells doubly infected with the SL-XT-1 and B-tropic ecotropic viruses. However, neither N- nor B-tropic Fv-1 determinant was demonstrated in any xenotropic viruses isolated from embryo cells of BALB/c, NZB, or DBA/2 mice, or Cas E #1-IU, and xenotropic-like virus isolated from a wild mouse.     

Techniques of embryo transfer and facility decontamination used to improve the health and welfare of transgenic mice

'Reduction' and 'Refinement' can be achieved in transgenic mouse studies by re-deriving transgenic mouse lines and subsequently maintaining them under high standards of husbandry in a unit with restricted access. This report describes the initial steps of a project to improve the health and welfare of transgenic mice at the European Molecular Biology Laboratory (EMBL), by re-deriving transgenic lines as microbiologically defined animals to be maintained in a barrier unit in a newly constructed animal facility. A pilot study showed that it was possible to transfer embryos obtained from contaminated donor mice in the old facility to specific pathogen free recipients housed in a ventilated cabinet in the new unit, without concomitant carry over of disease. The offspring born following embryo transfer were of high health status and did not show any evidence of contamination with any of the pathogens present in the mice in the old animal unit. Antibodies to various murine viruses (mouse hepatitis virus (MHV), rota virus, reo-3 virus, Theilers encephalomyelitis virus, adenovirus) and parasites were present in sentinel animals from the old animal house whereas the re-derived animals were found to be free of virus antibodies and parasites. Therefore the methods used were considered to be successful in terms of disease prevention and enhancement of welfare. The barrier unit was sterilized without the use of formaldehyde or related substances, to minimize the risks to personnel and to the environment from using potentially dangerous substances. From the results of in vitro and in vivo screening, the protocol for sterilization described here was found to be effective in achieving microbiological sterility of the barrier unit and was cost effective.     

Mouse strain resistant to N-, B-, and NB-tropic murine leukemia viruses.

Mouse strain G was studied for its susceptibility to various strains of murine leukemia and sarcoma viruses. Both N- and NB-tropic Friend leukemia viruses neither induced splenomegaly nor grew efficiently in strain G mice. Using the XC test, cultured embryo cells were found to be resistant, but not absolutely, to all the tested viruses, N-tropic AKR virus, N- and NB-tropic Friend leukemia viruses, NB-tropic Rauscher leukemia virus, B-tropic WN1802B virus, NB-tropic Moloney leukemia and sarcoma viruses, and N-tropic Kirsten sarcoma virus, although the resistance to Moloney leukemia and sarcoma viruses is sometimes not as strong as that for other viruses. Thus, the strain G mice are unique among mouse strains because they show resistance that is not related to the N-B tropism of murine leukemia viruses.