Friend leukemia and mouse endogenous viruses: II. Proteins of Friend leukemia and mouse endogenous C-type particles produced by BALBc mouse cell cultures

The polypeptide patterns of con A-isolated Friend leukemia virus and other particles of density 1.18 that are spontaneously produced by Friend-infected and uninfected $\text{BALB}\text{c}$ mouse cells are analyzed in high-resolution SDS-acrylamide gradient gels. Agglutination of virions by con A and fractionation of α-methyl mannose-dissociated complexes in sucrose equilibrium gradients yields a pattern associated with FLV (density 1.16) that contains at least 20 polypeptides of MW 30,000–218,000. The pattern includes the major structural polypeptide of MuLV, MW 34,000, and, compared to published patterns, it has a lower proportion of smaller polypeptides. Relative to serum albumin, a major component of culture medium, virion polypeptides are purified at least 50,000-fold, and the specific leukemia virus infectivity of isolates is approximately 1 PFU/800 particles.Though they are morphologically indistinguishable from FLV, and similarly contain viral 60–70 S RNA, spontaneously arising $\text{BALB}\text{c}$ endogenous viruses (density 1.18) possess a recognizable complement of at least 26 polypeptides including a major one of MW 44,000. The pattern includes a glycopolypeptide of MW 14,000 that is not found in FLV. Like the FLV pattern, the majority of polypeptides are of MW 30,000–218,000. The greater number of polypeptides in endogenous virus suggests that the pattern may be derived from a mixture of particles, as suggested previously by others, and some evidence for mutual interaction between FLV and coproduced endogenous viruses is presented.     

Characterization of the immune response to the major glycoprotein (gp71) of friend leukemia virus III. Influence on endogenous MuLV-mediated pathogenesis

The response of AKR mice to immunization with purified gp71 of Friend murine leukemia virus (FLV)⁹ was characterized. A humoral response was detectable by radioimmune precipitation assay with FLV or radioimmunoassay with FLV gp71. In contrast, no reactivity with either the endogenous AKR virus or AKR gp71 was detectable. The humoral immune response to FLV gp71 was also detectable in complement-dependent cytotoxicity assays and again appeared type-specific for FLV-producing cell lines and could be specifically blocked by absorption with FLV gp71. In contrast, no specific neutralization of FLV was detectable with immune sera. Cell-mediated reactivity was examined by blastogenesis assays with FLV gp71 and in vitro cytotoxicity assays. Although no consistent cell-mediated cytotoxicity was demonstrable in vitro, speen cells from FLV gp71-immunized mice did undergo blastogensis when incubated with purified gp71. The ability of FLV gp71 immunization to protect AKR mice from spontaneous thymomas and irradiated C57B1/6 mice from radiation-induced thymomas was also examined. The results demonstrate that FLV gp71 immunization has no clear influence on induction of radiation thymomas in C57Bl/6 mice. In contrast, FLV gp71 immunization greatly increased the rate of mortality of AKR mice from thymic lymphomas without altering the time of onset of disease-related deaths. The possible mechanisms behind this enhancement of thymomas by FLV gp71 immunization, including the activation of the endogenous AKR virus, are considered.     

Properties of mouse leukemia viruses. VII. The major viral glycoprotein of friend leukemia virus. Isolation and physicochemical properties

The major viral glycoprotein of Friend leukemia virus was isolated by a procedure comprising density gradient centrifugation, Con A affinity chromatography, and Sephadex gel filtration. It was obtained in milligram amounts and found to be pure by physicochemical and seroimmunological methods. It has a buoyant density in sucrose of 1.18 g/cm³ and a molecular weight of 71,000 as determined by SDS-polyacrylamide gel electrophoresis. A molecular weight of 58,000 was calculated from s₂₀ = 4.05 S and D₂₀ = 6.5 · 10^?7 cm²/sec. The specific complement-fixing activity of the purified material was 50,000–100,000 complement-fixing units/mg protein.     

Herpes simplex virus proteins: DNA-binding proteins in infected cells and in the virus structure.

DNA-binding proteins have been isolated from $\text{BHK21}\text{C13}$ cells infected with herpes simplex virus type 1 strain 17 syn⁺ by native and denatured DNA affinity chromatography. Sixteen proteins have been identified as specific to the infected cell on SDS polyacrylamide gradient gel electrophoresis and their molecular weights range from 145 × 10³ to 10.5 × 10³. After infection with herpes simplex virus many of the mock infected cell specific DNA binding proteins are no longer produced and this “switch off” of host cell protein synthesis is rapid. The virus-induced DNA-binding proteins fall into two quantitative classes and there is evidence for temporal control of their synthesis. One of the virus-induced DNA-binding proteins has a molecular weight identical to a polypeptide found in full but not empty particles and another binds preferentially to denatured DNA.     

Interactions of chemical inducers and steroid enhancers of endogenous mouse type-C RNA viruses.

Biologically distinguishable endogenous type-C RNA viruses of $\text{BALB}\text{c}$ mouse cells are differentially affected by two classes of chemical inducers, halogenated pyrimidines and inhibitors of protein synthesis. In the present studies, the effects of these chemicals were compared on cells of genetic crosses involving $\text{BALB}\text{c}$, C58, and NIH Swiss mouse strains. Cycloheximide was found to activate xenotropic vius from those genetic crosses from which xenotropic virus was inducible by IdU. In contrast, NIH Swiss-tropic endogenous viruses of $\text{BALB}\text{c}$ and C58 cells were much more resistant to activation by inhibitors of protein synthesis. Steroids possessing glucocorticoid activity enhanced virus release by cells exposed to either class of inducers. Unlike the inducers, which inhibited type-C virus release by exogenously infected cells, steroids augmented chronic virus production. These findings indicate that the mechanisms of action of inhibitors of protein synthesis and halogenated pyrimidines involve the virus-activation process, while steroids enhance rather than initiate virus synthesis.     

Evidence for helper independent murine sarcoma virus. I. Segregation of replication-defective and transformation-defective viruses

In the conventional focus assay for murine sarcoma virus (Hartley and Rowe, 1966), the formation of a focus involves repeated rounds of infection, and, as is shown in this report, the possibility of alterations in the genome of the virus is thereby increased. Multiple rounds of infection were avoided by infecting cells in suspension, plating them sparsely, and allowing them to grow into colonies. XC cells were added to detect which colonies were producing leukemia virus. When cells were infected with the Moloney sarcoma-leukemia virus (M-MuSV(MuLV)), four types of colonies were seen: (1) morphologically normal with syncytia (XC⁺) or (2) without syncytia (XC^?), (3) morphologically transformed with no syncytia, (4) transformed with syncytia. The proportions infected by MuSV (transformed cells) or by MuLV (XC⁺) conformed to Poisson's distribution, and this allowed the calculation of the titers of MuSV and MuLV. Clones of chronically infected cells could readily be isolated.A clone of transformed cells called G8 was derived from JLS-V9 cells infected with M-MuSV(MuLV). The cells produced no MuLV detectable by cocultivation with XC cells, but they did produce sarcoma virus detected by the production of sarcomas in mice and morphological transformation of several lines of mouse cells in culture. The virus had a density of 1.16 g/cm³.The kinetics of focus formation were one-hit when assayed by the conventional assay. Virus picked from most ($\text{32}\text{38}$) of these foci consisted of a mixture of sarcoma virus and leukemia virus but some ($\text{4}\text{38}$) foci were found that produced sarcoma virus alone (presumably “competent” sarcoma virus, i.e., helper-independent). The presumed “competent” sarcoma virus was carried through 4 successive passages and each time, most of the foci were found to contain both MuSV and MuLV, but some produced MuSV only. In contrast, the original, chronically infected G8 cells did not release detectable MuLV through more than 30 passages. Leukemia virus or defective sarcoma virus segregated from the competent MuSV with low and equal frequencies only when new mouse cells were infected.Examination of the individual cells within foci formed by spread of viral infection showed that some cells produced only competent virus; other cells from the foci produced MuSV and MuLV; others were transformed nonproducers containing defective MuSV that could be rescued by superinfection with MuLV; and still others were transformed but MuSV could not be rescued from them.No evidence was found for the presence of a helper virus in excess of the concentration of sarcoma virus and competence appears to be a property of the virion itself. The data suggest that the competent MuSV is similar to the helper-independent strain of the Rous avian sarcoma virus. It is not known why this virus is negative in the XC assay, although the MuLV that segregates from it is positive in this assay.In the search for a helper virus, a form of MuSV was found that did not morphologically transform the cells it infected, nor was it produced by them, but both transformation and release of MuSV appeared on superinfection with MuLV.     

Group-specific cytolytic antibody directed against the major glycoprotein (gp70) of murine leukemia viruses in serum of mice with dormant FLV infections.

Serum of mice with dormant Friend leukemia virus (FLV) infections contains cytolytic activity against FLC-745 cells, a Friend erythroleukemic cell line. FLC-745 cells express only one cell surface FLV-coded antigen, shown by competition radioimmunoassay experiments to be FLV virion gp70. AKR virus blocks completely the cytolytic activity of serum but cannot block the precipitation of gp70 from detergent-disrupted ¹²⁵I-labeled cells. These results indicate that the FLC-745 cytolytic antibody in serum from mice with dormant FLV infections is directed against virion gp70 and is group specific. Furthermore, this serum contains a type specific gp70 antibody which is not lytic for FLC-745 cells.     

Activated endogenous viruses of tumor induced by human adenovirus: an electron microscope study.

Virus particles were examined electron microscopically in human adenovirus 12-induced cells of $\text{C3H}\text{BifB}$ mice. Specimens examined included conventional thin-sections, freeze-fracture-etched preparations and scanning electron microscope preparations. Both C type virus particles and intracisternal A particles were quite numerous in primary and transplanted tumors. Intracisternal A particles were usually found in the endoplasmic reticulum in the center of tumor cells and sometimes in the perinuclear cistern. C type particle were massed in the intercellular spaces. The C particle budding processes protruded from many portions of the tumor cell surface. Immature particles often had a tail and the core configuration suggested an icosahedral symmetry.     

Interferon inhibits induction of endogenous murine leukemia virus production.

In 5-iododeoxyuridine (IUDR) induction of murine leukemia virus (MLV) in Balb cells transformed by SV40 (Balb SV40) or AKR cells, interferon inhibited virus production. This could have been due to inhibition of the X- and N-tropic virus induced or to inhibition of subsequent reinfection of the mouse cells by the N tropic virus. Cycloheximide, however, activates $\text{KBalb}\text{3T3}$ cells ($\text{Balb}\text{3T3}$ cells transformed by Kirsten sarcoma virus) to produce only an X-tropic virus which could not reinfect mouse cells. In this system, interferon treatment also decreases virus yields and, therefore, an event in the virus induction process.     

Endocytosis by echinoid phagocytosis in vitro. I. Recognition of foreign matter

Endocytosis, with emphasis on phagocytic recognition was examined in the sea urchin $\text{Strongylocentrotus droebachiensis}$. Most cells (about 67%) in the coelomic fluid were phagocytes. There was also a linear uptake of colloidal gold with time in the cultured cells, presumably caused by pinocytosis. No opsonizing activity was found in the cell-free coelomic fluid, and the parallel phagocytic avidity $\text{in vivo}$ and $\text{in vitro}$ of different particles also indicates that humoral factors were absent or not essential for recognition of foreign surfaces by phagocytes. Untreated erythrocytes (RBC) were taken up at a low rate, while RBC treated with aldehyde, Fe²⁺, tannin, Con A, or IgM + mouse C5-deficient complement were phagocytosed efficiently. Phagocytosis was reduced when RBC were covered with specific antibodies. Thus, the echinoid phagocyte lacks an Fc receptor, but may have a C3b receptor on its membrane. Other particles with high phagocytic avidities were carbon, Sephadex, latex and $\text{Escherichia coli}$. Thus the cell membranes of phylogenetically distant phagocytes may exhibit common structural features responsible for foreign surface recognition.     

Purification and properties of tomato spotted wilt virus

Tomato spotted wilt virus was purified by chromatography on columns of calcium phosphate, precipitation with polyethylene glycol, density gradient centrifugation, and ascending zone electrophoresis in a sucrose density gradient. Examination of the virus by electron microscopy revealed particles of different shapes in different suspending media. Particles fixed with glutaraldehyde were spherical and somewhat flattened and had a corrected diameter of 85 nm.Antisera with a titer of $\text{1}\text{128}$ against the virus were prepared which did not react with plant antigens.The virus had a sedimentation coefficient s_20,w of 530 S and, when fixed in 0.5% glutaraldehyde, it had an electrophoretic mobility at pH 7 of ?19.8 × 10^-5 cm² V^?1 sec^?1.     

Partial characterization of virus-like particles in chloroplasts of plants infected with the U5 strain of TMV

Virus-specific, intraplastidial particles (VIP) were isolated from chloroplasts of tobacco leaves (Nicotiana tabacum L.) infected with the U5 strain of tobacco mosaic virus (TMV). Serologically specific electron microscopy (SSEM) revealed that the VIP were about $\text{1}\text{3}$ the normal length of TMV. In manually inoculated leaves and in infected, isolated leaf protoplasts the VIP exhibited linear growth to an average of about 100 nm over a period of several days after inoculation. Preparations of VIP from which nearly all TMV detectable by SSEM had been removed were minimally infectious. It was concluded that the VIP are not infectious TMV particles.     

A major genetic locus affecting resistance to infection with murine leukemia viruses. IV. Dose-response relationships in Fv-1-sensitive and resistant cell cultures.

The resistance to N- and B-tropic viruses controlled by the murine genetic locus Fv-1 was studied by dose-response analyses, using infectious center plating onto sensitive cells to determine the number of virus-producing cells. Three components contributing to the reduced plaquing efficiency on Fv-1 resistant cells were identified. First, the dose-response relations in Fv-1 resistant cells showed multiple-hit kinetics; most virus strains showed 2-hit kinetics, but the Gross Passage A virus gave 3-hit kinetics. This hitness factor is considered to represent the basic effect of Fv-1. Second, in many instances only a small fraction of Fv-1 resistant cells receiving the minimum required number of hits actually became virus producers. Third, only a fraction of the virus-producing cells registered as plaques when left in situ with resistant cells, due to the relative inefficiency of subsequent cycles of infection. Strain DBA/2 cells differed from those of other Fv-1ⁿ strains in showing only the hitness component. $\text{Fv-1}{}^{\mathrm{<mtext>n</mtext><mtext>b</mtext>}}$ hybrid cells showed multiple-hit dose-response curves with both N- and B-tropic viruses. Once infection has been established, virus yield from sensitive and resistant cells is similar.     

Regulation mechanism of simian virus 40 late gene expression in primary kidney cells and simian virus 40 transformed 3T3 cells.

Primary and continuous lines of mouse cells are nonpermissive for simian virus 40 (SV40). These cells, infected by the conventional virus absorption method, promote expression of the early but not of the late viral genes. Induction of V-antigen synthesis was found in a large number of primary mouse kidney cells and SV40-transformed $\text{Balb}\text{c}\text{-3T3}$ cells (SV-T2) infected with SV40-DNA component I by the microinjection technique. The proportion of SV40 V-antigen-synthesizing cells is correlated to the quantity of injected DNA molecules in both primary mouse kidney and SV-T2 cells.     

Purification and some properties of beet yellow virus

Beet yellows virus was purified by a method, based on ultracentrifuging at 35,000 g, that preserved the normal length of the virus particle and eliminated a uv-absorbing contaminant retained by previously described methods. The purified particles had a modal length in shadowed preparations of 1250 nm and sedimented in the analytical centrifuge usually as one component at 130 S. Purified preparations had an $\text{A}{}_{260}\text{A}{}_{280}$ ratio of 1.44 and an extinction coefficient at 260 nm of 2.9. In Cs₂SO₄ isopycnic banding, the virus had a density of 1.285 g/cm³, while in CsCl two bands were produced at 1.307 and 1.312 g/cm³.     

Pathogenic murine coronaviruses. III. Biological and biochemical characterization of temperature-sensitive mutants of JHMV.

JHMV is a neurotropic member of the hepatoencephalitis group of murine coronaviridae. The characteristics of the biology and intracellular viral RNA synthesis and the intracellular viral protein synthesis of JHMV are discussed in the two previous papers, respectively. This paper describes the neuropathogenesis of JHMV and the isolation and characterization of 34 temperature-sensitive mutants of JHMV. These mutants were selected for their inability to induce syncytia formation after low multiplicity infection (m.o.i. = 0.1 iU) in BALB/c 17CL-1 cells at 38.5° as compared to the induction of syncytia at 33°. N-Methyl-N′-nitrosoguanidine (14 mutants) and 5-fluorouridine (20 mutants) were used as mutagens at a concentration that reduced infectivity by 90–95%. Characterization of these mutants included: induction of syncytia; synthesis of JHMV-specific intracellular RNA; progeny yields at 33, 37, and 38.5°; synthesis of JHMV-specific antigens as determined by indirect immunofluorescence and sodium dodecyl sulfate-polyacrylamide gel electrophoresis; virion thermostability; neuropathogenesis including isolation of virus from infected brain, immunofluorescence of infected brain, and histopathology of brain and spinal cord by light and transmission electron microscopy; ability to protect mice from a lethal JHMV infection; and complementation. RNA-minus ($\text{17}\text{34}$), RNA-intermediate ($\text{14}\text{34}$), and RNA-plus ($\text{3}\text{34}$) groups were defined. One mutant, N3, produces chronic meningitis and demyelination without typical JHMV encephalitis in spite of the fact that neurons are infected as detected by immunofluorescence. This altered neuropathogenesis cannot be explained by “leakiness” or reversion. In addition, non-temperature-sensitive variants of JHMV have been selected for altered neuropathogenesis and are described.     

Studies on virus induction by 5-bromodeoxyuridine in nonproducer murine sarcoma virus-transformed 3T3 cells.

Treatment with 5-bromodeoxyuridine (BrdU) of nonproducer $\text{BALB}\text{3T3}$ cells (KA31 cells) transformed by the Kirsten strain of murine sarcoma virus (Ki-MSV) induced the synthesis of DNA polymerase-containing particles which band in sucrose gradients at a density of 1.16 and utilize poly(I) (dC)_12–18, a template-primer specific for RNA-directed DNA polymerase. In order to establish optimal conditions for virus induction, the induction process was studied by analysis of the culture fluid for polymerase activity and the induced cells for viral proteins by immunofluorescence and virus particles by electron microscopy. By varying the concentration and time of exposure to BrdU, it was found that the maximal level of polymerase activity was induced when cells were treated with 20 μg of BrdU for a 24-hr period, 24 hr after seeding. Under these conditions about 15% of the cells were induced to synthesize viral antigens detected with anti-Moloney MSV rat serum. The level of polymerase activity in the culture fluid and viral antigen in the cells became maximal at 3 days after BrdU treatment and decreased rapidly. Electron microscopy showed a large increase in the proportion of cells containing incomplete virus particles and in the number of particles per cell at 2 and 3 days after BrdU treatment. At 3 days after treatment, 82% of the cells contained incomplete virus particles within dilated vesicles of the endoplasmic reticulum as compared to 18% for untreated cultures.A high level of virus production could be maintained for at least 15 days by continuous maintenance of cells on 4 μg/ml of BrdU. It is not clear whether the mechanism involves continuous reinduction of virus or overcoming the normal inhibition of virus multiplication in these cells. Under these conditions, BrdU reversed the rounded, transformed appearance of KA31 cells to a flat fibroblastic morphology.     

Conversion of restrictive mouse cells to permissiveness during sequential and mixed double infection by murine leukemia viruses.

Conversion from restrictive (two-hit) to permissive (one-hit) kinetics was observed when N- or B-tropic murine leukemia viruses were titrated on mouse embryo fibroblasts of nonpermissive Fv-1 genotype that had previously been nonproductively infected with the same virus at an average multiplicity of one. The effect was transient, disappearing within about 24 hr after the first infection, and was abrogated by exposure of the first virus preparation to increasing doses of ultraviolet light, with a D₃₇ inactivation dose of 2550 ergs/mm², about three times that for inactivation of viral replication. Prior infection of nonpermissive mouse cells with the NZB xenotropic virus did not alter their restrictive response to later infection with ecotropic viruses. Low multiplicity infection of $\text{NIH}\text{3T3}$ cells with a B-tropic virus, followed by treatment with iodoeoxyuridine, failed to induce productive infection by endogenous or exogenous virus. Cells of F₁ hybrid Fv-1 genotype, which are restrictive for both N- and B-tropic viruses, were converted to permissiveness for virus of either tropism after low multiplicity infection with virus of the opposite tropism. No evidence of NB-tropic recombinant progeny could be detected under these experimental conditions. The implications of these experimental observations with respect to the mechanism of restriction governed by the Fv-1 gene are discussed.