Cell fusion induced by the murine leukemia virus envelope glycoprotein.

To determine whether ecotropic murine leukemia virus (MuLV) envelope glycoproteins are sufficient to cause cell-to-cell fusion when expressed in the absence of virus production, we used an ecotropic MuLV, AKV, to construct env expression vectors that lack the gag and pol genes. The rat cell line XC, which undergoes cell-to-cell fusion upon infection with ecotropic MuLV, was transfected with wild-type env expression vectors, and high levels of syncytium formation resulted. Transfection of the murine cell line NIH 3T3 with expression vectors containing the wild-type or mutated env region did not result in syncytium formation. Immunoprecipitation analysis of the envelope glycoproteins expressed in NIH 3T3 and XC cells showed that the mature surface glycoprotein expressed in XC cells was of a much lower apparent molecular weight than that expressed in NIH 3T3 cells. Further characterization showed that most if not all of this difference was the result of differences in glycosylation. Finally, site-directed mutagenesis was used to introduce several conservative and nonconservative changes into the amino-terminal region of the transmembrane protein. Analysis of the effect of these mutations confirmed that this region is a fusion domain.

3Y1 rat cells are defective in processing of the envelope precursor protein of AKR virus.

Rat 3Y1 cells were infected by AKR virus through microinjection of molecularly cloned proviral DNA. Based on a strong immunofluorescence using anti-p30 as an antiserum a cell clone (RESA-2) was selected that had a high expression of viral antigens. Subsequent restriction analysis of its DNA revealed that the RESA-2 clone contained at least 30 apparently intact integrated proviruses per genome. There was an apparently normal synthesis and processing of gag and pol gene products. The viral envelope precursor polyprotein gPr82env, however, did not yield the major envelope glycoprotein gp70. The gag precursor polyprotein, Pr65gag, as well as the gPr82env from RESA-2 cells were identified as AKR viral proteins by gel electrophoresis of hydroxylamine cleavage fragments. The virions formed by RESA-2 cells lacked gp70 and were noninfectious. After fusion of RESA-2 cells and mouse cells an infectious N-tropic virus was produced. The results indicate that rat 3Y1 cells lack (a) factor(s) necessary for the correct processing of gPr82env. The high incidence of abortive infections of murine leukemia virus (MLV) in susceptible rat cells reported by others is therefore probably due to defective particles in the virus stock and/or to the lack of (a) cellular factor(s) necessary for reverse transcription and subsequent integration of the viral genome.

Metabolism of viral RNA in murine leukemia virus-infected cells; evidence for differential stability of viral message and virion precursor RNA.

Molecular hybridization techniques were used to examine the stability of viral message and virion precursor RNA in murine leukemia virus-infected cells treated with actinomycin D. Under the conditions used, viral RNA synthesis was inhibited, but viral protein synthesis continued, and the cells produced noninfectious particles (actinomycin D virions) lacking genomic RNA (J. G. Levin and M. J. Rosenak, Proc. Natl. Acad. Sci. U.S.A. 73:1154-1158, 1976). Analysis of total RNA in virions revealed that the amount of hybridizable viral RNA decreased steadily after the addition of actinomycin D and by 8 h was 10% of the control value. Studies on fractionated viral RNA showed that this low level of hybridization is due to residual 70S RNA in the virion population. The results indicated that viral RNA which is destined to be encapsidated into virions has a half-life of approximately 3 to 4 h. In contrast, other intracellular virus-specific RNA molecules appeared to be quite stable and persisted for a long period of time, with a half-life of at least 12 h. These observations support the idea that two independent functional pools of 35S viral RNA exist within the infected cell: one serving as message and the other as precursor to virion RNA. The existence of two viral RNA pools was further documented by the finding that 12 h after the addition of actinomycin D, when virion precursor RNA was depleted, 35S and 21S viral nRNA species could be identified in polyribosomal RNA as well as in total polyadenylated cell RNA. Surprisingly, 35S and mRNA declined more rapidly than did 21S mRNA, which appeared to be increased in amount.

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.

Selective packaging of host tRNA's by murine leukemia virus particles does not require genomic RNA.

The 4S RNA contained in RNA tumor virus particles consists of a selected population of host tRNA's. However, the mechanism by which virions select host tRNA's has not been elucidated. We have considered a model which specifies that 35S genomic RNA determines which tRNA's are to be encapsidated as well as the relative amounts of these tRNA's within the virion. The model was tested by comparing the free 4S RNA composition of normal murine leukemia virus (MuLV) particles and noninfectious virions from actinomycin D (ActD)-treated cells, which are deficient in genomic RNA (ActD virions). Viral 4S RNA was analyzed by two-dimensional polyacrylamide gel electrophoresis. Surprisingly, the patterns obtained for control and ActD 4S RNA were identical to each other and were clearly distinct from the cell 4S RNA pattern. The viral patterns had three prominent areas of radioactivity. One of the spots was identified on the basis of its oligonucleotide fingerprint as tRNA (Pro), the primer for MuLV RNA-directed DNA synthesis. These results were obtained with two different MuLV strains, AKR and Moloney, each grown in SC-1 cells. The demonstration that ActD virions contain primer tRNA and in general exhibit the characteristic MuLV tRNA pattern rather than the complete representation of cell 4S RNA leads to the conclusion that genomic RNA is not the major determinant in selective packaging of host tRNA's. A possible role for one or more viral proteins, including reverse transcriptase, is suggested.

Characterization by molecular hybridization of two viral populations derived from a radiation leukemia virus.

Two leukemogenic viral populations were derived from a radiation leukemia virus of the C57BL mouse. One (FB), in which only B-tropic virus could be detected, was obtained in vivo by serial passage of cell-free extract in newborn rats. The second (3C), a complex containing at least B-tropic and xenotropic viruses, was produced in vitro by a permanent cell line (13-3C) established from the spleen of a virus-infected C57BL mouse. In molecular hybridization experiments, the 70S RNA of Gross leukemia virus hybridized 96 and 78% of FB and 3C radioactive complementary DNA's, respectively, with a relatively high thermal stability of the duplexes formed. In contrast, the 70S RNA of Rauscher leukemia virus hybridized 23 and 20% of the FB and 3C DNA probes, respectively, with a low thermal stability. The rat-grown FB virions exhibited 50% genome homology with the viruses produced in vitro on the 13-3C cells. Finally, hybridizing the FB and 3C probes with normal or leukemic mouse spleen DNA's resulted in 89 to 100% homology. The rat-grown virions did not appear to contain detectable rat cellular DNA sequences, while about 20 complete copies of their nucleotide sequences were detected in covalent linkage with FB-infected rat spleen DNA. These findings strongly support the endogenous murine origin of the investigated virions.

Synthesis of murine leukemia virus proteins associated with virions assembled in actinomycin D-treated cells: evidence for persistence of viral messenger RNA.

Murine leukemia virus particles assembled in actinomycin D-treated cells were detected by determination of reverse transcriptase [RNA-dependent DNA polymerase (nucleotidyltransferase)] activity and by radioimmunoassay of the major virion protein, p30. The levels of enzyme activity and p30 protein were both 30-40% relative to the control over an 8 hr period, whereas after 3 or 4 hr infectivity was reduced by 95%. Thus, virions produced in the absence of RNA synthesis represent a fairly homogeneous population of defective particles. Although RNA synthesis is not necessary for virus assembly, protein synthesis is required. Treatment of cells with 10 mug/ml of cycloheximide reduced virus production by 80-85% within 2 hr, and by greater than 95% at later times. As might be expected from this finding, viral protein synthesis accompanies virus assembly in actinomycin D-treated cells. Newly synthesized proteins associated with the defective particles were identical with those found in standard virions and were present in the correct proportions. The results demonstrate that viral mRNA persists in cells in which RNA synthesis is blocked and continues to direct viral protein synthesis with a functional half-life of approximately 6-8 hr. Since viral mRNA is not packaged in virions even when viral RNA synthesis is shut off [Levin et al. (1974) J. Virol. 14, 152-161], we propose that murine leukemia virus-infected cells contain two nonequilibrating pools of intracellular viral RNA molecules, one associated with polyribosomes and one which is encapsidated into extracellular particles.