Recovery of Rauscher Leukemia Virus from Large Volumes of Seeded Cow''s Milk and from Infected Murine Spleens

Rauscher murine leukemia virus was used as an indicator agent to develop a methodology for the extraction and concentration of a theoretical leukemia virus from bovine milk and tissues. The indicator virus was seeded into cow's milk or was recovered from infected murine spleens. The tissue homogenates and the defatted milk were processed in a B-XVI rotor of a Spinco L-4 ultracentrifuge at a flow rate of 3 liters/hr. The efficiency of Rauscher virus recovery was greatest when the rotor was used without a gradient. A loss of between 0.6 and 0.7 log of total infectious virus, as determined by the spleen assay method, resulted when the seeded milk and murine spleens were processed. The procedures developed are presently being used in transmission experiments in an attempt to induce leukemia in the bovine.     

Recovery of Murine Leukemia Virus from Large Volumes of Freshly Harvested Culture Fluids by Using a Single Density Gradient

Concentration and purification of murine leukemia virus for use as complement fixation antigens was accomplished by using a single density gradient in the model RK ultracentrifuge.     

Defectivity of Rauscher Murine Erythroblastosis Virus

THE Rauscher leukaemia virus (RLV) induces a rapidly developing fatal erythroblastosis in mice, characterized by splenomegaly and a highly increased number of nucleated red cells in the peripheral blood¹. The first sign of action of the virus is the production of erythroblast foci in the spleen within a week². A linear dose-response relationship for virus and number of foci has been observed in several mouse strains, suggesting that a colony can be initiated by a single infective unit².     

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.     

Characterization of Glycos-aminoglycans Associated with Rauscher Murine Leukemia Virions

Host-derived sulfated components that copurify and are physically associated with the envelope of Rauscher murine leukemia virions grown in JLS-V9 cells were characterized by digestion with chondroitinase ABC and chondroitinase AC II, as well as nitrous acid degradation. A dermatan-sulfate-chondroitin-sulfate copolymer and heparin or heparan sulfate were shown to be associated with the virions. Competitive binding studies indicated a specificity of the virions for association with heparan sulfate. The physiological importance of the association is discussed.     

Moloney Murine Leukemia Virus Integrase Protein Augments Viral DNA Synthesis in Infected Cells

Mutations in the IN domain of retroviral DNA may affect multiple steps of the virus life cycle, suggesting that the IN protein may have other functions in addition to its integration function. We previously reported that the human immunodeficiency virus type 1 IN protein is required for efficient viral DNA synthesis and that this function requires specific interaction with other viral components but not enzyme (integration) activity. In this report, we characterized the structure and function of the Moloney murine leukemia virus (MLV) IN protein in viral DNA synthesis. Using an MLV vector containing green fluorescent protein as a sensitive reporter for virus infection, we found that mutations in either the catalytic triad (D184A) or the HHCC motif (H61A) reduced infectivity by approximately 1,000-fold. Mutations that deleted the entire IN (DeltaIN) or 34 C-terminal amino acid residues (Delta34) were more severely defective, with infectivity levels consistently reduced by 10,000-fold. Immunoblot analysis indicated that these mutants were similar to wild-type MLV with respect to virion production and proteolytic processing of the Gag and Pol precursor proteins. Using semiquantitative PCR to analyze viral cDNA synthesis in infected cells, we found the Delta34 and DeltaIN mutants to be markedly impaired while the D184A and H61A mutants synthesized cDNA at levels similar to the wild type. The DNA synthesis defect was rescued by complementing the Delta34 and DeltaIN mutants in trans with either wild-type IN or the D184A mutant IN, provided as a Gag-IN fusion protein. However, the DNA synthesis defect of DeltaIN mutant virions could not be complemented with the Delta34 IN mutant. Taken together, these analyses strongly suggested that the MLV IN protein itself is required for efficient viral DNA synthesis and that this function may be conserved among other retroviruses.     

Electron Microscopy of Friend Murine Leukemia Virus in the Mid-Gut of Experimentally Infected Mosquitoes

After 30 and 78 hr, Friend murine leukemia virus (FLV) particles were detected by electron microscopy in the mid-gut lumen of the mosquitoes Aedes aegypti (Linnaeus) and Anopheles stephensi Liston which had fed on leukemia BALB/c mice infected with FLV. Various developmental stages of the virions were observed within and on the surface of ingested blood cells, particularly young erythroblasts, as well as free in the lumen after budding. These preliminary findings indicate that FLV continues to multiply in the mid-gut of these species for at least 3 days despite the action of digestive enzymes. Detailed studies are in progress to determine the fate of FLV in these mosquito species.     

Transfection of Fibroblasts by Cloned Abelson Murine Leukemia Virus DNA and Recovery of Transmissible Virus by Recombination with Helper Virus

A cloned, permuted DNA copy of the Abelson murine leukemia virus (A-MuLV) genome was capable of eliciting the morphological transformation of NIH/3T3 fibroblasts when applied to cells in a calcium phosphate precipitate. The efficiency of the process was extremely low, yielding approximately one transformant per microgram of DNA under conditions which give 10(4) transfectants per microgram of other DNAs (e.g., Moloney sarcoma virus proviral DNA). The DNA was able to induce foci, even though the 3' end of the genome was not present. The transforming gene was thus localized to the 5' portion of the genome. The transformed cells all produced viral RNA and the virus-specific P90 protein. Transmissible virus could be rescued from these cells at very low frequencies by superinfection with helper virus; the rescued A-MuLV virus had variable 3' ends apparently derived by recombination with the helper. Dimerization of the permuted A-MuLV cloned genome to reconstruct a complete provirus did not improve transformation efficiency. Virus could be rescued from these transformants, however, at a high efficiency. Cotransfection of the permuted A-MuLV DNA with proviral M-MuLV DNA yielded a significant increase in the efficiency of transformation and cotransfection of dimeric A-MuLV and proviral M-MuLV resulted in a high-efficiency transformation yielding several thousand more transformants per microgram than A-MuLV DNA alone. We propose that helper virus efficiently rescues A-MuLV from transiently transfected cells which would not otherwise have grown into foci. We hypothesize that multiple copies of A-MuLV DNA introduced into cells by transfection are toxic to cells. In support of this hypothesis, we have shown that A-MuLV DNA sequences can inhibit the stable transformation of cells by other selectable DNAs.     

Protein Kinase and Phosphate Acceptor Proteins in Rauscher Murine Leukaemia Virus

Rauscher murine leukaemia virus contains both a protein kinase and substrate proteins. The proteins phosphorylated seem to include the major structural components of the virus. The enzyme and substrate proteins are also found in other membrane-maturing viruses, including vesicular stomatitis virus.     

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.     

Reversible Inactivation of the Deoxyribonucleic Acid Polymerase of Rauscher Leukemia Virus

Rauscher leukemia virus deoxyribonucleic acid polymerase is reversibly inactivated by 6 m guanidine-hydrochloride. Gel filtration in 6 m guanidine-hydrochloride reveals that the viral deoxyribonucleic acid polymerase consists of a single polypeptide chain of approximately 70,000 molecular weight.     

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.     

Striatal Met-Enkephalin and Substance P Levels Are Decreased in Mice Infected with the LP-BM5 Murine Leukemia Virus

Abstract: Mice infected with the LP-BM5 murine leukemia virus mixture develop severe immunosuppression and an encephalopathy characterized by spatial learning deficits. Twelve weeks after infection of C57BL/6J mice with LP-BM5, significant (50–60%) reductions in Met-enkephalin and substance P levels were observed in the striatum, whereas somatostatin levels were unchanged. In addition, a 39% decrease in hypothalamic substance P concentrations was observed, with no alteration in Met-enkephalin levels. The apparent selectivity of the decrease in neuropeptide concentrations indicates that a functional alteration of the primary striatal efferent neurons occurs in this infection, which may contribute to the impairment of spatial learning observed in these mice. Moreover, this decrease in striatal neuropeptide levels is similar to the neuropathological changes in basal ganglia observed in HIV-infected individuals and is consistent with previous studies suggesting that the LP-BM5-infected mouse may serve as a useful model of AIDS dementia.     

Purification of the Moloney and Rauscher Murine Leukemia Viruses by Use of Zonal Ultracentrifuge Systems

The B-IV and B-IX zonal ultracentrifuge rotors were applied to the concentration and purification of the Moloney and Rauscher murine leukemia viruses from large volumes of infected tissue culture fluids and animal materials. Potassium tartrate, potassium citrate and sucrose gradients were used to obtain viral concentrates from the density 1.16 to 1.18 zone. Proteolytic enzyme digestion of tissue culture preparations prior to zonal ultracentrifuge processing was effective in releasing virus from cell debris and producing highly purified, though nonleukemogenic, viral concentrates. Infected Rauscher mouse plasma was processed to give highly purified infectious virus fractions. A single centrifugation of crude Rauscher mouse spleen homogenates resulted in partially purified infectious concentrates with high virus particle counts.     

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.     

Pseudotype Formation of Moloney Murine Leukemia Virus with Sendai Virus Glycoprotein F

Mixed infection of cells with both Moloney murine leukemia virus (MoMLV) and related or heterologous viruses produces progeny pseudotype virions bearing the MoMLV genome encapsulated by the envelope of the other virus. In this study, pseudotype formation between MoMLV and the prototype parainfluenza virus Sendai virus (SV) was investigated. We report for the first time that SV infection of MoMLV producer cells results in the formation of MoMLV(SV) pseudotypes, which display a largely extended host range compared to that of MoMLV particles. This could be associated with SV hemagglutinin-neuraminidase (SV-HN) glycoprotein incorporation into MoMLV envelopes. In contrast, solitary incorporation of the other SV glycoprotein, SV fusion protein (SV-F), resulted in a distinct and narrow extension of the MoMLV host range to asialoglycoprotein receptor (ASGP-R)-positive cells (e.g., cultured human hepatoma cells). Since stably ASGP-R cDNA-transfected MDCK cells, but not parental ASGP-R-negative MDCK cells, were found to be transduced by MoMLV(SV-F) pseudotypes and transduction of ASGP-R-expressing cells was found to be inhibited by ASGP-R antiserum, a direct proof for the ASGP-R-restricted tropism of MoMLV(SV-F) pseudotypes was provided. Cultivation of ASGP-R-positive HepG2 hepatoma cells on Transwell-COL membranes led to a significant enhancement of MoMLV(SV-F) titers in subsequent flowthrough transduction experiments, thereby suggesting the importance of ASGP-R accessibility at the basolateral domain for MoMLV(SV-F) pseudotype transduction. The availability of such ASGP-R-restricted MoMLV(SV-F)-pseudotyped vectors opens up new perspectives for future liver-restricted therapeutic gene transfer applications.