Activation of Nonexpressed Endogenous Ecotropic Murine Leukemia Virus by Transfection of Genomic DNA into Embryo Cells

We studied the infectivity of endogenous ecotropic murine leukemia virus genomes contained in high-molecular-weight DNA prepared from virus-free cells of the AKR-2B line, and from RF, BALB/c, B6, and (BALB/c x B6)F(1) mouse embryo cells. When DNA prepared from virus-free AKR-2B cells was transfected into NIH-3T3 cells, no virus-positive cultures were observed, a result consistent with previous reports. However, when DNAs from virus-free AKR-2B cells or virus-free cells containing the RF/J or BALB/c ecotropic proviruses were transfected into chicken embryo cells that were then cocultivated with SC-1 (mouse) cells, virus-positive cultures were recovered. The specific infectivities of the AKR provirus(es) contained in virus-free cells and the molecularly cloned Akv-1 provirus were similar when chicken embryo cells were used as primary recipients. Virus-positive cultures were also observed when secondary mouse embryo cells were used as recipients for DNA from virus-free AKR-2B and RF/J cells. The transfected chicken embryo-SC-1 cultures produced XC-positive murine leukemia virus that is N-tropic. Virus-positive recipient cultures were observed 10- to 100-fold more frequently when AKR-2B DNA was used than when BALB/c DNA was used as the donor DNA. Our studies indicate that some nonexpressed ecotropic murine leukemia virus proviruses are activated upon transfection into chicken embryo cells. Such studies suggest that there are different factors governing the expression of murine leukemia virus after transfection into established cell lines (NIH-3T3) and into nonestablished secondary cultures (chicken and mouse).     

Effect of Phleomycin on Polyoma Virus Synthesis in Mouse Embryo Cells

The addition of phleomycin (25 mug) to primary mouse embryo cells infected with polyoma virus was found to cause 96% inhibition of the synthesis of infectious virus. When ribonucleic acid and protein synthesis was investigated in these cells by use of isotope incorporation, it was found that neither was inhibited drastically. Immunofluorescent staining studies with the use of antibody directed to the viral structural proteins showed that proteins were synthesized in the presence of the antibiotic. However, when deoxyribonucleic acid (DNA) synthesis was investigated, it was found that DNA synthesis in uninfected cells was completely inhibited within the initial 10 hr of phleomycin addition, whereas DNA synthesis in infected cells proceeded at a reduced rate. Selective DNA extraction (Hirt method) of phleomycin-treated infected cells demonstrated that synthesized viral DNA was salt-extractable, similar to that in infected control cells lacking phleomycin. This extracted DNA was further fractionated by ethidium bromide-cesium chloride density gradient equilibrium centrifugation. The phleomycin-treated preparations revealed twice as much component II (circular nicked and linear) as component I (supercoiled) DNA, whereas the DNA from normally infected control cells showed the reverse picture. It was also demonstrated that viral particles synthesized in the presence of phleomycin did not contain component I DNA. This packaged DNA was found to consist of fragments of both the host and viral types. Cells that were prelabeled with (3)H-thymidine and then treated with phleomycin demonstrated host DNA degradation. However, fragments formed from prelabeled host DNA were not encapsidated into viral particles.     

Replication of Radiation-Induced Murine Leukemia Virus in Normal and Transformed Mouse Cells

Autonomous radiation-induced leukemia virus (RadLV) replication could be detected in mouse 3T3 cells by the development of interference with murine sarcoma virus (MSV), the appearance of covert helper activity for defective MSV, and by the induction of cytopathic effect type foci in MSV-transformed, leukemia virus-negative (S+L-) cells. A chronic infection of either 3T3 or S+L- cells with RadLV could be established. Both RadLV infectivity and helper activity were demonstrated in the same peak at a buoyant density of 1.16 g/cm(3). Additionally a soluble inhibitor of MSV focus formation was found which could be separated from infectious RadLV. Examination of cell clones derived from chronically infected 3T3 cells showed that essentially every cell was infected and produced both infectious RadLV and low levels of inhibitor. Quantitative comparisons of autonomously replicating RadLV in normal 3T3 and S+L- cells suggested that RadLV may consist of several populations of virus of varying replicative potential. Apparently 99% of RadLV can be assayed only as helper units in normal cells or as replicative units in S+L- cells. To explain the atypical results, a model for RadLV deficiency is proposed.     

Focus Formation by a Murine Sarcoma-Leukemia Virus Complex: II. Quantitative Aspects of the Interaction Between Radiation Leukemia Virus and Its Murine Sarcoma Virus Pseudotype in Strain C57BL Mouse Embryo Cells

A quantitative study has been made of the interactions between radiation leukemia virus (RadLV), its murine sarcoma virus pseudotype, and their C57BL host cells. The elimination of interference phenomena by delayed infection of cells with RadLV made possible the quantitative determination of the pseudotype in terms of defective sarcoma and endogenous RadLV particles. This in turn permitted the quantitative assessment of RadLV helper activity and of the various factors which influence the accuracy and sensitivity of the helper assay.     

Electron Microscopic Studies of Tumor Viruses I. Entry of Murine Leukemia Virus into Mouse Embryo Fibroblasts

Entry of Rauscher leukemia virus into mouse embryo fibroblasts was studied by electron microscopy. The polycation diethylaminoethyl-dextran enhanced viral attachment and subsequent entry. At the site of viral attachment to the cell membrane, three distinct interactions occurred between the viral envelope and cell membrane, namely, (i) dissolution of viral envelopes on the cell membrane, which itself remained unaltered; (ii) simultaneous dissolution of both the envelope and cell membrane, resulting in passage of viral nucleoids directly into the cytoplasm; and (iii) dissolution of the cell membrane with direct penetration of intact enveloped particles into the cytoplasm, followed by intracytoplasmic disruption of the envelope, resulting in release of nucleoids into the cytoplasm. These interactions occurred with both mature and immature C-type particles. At no time was fusion of viral envelopes with the cell membrane observed. The mechanism of these interactions is discussed.     

Host-Range Restrictions of Murine Leukemia Viruses in Mouse Embryo Cell Cultures

Murine leukemia virus strains fall into three categories with respect to their ability to propagate in cells of National Institutes of Health (NIH) Swiss and BALB/c mouse embryos. Cultures of NIH cells are 100- to 1,000-fold more sensitive to "N-tropic" strains than BALB/c cell cultures, but are 30- to 100-fold less sensitive to "B-tropic" strains. Some virus strains (dually tropic or "NB-tropic") propagate equally well in both cells. M-MSV pseudotypes show the host-range characteristics of the virus supplying the envelope, both in vitro and in vivo. The host-range characteristics appear to be genetically determined and could not be explained by host-induced modification or virus mixtures. There was no correlation between host range and Gross-AKR or FMR serotype.     

L6565小鼠白血病病毒诱发小鼠白血病

为探讨病毒与白血病发生的关系,我们用L6565小鼠白血病病毒(L6565MLV)悬液感染乳鼠,每周观察小鼠的发病情况及病理变化,并用逆转录一聚合酶链反应(RT-PCR)动态检测小鼠体内病毒核酸的分布.结果发现小鼠感染病毒后3～5周,其脾脏和淋巴结呈早期白血病的病理改变.至第10～12周小鼠发生淋巴细胞白血病,表现出耸毛、活动减少、腹膨胀等症状.病毒核酸于感染后第2周首先在小鼠胸腺、脾脏检测到,随时间延长,病毒核酸广泛分布在外周血、胸腺、脾脏、淋巴结等多种脏器组织中.本实验表明L6565小鼠白血病病毒可诱发小鼠白血病,其机制可能与病毒促使淋巴细胞向白血病细胞转化有关.     

L6565小鼠白血病病毒诱发小鼠白血病

为探讨病毒与白血病发生的关系,我们用L6565小鼠白血病病毒（L6565MLV）悬液感染乳鼠,每周观察小鼠的发病情况及病理变化,并用逆转录一聚合酶链反应（RT－PCR）动态检测小鼠体内病毒核酸的分布,结果发现：小鼠感染病毒后3－5周,其脾脏和淋巴结呈早期白血病的病理改变,至第10－12周小鼠发生淋巴细胞白血病,表现出耸毛、活动减少、腹膨胀等症状。病毒核酸于感染后第2周首先在小鼠胸腺、脾脏检测到,随时间延长,病毒核酸广泛分布在外周血、胸腺、脾脏、淋巴结等多种脏器组织中。本实验表明L6565小鼠白血病病毒可诱发小鼠白血病,其机制可能与病毒促使淋巴细胞向白血病细胞转化有关。     

Influence of Diethylaminoethyl-Dextran on Uptake and Degradation of Polyoma Virus Deoxyribonucleic Acid by Mouse Embryo Cells

The uptake of (32)P-labeled polyoma virus deoxyribonucleic acid (DNA) (I and II + III) by mouse embryo cells was increased from two- to fivefold in the presence of 500 mug of diethylaminoethyl-dextran (DEAE-D) per ml. This concentration of DEAE-D gives maximal enhancement of infectivity; however, the increase is many thousand-fold. As the DEAE-D concentration was increased from 0 mug/ml, uptake and infectivity increased to flat maxima and then decreased in a similar manner, except that at low DEAE-D concentrations uptake was relatively much greater than infectivity. Several other polycations also increased DNA uptake but did not enhance infectivity, and uptake of viral DNA was unaffected by the presence of mouse DNA, although infectivity was reduced. Thus, increased uptake is not the sole basis for the enhancement of infectivity produced by DEAE-D. The possibilities that DNA complexed with DEAE-D penetrates more rapidly or is stabilized against degradation do not completely account for enhancement since complexes formed in mixtures of DNA and DEAE-D, which sedimented heterogeneously from 40 to 60S, were infectious only for monolayers treated with DEAE-D. A more likely factor in enhancement is inhibition of the cellular nuclease activity detected, since virus DNA exposed to cells was much more degraded in the absence than in the presence of DEAE-D. The nuclease activity produced single-strand breaks in double-stranded DNA. Treatment of monolayers with deoxyribonuclease after adsorption of DNA in the presence of DEAE-D reduced cell-associated radioactivity by about 70%, although the number of plaques formed was not affected. In the absence of DEAE-D, 90 to 100% was removed by deoxyribonuclease. Thus, in both cases most of the DNA was adsorbed extracellularly. The greater deoxyribonuclease-resistant fraction in the presence of DEAE-D would be consistent with another possibility: that enhancement results from an increase in DNA penetration rate due to some action of DEAE-D on the cell.     

Deoxyribonucleotide Pools and Deoxyribonucleic Acid Synthesis in Mouse Embryo Cells Infected with Three Classes of Polyoma Virus Particles

Polyoma virus particles were purified by equilibrium centrifugation in CsCl. Particles from three regions of the density gradient were examined for infectivity, for their ability to induce expanded pools of deoxyribonucleic acid (DNA) precursors, and for their ability to stimulate the synthesis of DNA. The most infectious population of particles, the virions, having a buoyant density of 1.33 g/ml, gave the greatest stimulation of the DNA-synthesizing apparatus of mouse embryo cells. Empty particles at density 1.29 g/ml had no DNA stimulatory activity. A population of particles of intermediate density, referred to as pseudovirions, was also much less active than virions in stimulating DNA synthesis, and the limited stimulatory activity of the latter fraction may be accounted for by its measured contamination with infective particles.     

Activation of a Latent Measles Virus Infection in Hamster Cells

The characteristics of infectious measles virus released from latently infected hamster embryo fibroblast cells are described. Low levels of virus were released spontaneously when the cultures were incubated at 37 C; this phenomenon was observed 19 passages after the cells had been exposed to the virus and has continued through cell passage 45. The virus yield could be significantly increased by cocultivation of the hamster cells with BSC-1 cells or incubation of the latently infected cells at 33.5 C rather than at 37 C. Measles virus released after cocultivation demonstrated increased cytopathology in cell culture and reduced temperature sensitivity when compared to the virus released at 33.5 C. After cell passage 45, there was an increase in spontaneous release of virus. However, the viruses recovered by cocultivation or temperature release after cell passage 45 were nearly identical. These observations suggest a possible mechanism for measles virus activation in cells latently infected with this virus.     

流感疫苗中间品-鸡胚尿囊病毒液质量控制的研究

为建立流感疫苗中间品-鸡胚尿囊病毒液质量控制点提供依据。用鲎试剂法、微生物限度检查法、沙门菌检查法检测流感疫苗中间品一鸡胚尿囊病毒液的细菌内毒素含量、微生物限度及沙门菌。在300份鸡胚尿囊病毒液中,细菌内毒素含量大于5EU／mL的阳检率为5％;微生物限度检查中小于lO个／mL细菌菌落数的占78．67％,小于10个／mL霉菌菌落数的占80．67％,小于10个／mL酵母菌菌落数的占88．67％。沙门菌属的阳检率为4％,其中未检出A～F群的沙门菌。对流感疫苗中间品-鸡胚尿囊病毒液进行细菌内毒素,微生物限度及沙门菌检测可避免不合格尿囊病毒液进入后续生产,污染后续中间品。     

Fate of Unintegrated Viral DNA in Fv-1 Permissive and Resistant Mouse Cells Infected with Murine Leukemia Virus

We have found that levels of unintegrated linear viral DNA were nearly identical in several Fv-1 resistant cell lines, whereas levels of closed circular viral DNA are markedly reduced in these resistant cells, to the same extent as virus production (P. Jolicoeur and E. Rassart, J. Virol. 33:183-195, 1980). To determine the fate of linear viral DNA made in resistant cells we performed pulse-chase experiments, labeling viral DNA with 5-bromodeoxyuridine and following it with a thymidine chase. 5-Bromodeoxyuridine-labeled viral DNA (HH) recovered by banding on cesium chloride gradients was sedimented on neutral sucrose density gradients or separated by the agarose gel-DNA transfer procedure and detected by hybridization with complementary DNA. Levels of linear viral DNA made in Fv-1^b/b (JLS-V9 and SIM.R) and Fv-1^n/n (NIH/3T3 and SIM) cells were found to decrease during the chase period at about the same rate in permissive and nonpermissive conditions, indicating that linear viral DNA is not specifically degraded in Fv-1 resistant cells. Levels of the two species of closed circular viral DNA made in Fv-1 permissive cells increased relative to the levels of linear DNA during the chase period. This confirmed the precursor-product relationship between linear DNA and the two species of circular DNA. In Fv-1 resistant cells, this apparent conversion of linear viral DNA into circular forms was not seen, and no supercoiled viral DNA could be detected. To determine whether the transport of linear viral DNA from the cytoplasm into the nucleus was prevented by the Fv-1 gene product, SIM.R cells were fractionated into cytoplasmic and nuclear fractions, and viral DNA was detected in each fraction by the agarose gel-DNA transfer procedure. Levels of linear viral DNA were nearly identical in both cytoplasmic and nuclear fractions of permissive or resistant cells. Circular viral DNA could be detected in the nuclear fraction of permissive cells, but not in that of resistant cells. A pulse-chase experiment was also performed with SIM.R cells. During the thymidine chase period, linear viral DNA was seen to accumulate in nuclei of both permissive and resistant cells, whereas supercoiled viral DNA accumulated only in nuclei of permissive cells. These results indicate that the Fv-1 gene product does not interfere with the transport of linear viral DNA into the nucleus. Our data also suggest that the Fv-1 restriction does not operate through a degradation process. Therefore, the Fv-1 gene product could either block the circularization of linear viral DNA directly or promote the synthesis of a faulty linear viral DNA whose defect (yet undetected) would prevent its circularization.     

Adaptation and Infection of Mouse Bone Marrow (JLS-V9) Cells in Suspension Culture for Production of Rauscher Leukemia Virus

JLS-V9 mouse bone marrow cells were readily adapted to suspension culture, chronically infected with Rauscher leukemia virus (RLV), and subsequently grown in 7.5- and 14-liter New Brunswick fermentors. The suspension-type cell system can be modified to produce virus with clearly defined properties, such as high ribonucleic acid-dependent deoxyribonucleic acid polymerase (RDDP) activity, high particle count, and high infectious particle count. Biological and biophysical properties of suspension-produced RLV were not affected by concentration and purification employing continuous-flow and rate-zonal centrifugation procedures. The RDDP assay was standardized and showed a linear incorporation of (3)H-thymidine 5'-monophosphate ((3)H-TMP) up to 30 min. Further characterization indicated that a high percentage of (3)H-TMP incorporation was due to RDDP.     

Evidence for Cooperation between Murine Leukemia Virus Env Molecules in Mixed Oligomers

A retroviral Env molecule consists of a surface glycoprotein (SU) complexed with a transmembrane protein (TM). In turn, these complexes are grouped into oligomers on the surfaces of the cell and of the virion. In the case of murine leukemia viruses (MuLVs), the SU moieties are polymorphic, with SU proteins of different viral isolates directed towards different cell surface receptors. During maturation of the released virus particle, the 16 C-terminal residues of TM (the R peptide or p2E) are removed from the protein by the viral protease; this cleavage is believed to activate the membrane-fusing potential of MuLV Env. We have tested the possibility that different MuLV Env proteins in the same cell can interact with each other, both physically and functionally, in mixed oligomers. We found that coexpressed Env molecules can be precipitated out of cell lysates by antiserum which reacts with only one of them. Furthermore, they can evidently cooperate with each other: if one Env species lacks the R peptide, then it can apparently induce fusion if the SU protein of the other Env species encounters its cognate receptor on the surface of another cell. This functional interaction between different Env molecules has a number of implications with respect to the mechanism of induction of membrane fusion, for the genetic analysis of Env function, and for the design of targeted retroviral vectors for gene therapy.     

Enhanced Virus Transformation of Hamster Embryo Cells In Vitro

Since transformation of hamster cells in vitro by simian virus 40 (SV40) is a rare event in a homogeneously infected cell population, physiochemical studies of the events of virus transformation are difficult. Similarly, other deoxyribonucleic acid-containing oncogenic viruses produce transformed-cell foci in vitro with low efficiency. Sublethal doses of X-ray irradiation, as well as preincubation of hamster embryo cells with the radiomimetic analogue, 5-iododeoxyuridine, markedly sensitized hamster embryo cells to SV40 in vitro. Agents were used at dosages which neither produced lethality nor caused neoplastic transformation in the absence of virus. Embryo cells maintained in vitro for long periods of time became increasingly more sensitive to SV40 transformation. Radiation also stimulated transformation by adenovirus 31. Delay in the addition of virus to preirradiated cells reduced the susceptibility to transformation by SV40 which was observed for cells infected immediately after irradiation, suggesting that radiation repair mechanisms or, possibly, release from radiation-induced "mitotic delay" may interfere with the process of neoplastic conversion by SV40.