Restriction of Moloney murine leukemia virus replication in Moloney murine sarcoma virus-infected cells

MuSV349 is a TB-cell line which produces infectious MuSV with little or no MuLV detectable by the XC assay. The apparent restriction of MuLV replication in MuSV349 cells was investigated. A replication-competent helper virus, with protein composition nearly identical to that of Mo-MuLV was isolated from the viruses produced by MuSV349 cells. This helper MuLV after it was separated from MuSV and upon infection of TB cells produced viral titer similar to that of Mo-MuLV-infected TB cells indicating that its replication might have been restricted in the MuSV349 cells. To find out whether the suppression of the helper virus replication is due to the genetic peculiarities of the virus or MuSV349 cells, the relative amounts of MuLV and MuSV produced by several distinct clonal MuSV isolates (derived from a common progenitor) upon superinfection with Mo-MuLV were determined. The results of these experiments showed that while both SV7 and SV15F on coinfection with Mo-MuLV produced MuSV in excess over MuLV; and ts110 and tsSV13 on coinfection with Mo-MuLV produced MuLV in excess over MuSV. Since the same Mo-MuLV is used in these experiments and since upon transfer to a different cell, SV7, SV15F, and ts110 retain the property to restrict or not restrict MuLV replication it appears that the above property is determined by the genetics of the MuSV.     

Hexagonal organization of Moloney murine leukemia virus capsid proteins

To help elucidate the mechanisms by which retrovirus structural proteins associate to form virus particles, we have examined membrane-bound assemblies of Moloney murine leukemia virus (M-MuLV) capsid (CA) proteins. Electron microscopy and image reconstruction techniques showed that CA dimers appear to function as organizational subunits of the cage-like, membrane-bound protein arrays. However, new three-dimensional (3D) data also were consistent with hexagonal (p6) assembly models. The p6 3D reconstructions of membrane-bound M-MuLV CA proteins gave unit cells of a = b = 80.3 A, c = 110 A, gamma = 120 degrees, in which six dimer units formed a cage lattice. Neighbor cage hole-to-hole distances were 45 A, while distances between hexagonal cage holes corresponded to unit cell lengths (80.3 A). The hexagonal model predicts two types of cage holes (trimer and hexamer holes), uses symmetric head-to-head dimer building blocks, and permits the introduction of lattice curvature by conversion of hexamer to pentamer units. The M-MuLV CA lattice is similar to those formed in helical tubes by HIV CA in that hexamer units surround cage holes of 25-30 A, but differs in that M-MuLV hexamer units appear to be CA dimers, whereas HIV CA units appear to be monomers. These results suggest that while general assembly principles apply to different retroviruses, clear assembly distinctions exist between these virus types.     

The effect of cerulenin on Moloney murine leukemia virus morphogenesis

Cerulenin is an antibiotic that interferes with fatty acid synthesis in eukaryotic cells. It had been shown by Schultz and Oroszlan (1983), that murine leukemia virus (MuLV) Pr65gag, the polyprotein precursor to the virion core proteins contains the fatty acid myristate at its NH2 terminus. We showed that when 20 micrograms/ml of cerulenin is added for 3 h to mouse fibroblasts chronically infected with Moloney (M)-MuLV it causes a greater than 4-fold decrease in virus production. This is accompanied by an accumulation of uncleaved Pr65gag in the infected cells. Further, thin-section electron micrographs of cerulenin-treated cells show a 2-fold increase in the number of nascent-budding forms, as well as the appearance of aberrant viral forms at the cell membrane. This suggests that the failure to add myristic acid to Pr65gag prevents their proper assembly into viral particles.     

Studies of two temperature-sensitive mutants of Moloney murine leukemia virus

Events associated with the replication of two spontaneous temperature-sensitive mutants (ts1 and ts3) of Moloney murine leukemia virus were studied under non-permissive and permissive conditions of infection. From temperature-shift experiments ts1 appears to have a temperature-sensitive step occurring early in the growth cycle and in an unidentified function. The temperature-sensitive defect of ts3 occurs late in the replicative cycle. Electron microscope studies showed that although budding partially occurs at the nonpermissive temperature there is no release of virus particles from the cell membrane. The defect of ts3 was also found to be in one of the factors required for the rescue of MuSV.     

Large-scale purification of gp70 from Moloney murine leukemia virus

The external envelope glycoprotein, gp70, of the Moloney murine leukemia virus was extracted from NIH 3T3 cells utilizing the detergent n-octyl-beta-D-glycopyranoside. The extracted gp70 was sequentially purified utilizing lectin-affinity, anion-exchange, and molecular-exclusion chromatography techniques. Approximately 10 mg of gp70 was purified by this method and shown to be 95% homogeneous, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The presence of purified gp70 from Moloney murine leukemia virus was confirmed by amino acid analysis, amino-terminal sequencing, and immunoreactivity with a monoclonal antibody raised against gp70. The procedure is rapid, utilizes commercially available media, and can be used to purify large amounts of retroviral envelope glycoprotein from virus.     

Rapid purification of extracellular and intracellular Moloney murine leukemia virus

Summary The present study demonstrates the advantages of a combination of concentration by polyethylene glycol-6000 and Sepharose Cl-4B chromatography as a rapid procedure for retroviruses purification. This procedure can be completed within 3 hours, providing a high degree of virus purification with minimal damage to its structural and biological properties. Using transmission electron microscopy we observed many intracellular type-C virions in cytoplasmic vacuoles of 3T3/NIH cells chronically infected with Moloney murine leukemia virus. There intracellular virions could be isolated from postmitochondrial cytoplasmic fractions prepared from the infected cells by a procedure which minimized its contamination by extracellular free or membrane-bound virions. SDS-polyacrylamide gel electrophoresis showed that the intracellular and extracellular virions contained similar protein composition.With 5 Figures     

Localization of actin in Moloney murine leukemia virus by immunoelectron microscopy.

Immunoelectron microscopy was used to detect actin in wild-type (wt) Moloney murine leukemia virus (MoMuLV) and in virus-like particles (VLP) produced by recombinant Semliki Forest virus expressing only the MoMuLV gag polyprotein. Gold immunolabeling revealed the presence of actin on the surface of delipidized VLP and delipidized wt virus particles. Statistical evaluation of the number of colloidal gold particles per VLP revealed a large range of values and a prevalence of VLP with small numbers of gold particles. Labeling for actin was lost after prolonged treatment of VLP with 1% Nonidet-P40, high-pH buffer, or gelsolin. Gold immunolabeling with antibodies to gag proteins p15 (MA) and p12 and p30 (CA) was abundant and was not affected by treatment of VLP or wt virus with 1% Nonidet or gelsolin. VLP treated with a mixture of detergent and aldehyde fixatives showed more uniform and consistent labeling for actin than without fixatives. Negative staining or heavy metal shadowing revealed a globular surface of delipidized VLP. Stereomicrographs of gold-immunolabeled VLP showed that p15gag and p12gag were associated with the globular projections. Delipidized VLP were also well labeled with antibody to p30gag, which indicated that the gag shell permitted access of antibodies to p30gag and was therefore not a closely packed structure. Labeling for actin-binding proteins moesin and ezrin was negative in both the wt virus and the VLP. The absence of Gaussian distribution of actin in the sample of VLP suggests that actin is not a structural protein and its presence in MuLV virus particles may be fortuitous. This, however, does not rule out any possible role of actin in transport, assembly, budding, or release of virus particles, events which take place in the cytoplasm or at the plasma membrane. The site of actin in VLP is discussed in relation to the present knowledge of the molecular organization of the MuLV gag shell.     

Mutational analysis of the N-terminus of Moloney murine leukemia virus integrase

The retroviral integrase (IN) carries out the integration of viral DNA into the host genome. The IN protein consists of three domains: the N-terminal HHCC motif, the catalytic core region, and the C-terminus. The Moloney murine leukemia virus (M-MuLV) IN encodes a unique 45-amino-acid domain N-terminal to the HHCC motif. The function of the N-terminus of M-MuLV IN was studied through deletional and mutational analyses. The IN 1-105 domain was dissected into two halves expressing either the unique N-terminus or the HHCC domain. Although the parental IN 1-105 could functionally complement the core-C-terminus for integration reactions, neither half of the N-terminus was sufficient. Partial complementation of strand transfer, but not 3prime prime or minute processing, could be obtained through mixing the two halves. The dimerization of the M-MuLV N-terminus was dependent on the expression of the intact 1-105. Critical basic amino acids within the HHCC domain which are required for 3' processing and strand transfer reactions were identified through alanine mutagenesis. Loss of in vitro strand transfer activity correlated with loss of viral titer in vivo for this cluster of basic amino acids within the HHCC domain.     

Dependence of Moloney murine leukemia virus production on cell growth.

Production of Moloney murine leukemia virus (MuLV) ceases in cultures of infected mouse fibroblasts made stationary by growth in limiting medium. Stimulation of the cells to reenter the growth cycle synchronously was followed by two waves of virus release. The first wave, occurring within 4 hr after the cells were released from G₀, was unaffected by 0.1 μg/ml of actinomycin D. The second wave, coinciding roughly with the mitotic period of the following cell cycle, was prevented by actinomycin D. Both waves of virus release were inhibited by 10 μg/ml of cycloheximide. The first wave of released virus appears to contain viral RNA already present in the resting cells, while the second wave of released virus particles carry newly formed viral RNA.     

Differential multimerization of Moloney murine leukemia virus integrase purified under nondenaturing conditions

Retroviral integrases (IN) catalyze the integration of the reverse-transcribed viral DNA into the host genome, an essential process leading to virus replication. For Moloney murine leukemia virus (M-MuLV) IN, the limited solubility of the recombinant protein has restricted the development of biophysical and structural analyses. Herein, recombinant M-MuLV IN proteins, either full length or two nonoverlapping domain constructs, were purified under non-denaturing conditions from solubilized bacterial extracts by Ni(2+)-NTA resins. Additionally, WT IN was further purified by heparin chromatography. All of the purified proteins were shown to be active and stable. WT M-MuLV IN chromatographed with a peak corresponding with a dimer by gel filtration chromatography. In contrast, the single point mutant C209A IN migrated predominantly as a tetramer. For both proteins, fractions in equilibrium between dimers and tetramers were competent to assemble concerted two-end integrations and yielded a unique strand-transfer profile in the presence of a 28-mer U5 oligonucleotide substrate, indicative of a distinct conformation within the synaptic complex. This specific target-site selection was not observed with a shorter 20-mer U5 substrate. These studies provide the foundation for biophysical and structural analysis on M-MuLV IN and the mechanism of retroviral integration.     

Membrane-proximal cytoplasmic domain of Moloney murine leukemia virus envelope tail facilitates fusion

Removal of the R peptide (residues 617-632) from the Moloney murine leukemia virus (MoMuLV) envelope protein (Env) cytoplasmic tail potentiates fusion. We examined the role of the membrane-proximal cytoplasmic domain (598-616) of the MoMuLV Env in the Env-mediated membrane fusion and incorporation. The Env truncated at 616 exhibits maximum fusogenicity in cell-to-cell fusion assay. By comparison, full tail Env (632) and the Env truncated to residue 601 mediated fusion at 40%. The Envs truncated to residues 598 or 595 are not fusogenic. Progressive cytoplasmic tail truncation correlated with decreased Env incorporation into virions. Substitution of the domain 598-616 with an amphiphilic alpha-helix from melittin results in maximally fusogenic Envs that efficiently incorporated into transduction competent virions. However, substitution of the domain 598-616 with random or hydrophilic sequences caused loss of the Env fusogenicity and titer while retaining incorporation. Further, a secondary structure prediction analysis of 27 unrelated Env cytoplasmic tails indicates a common (23/27) propensity for an amphiphilic alpha-helical domain at immediate proximity to the viral membrane. These results support the suggestion that viral fusion is enhanced by a membrane-proximal cytoplasmic amphiphilic alpha-helix in Env tail. The model of its action is proposed.     

Expression of the Moloney murine leukemia virus and human immunodeficiency virus integration proteins in Escherichia coli

We have constructed expression plasmids containing the genes for the MuLV and the HIV integration proteins. When introduced into Escherichia coli, these plasmids cause the production of proteins of the expected molecular weight (43K for MuLV, 31K for HIV). The wild-type MuLV coding region induces the synthesis of large amounts of integration protein; to obtain large amounts of the full-length HIV integration protein in E. coli, it was necessary to modify the coding region to disrupt a sequence that promotes efficient internal translational initiation in E. coli. It is possible to disrupt this sequence without altering the encoded amino acids; the modified plasmid makes large amounts of the full-length protein and small amounts of the internally initiated protein. Both the MuLV and HIV integration proteins require SDS to be solubilized in E. coli extracts; however, following solubilization with SDS and transfer to a nitrocellulose filter, both integration proteins bind DNA.     

Moloney murine sarcoma virus encoded p37mos expressed in yeast has protein kinase activity

We describe the expression of the Moloney murine sarcoma virus env-mos protein (p37mos) in yeast under the control of the yeast GAL1 promoter. Consistent with our previous results concerning the p37mos protein kinase made in virus infected mouse cells, p37mos produced in yeast possesses autophosphorylation activity in an immune complex kinase assay using anti-mos (37-35) serum.     

Generation of thymotropic envelope gene recombinant virus and induction of lymphoma by ecotropic Moloney murine leukemia virus

Biologically cloned pure ecotropic Moloney MuLV was used to infect Balb/c and AKR mice to determine the replication of ecotropic virus, the possible generation of recombinant viruses, and the induction of disease. Infectious cell center (ICC) experiments carried out with lymphoid cells of individual Balb/c mice showed that e-M-MuLV rapidly infected up to 30% of lymphoid cells in liver, spleen, and especially in the thymus. No recombinant virus was seen until about Day 35 when a burst of RM-MuLV was observed only in the thymus. New RM-MuLV was found in all 32 preleukemic and leukemic mice tested and persisted at low levels until death. The RM-MuLV recovered early in the preleukemic phase had an env-related M-MuLV but grew very poorly. Cells from a late tumor which grew and cloned readily were examined to see whether the new RM-MuLV was present in every clone. Overtly, most tumor cells did not seem to contain RM-MuLV, but when "unmasking" was performed, every tumor cell contained identical RM-MuLV. In AKR mice, both e-M-MuLV and recombinant M-MuLV caused an acceleration of lymphoma. De novo appearance of a thymotropic RM-MuLV, which was of the Moloney RM-MuLV type and the absence of early detectable endogenous AKR-MCF-type recombinants, suggested that the early lymphoma was due to the induction of a new disease. Several theoretical approaches dealing with viral env-gene permutations are discussed.     

Point mutations in the P30 domain of the gag gene of Moloney murine leukemia virus

A series of point mutations in the P30 domain of the Moloney murine leukemia virus gag gene was generated by bisulfite treatment of heteroduplex DNAs containing a single-stranded region in the gag gene. One virus bearing such a mutation exhibited a coordinate defect in gag and pol function, and was similar to previously described deletion mutants with alterations in this gene. One mutant virus displayed a different phenotype: it could assemble virion particles and provide pol function, but the particles were defective in the early stages of infection. The continued concordance of the mutants' failure or ability to both assemble virions and provide pol lends further support to the proposal that similar parts of the gag gene are required for these two processes.