Binding kinetics of ecotropic (Moloney) murine leukemia retrovirus with NIH 3T3 cells.

A quantitative analysis of the binding kinetics of intact Moloney murine leukemia retrovirus (MoMuLV) particles with NIH 3T3 cells was performed with an immunofluorescence flow cytometry assay. The virus-cell binding equilibrium dissociation constant (KD), expressed in terms of virus particle concentration, was measured to be 8.5 (+/- 6.4) x 10(-12) M at 4 degrees C and was three- to sixfold lower at temperatures above 15 degrees C. The KD of virus binding is about 1,000-fold lower than the KD of purified MoMuLV envelope. The association rate constant was determined to be 2.5 (+/- 0.9) x 10(9) M-1 min-1 at 4 degrees C and was 5- to 10-fold higher at temperatures above 15 degrees C. The apparent dissociation rate constant at 4 degrees C was 1.1 (+/- 0.4) x 10(-3) min-1 and was doubled for every 10 degrees C increase in temperature over the range tested (15 to 37 degrees C).     

Intracellular production of virus particles and viral components in NIH/3T3 cells chronically infected with Moloney murine leukemia virus: effect of interferon.

The effect of interferon on the biochemical properties and the maturation process of intracellular viral particles isolated from the cytoplasmic fraction of NIH/3T3 cells chronically infected with Moloney murine leukemia virus was investigated. By labeling these virions with either [35S]methionine or [3H]glucosamine, we demonstrated that they contain the same viral proteins and glycoproteins found in extracellular virions. Interferon treatment was found to reduce the rate of intracellular virus assembly. This effect was not a consequence of an interferon inhibition of viral RNA synthesis or its translation or a consequence of an interference with the posttranslational cleavage processing of viral precursor proteins, since all of these steps were not affected by interferon. However, the reduced rate of virus assembly could be attributed to the inhibition of viral protein glycosylation observed in interferon-treated cells. Nevertheless, despite this reduced rate, virus particles accumulated in interferon-treated cells. This accumulation was probably due to the strong inhibition of their final release from such cells.     

The requirements for viral entry differ from those for virally induced syncytium formation in NIH 3T3/DTras cells exposed to Moloney murine leukemia virus.

Moloney murine leukemia virus (Mo-MuLV) has the unique ability to infect different cells via either a low-pH-dependent or a pH-independent entry pathway. Only the pH-independent mechanism of Mo-MuLV entry has been associated with Mo-MuLV-induced syncytium formation. We have now identified a transformed cell line (NIH 3T3/DTras) which efficiently forms syncytia when exposed to Mo-MuLV, yet is low pH dependent for Mo-MuLV entry. Treatment of NIH 3T3/DTras cells with chloroquine, an agent which raises endosomal pH, blocks Mo-MuLV entry, but not Mo-MuLV-induced syncytium formation. This demonstrates that fusion which accompanies viral entry and fusion which is responsible for syncytium formation occur as independent processes in these cells. In addition, we determined that neither inherent differences in the Mo-MuLV receptor nor reduced affinity for Mo-MuLV gp70 can account for resistance of NIH 3T3 cells to Mo-MuLV-induced syncytium formation.     

Integration of human immunodeficiency virus types 1 and 2 DNA in vitro by cytoplasmic extracts of Moloney murine leukemia virus-infected mouse NIH 3T3 cells.

An essential step in the life cycle of the human immunodeficiency virus (HIV) is integration of a DNA copy of the viral RNA into the genome of the infected cell. We show here that this step can be faithfully accomplished in vitro by the enzymatic machinery of another retrovirus, Moloney murine leukemia virus (MoMLV). Mini-HIV substrates, which are linearized plasmids with long terminal repeat sequences at their ends, were incubated with cytoplasmic extracts of MoMLV-infected NIH 3T3 cells and target DNA. The MoMLV integration apparatus carried out integration of the mini-HIV substrates correctly; the terminal nucleotides of the viral substrate were removed, and a 4-base-pair duplication of the target DNA flanked the inserted viral DNA (C. Shoemaker, S. P. Goff, E. Gilboa, M. Paskind, S. W. Mitra, and D. Baltimore, Proc. Natl. Acad. Sci. USA 77:3932-3936, 1980). Our experiments show that the substrate sequence requirements for integration in vitro were limited to a few nucleotides, as the similarity between HIV and MoMLV long terminal repeat ends is minimal.     

A chimeric human T-cell lymphotropic virus type 1 with the envelope glycoprotein of Moloney murine leukemia virus is infectious for murine cells

We constructed a chimeric human T-cell lymphotropic virus type 1 (HTLV-1) provirus in which the original envelope precursor sequence was replaced by that of ecotropic Moloney murine leukemia virus (Mo-MuLV). Chimeric particles produced by transient transfection of this chimeric provirus were infectious for murine cells, such as NIH 3T3 fibroblasts, lymphoid EL4 cells, and primary CD4(+) T lymphocytes, whereas HTLV-1 particles were not. The infectivity of chimeric particles increased 10 times when the R peptide located at the carboxy terminus of the MuLV envelope glycoprotein was deleted. Primary murine CD4(+) T lymphocytes, infected by the Delta R chimeric virus, released particles that could spread the infection to other naive murine lymphoid cells. This chimeric virus, with the Mo-MuLV envelope glycoprotein and the replication characteristics of HTLV-1, should be useful in studying the pathogenesis of HTLV-1 in a mouse model.     

Selection of optimal polypurine tract region sequences during Moloney murine leukemia virus replication

Retrovirus plus-strand synthesis is primed by a cleavage remnant of the polypurine tract (PPT) region of viral RNA. In this study, we tested replication properties for Moloney murine leukemia viruses with targeted mutations in the PPT and in conserved sequences upstream, as well as for pools of mutants with randomized sequences in these regions. The importance of maintaining some purine residues within the PPT was indicated both by examining the evolution of random PPT pools and from the replication properties of targeted mutants. Although many different PPT sequences could support efficient replication and one mutant that contained two differences in the core PPT was found to replicate as well as the wild type, some sequences in the core PPT clearly conferred advantages over others. Contributions of sequences upstream of the core PPT were examined with deletion mutants. A conserved T-stretch within the upstream sequence was examined in detail and found to be unimportant to helper functions. Evolution of virus pools containing randomized T-stretch sequences demonstrated marked preference for the wild-type sequence in six of its eight positions. These findings demonstrate that maintenance of the T-rich element is more important to viral replication than is maintenance of the core PPT.     

Deletion mutants of Moloney murine leukemia virus which lack glycosylated gag protein are replication competent

A series of deletion mutations localized near the 5' end of the Moloney murine leukemia virus genome was generated by site-specific mutagenesis of cloned viral DNA. The mutants recovered from such deleted DNAs failed to synthesize the normal glycosylated gag protein gPr80gag. Two of the mutants made no detectable protein, and a third mutant, containing a 66-base pair deletion, synthesized an altered gag protein which was not glycosylated. All the mutants made normal amounts of the internal Pr65gag protein. The viruses were XC positive and replicated normally in NIH/3T3 cells as well as in lymphoid cell lines. These results indicate that the additional peptides of the glycosylated gag protein are encoded near the 5' end, that the glycosylated and internal gag proteins are synthesized independently, and that the glycosylated gag protein is not required during the normal replication cycle. In addition, the region deleted in these mutants apparently encodes no cis-acting function needed for replication. Thus, all essential sequences, including those for packaging viral RNA, must lie outside this area.     

Regulation of Moloney murine leukemia virus replication in chronically infected cells arrested at the G0/G1 phase.

The replication of Moloney murine leukemia virus (MMuLV) in chronically infected mouse cells arrested at the G0/G1 phase of the cell cycle by different procedures was investigated. MMuLV production was inhibited in glutamine- and isoleucine (Gln-Ile)-deprived G0/G1 cells. In contrast, butyric acid treatment, which efficiently arrested the cells at the G0/G1 phase of the cell cycle, did not inhibit MMuLV production. Furthermore, the inhibition of MMuLV production caused by either Gln-Ile deprivation or by interferon (IFN) treatment was overcome by butyric acid treatment. Thus, the replication of MMuLV could be dissociated from cell proliferation. The inhibition of MMuLV production in Gln-Ile-deprived cell cultures was compared to the inhibitory effect of IFN, which is known to affect budding and release of the virus. Rates of MMuLV protein synthesis were not affected in both the IFN-treated and Gln-Ile-deprived cells. However, processing of the viral polyprotein Pre65gag into p30 was blocked in the Gln-Ile-deprived cells. Furthermore, whereas in IFN-treated cells, MMuLV accumulated on the cell surface and could be released upon treatment with trypsin, in Gln-Ile-deprived cells, no virions were released by such treatment. These results indicate that in cells arrested by Gln-Ile deprivation, MMuLV is inhibited at a posttranslation step. This step appears to precede the anti-MMuLV block induced by IFN.     

Characterization of intracellular precursor polyproteins of Moloney murine leukemia virus.

Intracellular Moloney murine leukemia viral precursor polyproteins were compared with mature viral proteins by immunoprecipitation and tryptic peptide mapping experiments. The results were consistent with precursor roles for Pr65gag, Pr200gag-pol, Pr135pol, and gPr83env. The glycosylated gag gene product gPr85gag, although containing sequences characteristic of all four core proteins plus additional sequences not found in Pr65gag, lacked a major tyrosine-containing p30 tryptic peptide, suggesting that gPr85gag is not processed to p30.     

Genetic studies of the ploidy of Moloney murine leukemia virus.

An assay for Moloney murine leukemia virus was developed that made use of the production of morphologically altered foci in nonproducer mouse cells (15F) carrying murine sarcoma virus. Wild-type (wt) virus gave a ratio of titers at 39 degrees C/34degrees C = 1.05 +/- 0.45 (standard deviation;n = 20). A spontaneous, thermosensitive (ts) mutant of Moloney murine leukemia virus, ts3, defective in a late viral function, gave 39 degrees C/34degrees C = 0. A murine cell line (TB) was mixedly infected with ts3 and wt (multiplicities of infection, 7.8:4.3), cloned after infection, and shown to be infected by both viruses. At 34 degrees C it produced wt, ts, and particles of mixed parentage. The heterozygotes (hz) had ratios of assays 39 degrees C/34 degrees C = 0.06 to 0.84 (mean, 0.36). To eliminate possible interference by multiploid particles with determination of the proportions of the three types of particles, the virus produced by the mixedly infected, cloned cell line at 34 degrees C was distributed by velocity sedimentation in a sucrose gradient, and virus was picked from the lightest part of the gradient. The proportions of ts, wt, and hz were 0.27, 0.26, and 0.47. Those particles identified as hz segreated ts, wt, and hz in the proportions 0.24, 0.27, and 0.49, respectively. These values were not significantly different from those predicted from a diploid model of the genome.     

Homomeric interactions between transmembrane proteins of Moloney murine leukemia virus.

We have studied homomeric interactions between transmembrane proteins (TM) of the Moloney murine leukemia virus envelope using the Saccharomyces cerevisiae two-hybrid system. TM interacts strongly with itself but not with various control proteins. Deletional and mutational analyses indicated that the putative leucine zipper motif in the extracellular domain of TM is essential and sufficient to mediate the binding. The first three repeats of the leucine zipper-like motif are the most important in mediating the interaction. The TM-TM interaction detected in this system may play a role in several stages of viral replication.     

Maturation of dimeric viral RNA of Moloney murine leukemia virus.

We have analyzed the dimeric RNA present in Moloney murine leukemia virus (MoMuLV) particles. We found that the RNA in newly released virions is in a conformation different from that in mature virions, since it has a different electrophoretic mobility in nondenaturing agarose gels and dissociates into monomers at a lower temperature. On the basis of these results, we suggest that the RNA initially packaged into nascent virions is already dimeric but that the dimer undergoes a maturation process after the virus is released from the cell. In further experiments, we tested the possibility that this maturation event is linked to the maturation cleavage of the virion proteins, which is catalyzed by the viral protease (PR). We found that the dimeric RNA isolated from PR- mutant virions resembles that from immature virions: it has a lower electrophoretic mobility and a lower sedimentation rate, and it also dissociates at a lower temperature than does RNA from mature wild-type virions. When Kirsten sarcoma virus is rescued by a PR- mutant or by a somewhat leaky cysteine array mutant of MoMuLV, its RNA also exhibits a electrophoretic mobility lower than that in the wild-type pseudotype. These results suggest that the maturation of dimeric RNA in released virus particles requires the cleavage of the Gag precursor and the presence of an intact cysteine array in the released nucleocapsid protein.     

Subcellular distribution of newly synthesized virus-specific polypeptides in Moloney murine leukemia virus infected cells.

Immune precipitation analysis of pulse-labeled proteins present in subcellular fractions of mouse embryo cells infected with Moloney murine leukemia virus showed the presence of anti-gp70 serum-precipitable viral envelope gene products mainly in the microsomal fractions of these cells. In contrast, anti-p30 serum-specific gag (group specific antigen) gene products were found to be distributed in similar amounts in both the microsomal and postmicrosomal supernatant fractions of pulse-labeled cells.     

Methylation pattern of genomic RNA from Moloney murine leukemia virus.

32P- and methyl-3H-labeled 70S Moloney murine leukemia virus RNA was purified from virions produced in Moloney murine leukemia virus-infected mouse embryo cells. Primer-free RNA subunits obtained by heat treatment and zonal centrifugation were digested with RNase T2, and methylated oligonucleotides were chromatographed on DEAE-Sephadex in 7 M urea. Approximately one molecule of RNase T2-stable oligonucleotide (-5 charge) was isolated per subunit. Structural analysis indicated that the sequence of the oligonucleotide is m7GpppGmpCp. Analysis of the mononucleotide fraction isolated by DEAE-Sephadex chromatography of the RNase T2 digest identified 15 to 23 internal N6-methyladenylic acid molecules per subunit.     

Tissue-specific replication of Friend and Moloney murine leukemia viruses in infected mice.

We have studied the replication of ecotropic murine leukemia viruses (MuLV) in the spleens and thymuses of mice infected with the lymphocytic leukemia-inducing virus Moloney MuLV (M-MuLV), with the erythroleukemia-inducing virus Friend MuLV (F-MuLV), or with in vitro-constructed recombinants between these viruses in which the long terminal repeat (LTR) sequences have been exchanged. At 1 week after infection both the parents and the LTR recombinants replicated predominantly in the spleens with only low levels of replication in the thymus. At 2 weeks after infection, the patterns of replication in the spleens and thymuses were strongly influenced by the type of LTR. Viruses containing the M-MuLV LTR exhibited a remarkable elevation in thymus titers which frequently exceeded the spleen titers, whereas viruses containing the F-MuLV LTR replicated predominantly in the spleen. In older preleukemic mice (5 to 8 weeks of age) the structural genes of M-MuLV or F-MuLV predominantly influenced the patterns of replication. Viruses containing the structural genes of M-MuLV replicated efficiently in both the spleen and thymus, whereas viruses containing the structural genes of F-MuLV replicated predominantly in the spleen. In leukemic mice infected with the recombinant containing F-MuLV structural genes and the M-MuLV LTR, high levels of virus replication were observed in splenic tumors but not in thymic tumors. This phenotypic difference suggested that tumors of the spleen and thymus may have originated by the independent transformation of different cell types. Quantification of polytropic MulVs in late-preleukemic mice infected with each of the ecotropic MuLVs indicated that the level of polytropic MuLV replication closely paralleled the level of replication of the ecotropic MuLVs in all instances. These studies indicated that determinants of tissue tropism are contained in both the LTR and structural gene sequences of F-MuLV and M-MuLV and that high levels of ecotropic or polytropic MuLV replication, per se, are not sufficient for leukemia induction. Our results further suggested that leukemia induction requires a high level of virus replication in the target organ only transiently during an early preleukemic stage of disease.     

Coordinated disintegration reactions mediated by Moloney murine leukemia virus integrase.

The protein-DNA and protein-protein interactions important for function of the integrase (IN) protein of Moloney murine leukemia virus (M-MuLV) were investigated by using a coordinated-disintegration assay. A panel of M-MuLV IN mutants and substrate alterations highlighted distinctions between the intermolecular and intramolecular reactions of coordinated disintegration. Mispairing of the crossbone single-strand region and altered long terminal repeat (LTR) positioning affected the intermolecular, but not the intramolecular, reactions of coordinated disintegration. Partial components of the crossbone substrate were coordinated by M-MuLV IN, indicating a reliance on both LTR and target DNA determinants for substrate assembly. The intramolecular reaction was dependent on the presence of either the HHCC domain or a crossbone LTR 5' single-stranded tail. An M-MuLV IN mutant without the HHCC domain (Ndelta105) catalyzed reduced levels of double disintegration but not single disintegration. A separately purified HHCC domain protein (Cdelta232) stimulated double disintegration mediated by Ndelta105, suggesting a role of the N-terminal HHCC domain in stable IN-IN and IN-DNA interactions. Significantly, crossbone substrates lacking the LTR 5' tails were not recognized by the fingerless Ndelta105 protein. Collectively, these data suggest similar roles of the HHCC domain and 5' LTR tail in substrate recognition and modulation of IN activity.