Effect of Polycations on Sensitivity of BALB/3T3 Cells to Murine Leukemia and Sarcoma Virus Infectivity

Polybrene or diethylaminoethyl dextran, when added with murine leukemia or sarcoma virus inocula, significantly increased virus infectivity of BALB/3T3 cells. Residual polycation concentrations were nontoxic.     

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。     

Histones Stimulate Polyribonucleotide-Directed Polydeoxyribonucleotide Synthesis by Murine Leukemia Virus

The rate of homoribopolymer-directed DNA synthesis by detergent-disrupted Moloney murine leukemia virus can be stimulated or inhibited by histone, depending on the ratio of histone to template. Of the fractions which can be separated from the whole histone, f1 causes both the greatest stimulation and the greatest inhibition. The effect of histone f1 is qualitatively similar whether the template is polyadenylate (poly A), polycytidylate, or polyuridylate, but the stimulation is greatest with poly A. The pattern of stimulation and inhibition differs, however, for a different polymerase; the DNA polymerase of Micrococcus luteus is inhibited by histone concentrations which stimulate the viral enzyme and stimulated by concentrations which inhibit the viral enzyme. For the viral enzyme, the optimum histone concentration is unaffected by changes in the virus or primer concentration; but it varies in proportion to the template concentration, suggesting that histone acts by combining stoichiometrically with the template. These data raise the possibility that a histone-like protein may participate in the synthesis of the provirus of RNA tumor viruses.     

The AKV Murine Leukemia Virus Is Restricted and Hypermutated by Mouse APOBEC3

APOBEC3 proteins are potent restriction factors against retroviral infection in primates. This restriction is accompanied by hypermutations in the retroviral genome that are attributable to the cytidine deaminase activity of the APOBEC3 proteins. Studies of nucleotide sequence diversity among endogenous gammaretroviruses suggest that the evolution of endogenous retroelements could have been shaped by the mutagenic cytidine deaminase activity of APOBEC3. In mice, however, APOBEC3 appears to restrict exogenous murine retroviruses in the absence of detectable levels of deamination. AKV is an endogenous retrovirus that is involved in causing a high incidence of thymic lymphoma in AKR mice. A comparative analysis of several mouse strains revealed a relatively low level of APOBEC3 expression in AKR mice. Here we show that endogenous mouse APOBEC3 restricts AKV infection and that this restriction likely reflects polymorphisms affecting APOBEC3 abundance rather than differences in the APOBEC3 isoforms expressed. We also observe that restriction of AKV by APOBEC3 is accompanied by G→A hypermutations in the viral genome. Our findings demonstrate that APOBEC3 acts as a restriction factor in rodents affecting the strain tropism of AKV, and they provide good support for the proposal that APOBEC3-mediated hypermutation contributed to the evolution of endogenous rodent retroviral genomes.Viruses that are restrained to infect only a specific animal species, subspecies, or strain have acquired particular features that enable them to circumvent the immune defenses of that particular host. Conversely, the natural hosts for these pathogens are alive today because they have evolved strategies to restrain the infectivities of their own pathogens. A virus with a broad host tropism will typically have evolved under selective pressure from several host factors that it will have encountered and successfully evaded. Ecotropic murine retroviruses generally have a restricted host range, due not only to the limited availability of their cellular receptor, mCAT-1 (58), but also to the various intrinsic restriction factors present in a specific host (7). Fv1 and Fv4 are the expression products of defective endogenous retroviruses that are present as germ line integrations and can interfere with and even block the infectivities of ecotropic retroviruses (6, 25).Mouse APOBEC3 is another type of host-encoded intrinsic restriction factor that can display deoxycytidine deaminase activity on single-stranded DNA (16, 54). APOBEC3 proteins have a potent inhibitory effect on retroelements ranging from primate lentiviruses to murine retrotransposons (reviewed in reference 17). In humans and primates there are seven APOBEC3 genes, most of which have been proposed to act as restriction elements for viruses and retroelements. The most extensively characterized of the primate APOBEC3 proteins are APOBEC3F and APOBEC3G, which constitute powerful restriction factors for human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) (reviewed in reference 17). The evidence that these lentiviruses are targets for APOBEC3 action does not come just from in vitro experiments: tissue samples from HIV type 1 (HIV-1)-infected humans contain retroviral sequences exhibiting a pattern of G→A hypermutation that is characteristic of APOBEC3F/G-dependent deoxycytidine deamination (4, 12, 26, 56, 57).In contrast to primates, mice have only a single APOBEC3 gene. This murine APOBEC3 has been shown to be able to inhibit retrotransposition of mouse MusD and intracisternal A particle elements in cotransfection assays (22, 23). However, the lack of any obvious signs of disease, developmental defect, or infertility in APOBEC3-deficient mice indicates that APOBEC3 may not play an essential role in suppressing the transposition of endogenous retroelements in laboratory mice (38, 40). With regard to exogenous retroviruses, mouse APOBEC3 has been shown to hinder the in vivo infectivity of the betaretrovirus mouse mammary tumor virus (MMTV) as well as that of the gammaretrovirus Friend murine leukemia virus (MLV) (40, 55); its activity against Moloney MLV (MoMLV), another gammaretrovirus, is apparently considerably weaker—likely reflecting the fact that MoMLV may have found ways to avoid APOBEC3-mediated restriction (14, 34, 46, 61). In none of these cases, however, does it appear that mouse APOBEC3 hypermutates the retroviral replication intermediates, suggesting that deamination is not central to its mechanism of restricting these retroviruses. Notwithstanding this failure to observe hypermutation of mouse retroviruses by mouse APOBEC3, recent studies of nucleotide sequence diversity among endogenous gammaretroviruses have suggested that the evolution of endogenous retroelements has been shaped by the mutagenic cytidine deaminase activity of APOBEC3 (28, 42). Thus, the picture which emerges is that APOBEC3 acts as one of several restriction factors of mouse retroelements, with some viruses having found ways to avoid APOBEC3-mediated restriction.Different mouse strains exhibit different patterns of APOBEC3 expression (41, 47, 55). Thus, two major mouse APOBEC3 alleles have been identified: one encodes a protein whose sequence is similar to that of the allele expressed in C57BL/6 mice, and the other resembles that of BALB/c mice (47, 55). Two major splicing isoforms of APOBEC3, which either do or do not include exon 5, have also been detected: the relative abundance of these two isoforms differs between strains (36, 41, 47, 55). The restriction of Friend MLV and that of MMTV both appear to be dependent on the identity of the mouse strain, and it has been proposed that this reflects the polymorphism in the sequence and splicing isoforms of APOBEC3 (41, 47, 55).In the course of our work on mouse APOBEC3, we discovered that APOBEC3 was expressed only at a low level in AKR mice. The AKR mouse strain harbors several germ line insertions of an endogenous ecotropic MLV designated AKV, which belongs to the gammaretrovirus family (5, 15, 27, 44, 45). A complex set of recombination events between AKV and nonecotropic endogenous retroviruses results in the production of leukemogenic mink cell focus-inducing viruses that are responsible for inducing a lethal form of thymic lymphoma of T-cell origin in these mice (19, 53). We were interested in determining whether the susceptibility of AKR mice to AKV infection could in part be explained by a failure of the APOBEC3 allele expressed in AKR mice to restrict this virus.Here we show that endogenous murine APOBEC3 in C57BL/6 mice not only acts to restrict AKV infection but also hypermutates AKV replication intermediates, likely providing a powerful block to natural transmission of the virus between mouse strains. We find that the different isoforms of APOBEC3 (whether or not they include exon 5) are effective in AKV restriction and that the differential resistance of lymphocytes from different mouse strains/mutants to AKV infection correlates with the abundance of endogenous APOBEC3 mRNA. Our results indicate that APOBEC3 confers effective protection against germ line integration of retroviral pathogens in rodents, and they provide tangible support to the proposal that DNA editing by APOBEC3 may have participated in the evolution of endogenous retroviral genomes.     

Studies on morphological transformation of BALB/3T3-derived clones by murine leukemia virus.

We have described a cell line, UC1-B, derived spontaneously from BALB/3T3 mouse embryo cells, which, unlike the standard BALB/3T3, are morphologically transformed and produce bizarre viral forms in response to murine leukemia virus. Although UC1-B and BALB/3T3 are morphologically similar, and both form contact-inhibited monolayers at confluence, the UC1-B cells are partially transformed because: they grow to a slightly higher saturation density than 3T3 cells, they grow in medium lacking serum growth factors, and they produce tumors in mice. Another clone, 12A-3, derived from BALB/3T3, also transforms and produces bizarre viral forms after infection with murine leukemia virus. Unlike UC1-B cells, the 12A3-8 cells are identical in growth properties to BALB/3T3; therefore, a partially altered morphology is not required for the induction of transformation by murine leukemia virus.     

Selection of Revertants of Kirsten Sarcoma Virus Transformed Nonproducer BALB/3T3 Cells

Revertants of Kirsten sarcoma virus transformed nonproducer BALB/3T3 cells (KA31 cells) were isolated after exposing the transformed cells to 5-fluorodeoxyuridine at high cell density, or when suspended in methylcellulose. Revertants were also isolated by treating KA31 cells with the lectin, concanavalin A, which is manyfold more toxic to transformed cells than for normal cells. The revertants resemble BALB/3T3 cells in their morphology and growth characteristics in that they have a low saturation density, fail to grow in 1% calf serum or when suspended in methylcellulose, and cease to synthesize DNA after reaching their saturation density. Infection by murine leukemia virus rescues Kirsten sarcoma virus from only the concanavalin-A-selected variants, though all the revertants are susceptible to infection by leukemia virus. The concanavalin A revertants also become transformed after infection with murine leukemia virus. All the revertants can be transformed by Kirsten sarcoma virus but not by simian virus 40.     

Studies on the regulation of insulin receptors in cultured BALB/3T3 fibroblasts

The level of [¹²⁵I]insulin binding to BALB/ 3T3 fibroblasts was low in growing cells and high in stationary cells. Since frequent changes of medium (every 2 h) did not modify the hormone binding of the stationary cells, it is unlikely that serum factors directly regulate the number of insulin receptors. Cells were grown to different densities by plating them in different concentrations of serum. Insulin binding was low in dense cultures maintained actively growing by high serum concentration, while binding was high in sparse cultures which were growth-arrested due to serum depletion. Thus, cell density does not directly regulate the insulin receptors. The growth status of the cells is the only factor that explains consistently the variations of insulin binding in these and previous [1, 2] experiments. Synchronization of the cells by two different methods did not show a reproducible cellcycle dependence for the insulin receptors.     

A Chimeric Ty3/Moloney Murine Leukemia Virus Integrase Protein Is Active In Vivo

This report describes the results of experiments to determine whether chimeras between a retrovirus and portions of Ty3 are active in vivo. A chimera between Ty3 and a Neo^r-marked Moloney murine leukemia virus (M-MuLV) was constructed. The C-terminal domain of M-MuLV integrase (IN) was replaced with the C-terminal domain of Ty3 IN. The chimeric retroviruses were expressed from an amphotrophic envelope packaging cell line. The virus generated was used to infect the human fibrosarcoma cell line HT1080, and cells in which integration had occurred were selected by G418 resistance. Three independently integrated viruses were rescued. In each case, the C-terminal Ty3 IN sequences were maintained and short direct repeats of the genomic DNA flanked the integration site. Sequence analysis of the genomic DNA flanking the insertion did not identify a tRNA gene; therefore, these integration events did not have Ty3 position specificity. This study showed that IN sequences from the yeast retrovirus-like element Ty3 can substitute for M-MuLV IN sequences in the C-terminal domain and contribute to IN function in vivo. It is also one of the first in vivo demonstrations of activity of a retrovirus encoding an integrase chimera. Studies of chimeras between IN species with distinctive integration patterns should complement previous work by expanding our understanding of the roles of nonconserved domains.     

Analysis of the Fusion of XC Cells Induced by Homogenates of Murine Leukemia Virus-Infected Cells and by Purified Murine Leukemia Virus

The fusion of XC cells induced by murine leukemia virus (MuLV)-infected cells is also induced by homogenates prepared from the infected cells and by purified MuLV. The fusion-inducing factor appears to contain a heat-labile lipoprotein. No synthesis of specific macromolecules by the XC cells is necessary to obtain fusion. The results suggest that specific components of the viral particle are the activators for the fusion process and they may also be present in the membranes of infected cells.     

Suppression of Murine Retrovirus Polypeptide Termination: Effect of Amber Suppressor tRNA on the Cell-Free Translation of Rauscher Murine Leukemia Virus, Moloney Murine Leukemia Virus, and Moloney Murine Sarcoma Virus 124 RNA

The effect of suppressor tRNA's on the cell-free translation of several leukemia and sarcoma virus RNAs was examined. Yeast amber suppressor tRNA (amber tRNA) enhanced the synthesis of the Rauscher murine leukemia virus and clone 1 Moloney murine leukemia virus Pr200^gag-pol polypeptides by 10- to 45-fold, but at the same time depressed the synthesis of Rauscher murine leukemia virus Pr65^gag and Moloney murine leukemia virus Pr63^gag. Under suppressor-minus conditions, Moloney murine leukemia virus Pr70^gag was present as a closely spaced doublet. Amber tRNA stimulated the synthesis of the “upper” Moloney murine leukemia virus Pr70^gag polypeptide. Yeast ochre suppressor tRNA appeared to be ineffective. Quantitative analyses of the kinetics of viral precursor polypeptide accumulation in the presence of amber tRNA showed that during linear protein synthesis, the increase in accumulated Moloney murine leukemia virus Pr200^gag-pol coincided closely with the molar loss of Pr63^gag. Enhancement of Pr200^gag-pol and Pr70^gag by amber tRNA persisted in the presence of pactamycin, a drug which blocks the initiation of protein synthesis, thus arguing for the addition of amino acids to the C terminus of Pr63^gag as the mechanism behind the amber tRNA effect. Moloney murine sarcoma virus 124 30S RNA was translated into four major polypeptides, Pr63^gag, P42, P38, and P23. In the presence of amber tRNA, a new polypeptide, Pr67^gag, appeared, whereas Pr63^gag synthesis was decreased. Quantitative estimates indicated that for every 1 mol of Pr67^gag which appeared, 1 mol of Pr63^gag was lost.     

Activation of the Murine Sarcoma Virus Genome After Infection with the Murine Leukemia Virus as Determined by Cell Agglutination

Non-virus-producing NIH/3T3 cells transformed by the murine sarcoma virus are agglutinated by conconavalin A to the same low level as normal NIH/3T3 cells. Infection with the murine leukemia virus greatly increases the agglutination of transformed cells but not that of normal cells. These data suggest that the morphological expression of cell transformation and the surface alterations associated with increased cell agglutination are controlled by the expressions of different sarcoma virus genes.     

Transformation of mouse BALB 3T3 cells by enterobacterial plasmid misrepair gene mucAB.

The enterobacterial plasmid misrepair gene mucAB, ligated to the metal-inducible mammalian MT-1 promoter, was introduced into the genome of mouse BALB 3T3 cells. In the presence of zinc ions, MucA but not MucB protein was produced, and the whole-cell population of each mucAB+ clone started to show the transformation phenotype in a few days. Foci appeared in the transformed cell population after 4 weeks, and cells from the foci produced tumors in nude mice, indicating malignant transformation by the mucA product. Growth of mucAB+ cells was stimulated by zinc-induced expression of mucA. The transformation phenotype was reversed by removing zinc ions from the culture, indicating that the transformation was due not to MucA-mediated mutation in the mouse genome but to the direct transforming activity of MucA protein.     

Murine Mammary Tumor Virus Expression During Mammary Tumorigenesis in BALB/c Mice

Steady-state levels of murine mammary tumor virus (MuMTV) RNA were quantitated during mammary tumorigenesis in BALB/c mice by molecular hybridization with a representative MuMTV complementary DNA (cDNA) probe. Hyperplastic alveolar nodule (HAN) lines are preneoplastic mammary lesions that were induced in BALB/c mice by hormones alone or in combination with 7,12-dimethylbenz(a)anthracene and give rise to mammary tumors. The hormone-induced HAN lines D1 and D2 contained detectable amounts of hybridizable MuMTV sequences. MuMTV RNA sequences were also observed in five of the six transplanted BALB/c mammary tumors that were examined. Similar levels of hybridizable MuMTV RNA were observed between the D1 or D2 HAN line and mammary tumors derived from each HAN line. The D2 HAN line as well as D2, C4, and CD8 mammary tumors accumulated RNA that was apparently homologous to most of the MuMTV genome. Thermal denaturation of hybrids indicated extensive sequence homology between the MuMTV cDNA and hybridizable RNA in the BALB/c HAN lines and mammary tumors. A low level of type C viral RNA was observed in the BALB/c HAN lines and most mammary tumors by molecular hybridization with a cDNA to Moloney murine leukemia virus. These data demonstrate that MuMTV sequences are frequently expressed in hormone-induced BALB/c HAN lines and mammary tumors derived from HAN lines or ductal hyperplasias induced in BALB/c mice by hormones and/or a chemical carcinogen. The transition from the preneoplastic to the neoplastic state in BALB/c mice does not appear to be due to a change in the steady-state levels of MuMTV RNA since the hormone-induced HAN lines and mammary tumors had similar levels of hybridizable MuMTV RNA.     

Analysis of the Ribonucleic Acid of Murine Leukemia Virus

Cells producing the Rauscher strain of murine leukemia virus (MLV) were exposed to (3)H-uridine, and labeled virus was collected at hourly intervals. Ribonucleic acid (RNA) extracted from virions (vRNA) had a characteristic single peak when analyzed by electrophoresis in polyacrylamide-agarose composite gels. Exposure of vRNA to dimethyl sulfoxide, urea, formaldehyde, or heat altered the mobility to a faster moving form (vRNA'). This vRNA' sedimented more slowly than native vRNA in sucrose gradients. Incubation of labeled virions at 37 C resulted in fragmentation of viral RNA which was detectable only after denaturation. Also, large differences in the temperature required for the change from vRNA to vRNA' were seen with alterations in NaCl concentration. These experiments demonstrate that the vRNA of MLV is held in a specific conformation by hydrogen bonds distributed over a large part of the molecule. The possibility that an undefined factor is associated with viral RNA is discussed.     

Efficient Production of Xenotropic Murine Leukemia Virus Unintegrated Proviral DNA by Cocultivation

Cocultivation of virus-producing cells and homologous uninfected cells yielded greater than a 10-fold increase in linear and superhelical proviral DNAs as compared with previously published techniques.     

Structure and Leukemogenic Activity of a Murine Leukemia Virus

Purified Friend viruses obtained from chronically infected tissue cultures were studied under the electron microscope in an effort to correlate the fine structure of the particles to their leukemogenic activity under varied experimental conditions, i.e., temperature treatments and exposure to Tween 80, amyl acetate, or ether. It was observed that an intact viral envelope was a prerequisite to leukemogenic activity as tested by intraperitoneal inoculation of newborn mice. It was also noted that the percentage of C particles was not increased after heating for 1 hr at 45 C (treatment which, however, completely inactivated the viruses). Digestion with ribonuclease indicated the presence of ribonucleic acid within the nucleoids of "enveloped A particles," which shows that these are not immature particles. The significance of the simultaneous presence of "enveloped A" and C particles is discussed.     

Cell Line derived from Balb/3T3 that is Transformed by Murine Leukaemia Virus : a Focus Assay for Leukaemia Virus

Balb/3T3 derived cell line UC1-B transforms on MuLV infection, forming foci of transformants which can be used as an assay for MuLV. MuLV virus type C is produced as well as bizarre forms of uncertain origin.