Inhibition of cleavage of Moloney murine leukemia virus gag and env coded precursor polyproteins by cerulenin

Cerulenin, an inhibitor of de novo fatty acid (and cholesterol) biosynthesis, has been shown to significantly decrease (greater than 75%) the amount of Moloney murine leukemia virus (MMuLV) released into the culture medium of chronically infected mouse fibroblasts (I. Katoh, Y. Yoshinaka, and R.B. Luftig, 1986, Virus Res., in press). In order to clarify the mechanism by which this decrease in virus production occurs, we analyzed the kinetics of gag and env coded protein synthesis in M-MuLV infected, cerulenin-treated cells by immunoprecipitation with monospecific antisera to p30, p12, p10, gp70, and p15(E). We found that in pulse (15 min-2 hr)-chase (0-4 hr) experiments the cleavage of not only Pr65gag to p30 and other gag coded proteins but Pr80env to gp70 and Pr15(E) as well, was greatly reduced by cerulenin treatment. Further, since the total amount of label in the Pr65gag and Pr80env bands remained about the same or was slightly decreased in 2-hr pulsed, cerulenin-treated cells, this suggests that cerulenin decreases virus production, in part, by inhibiting the cleavage of both precursor gag and env coded polyproteins during virus assembly and budding at the cell membrane. We also observed that at longer chase periods (4 hr), the effect of cerulenin could be partially overriden in that minor amounts of cleaved gag and env coded polyproteins were produced and assembled into virion particles. However, these particles contained abnormally large amounts of the uncleaved precursor Pr65gag, suggesting that maturation was incomplete. The above results suggest two independent, but not exclusive, possible mechanisms of cerulenin action to block M-MuLV production, viz. cerulenin decreases the pool of fatty acids, thereby inhibiting fatty acid acylation of Pr65gag, as well as Pr80env, and thus preventing the interaction between gag (the p15 antigenic determinant on Pr65gag) and env [the p15(E) antigenic determinant of Pr15(E)] coded gene products at the cell membrane needed for efficient virus assembly (M. Satake and R. B. Luftig, 1983, Virology 124, 259-273), and cerulenin inhibits one or more proteolytic enzymes responsible for the cleavage of Pr65gag and Pr80env.     

Fusion activity of virions of murine leukemia virus.

The fusion activity associated with many strains of murine leukemia virus (MLV) is commonly used to measure MLV infectivity. Other strains of MLV lack fusion activity and some of these can produce variants which have fusion activity. The SC-1/uv-XC fusion assay (Rowe et al., 1970) measures the infectivity of MLV grown in SC-1 cells by the XC cell fusion activity of the progeny virus. The fusion activity of parental MLV in XC cells can be measured directly within a few hours after infection. No new macromolecular synthesis (early after infection) is required for MLV-induced fusion. Treatment of MLV virions with proteolytic enzymes destroys their ability to fuse XC cells. The fusion activity of MLV, like the infectivity of MLV, is specifically inhibited by anti-MLV gp70 serum but not by antiserum against other MLV polypeptides. The infectivity of MLV is much more susceptible to uv irradiation than viral fusion activity and noninfectious (uv irradiated) MLV can cause XC cell fusion. When MLV virions are disrupted with NP-40 detergent, the fusion activity can be restored after high-speed centrifugation and dialysis of the detergent. The fusion activity of NP-40 disrupted MLV (after high-speed centrifugation and dialysis) is inhibited by antiserum against MLV gp70. Thus, the fusion activity of MLV is associated with the virion envelope glycoprotein.     

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.     

Interferon inhibits induction of endogenous murine leukemia virus production.

In 5-iododeoxyuridine (IUDR) induction of murine leukemia virus (MLV) in Balb cells transformed by SV40 (Balb SV40) or AKR cells, interferon inhibited virus production. This could have been due to inhibition of the X- and N-tropic virus induced or to inhibition of subsequent reinfection of the mouse cells by the N tropic virus. Cycloheximide, however, activates $\text{KBalb}\text{3T3}$ cells ($\text{Balb}\text{3T3}$ cells transformed by Kirsten sarcoma virus) to produce only an X-tropic virus which could not reinfect mouse cells. In this system, interferon treatment also decreases virus yields and, therefore, an event in the virus induction process.     

Methylcellulose media for plaque assay of murine leukemia virus.

When ecotropic murine leukemia virus was assayed by a methylcellulose-XC cell procedure, plaque titers showed less test-to-test variation, more uniform dose-response curves, and larger plaque sizes, as compared with results of the conventional liquid overlay-XC cell test system. This assay therefore seems to be reliable and useful for the titration of ecotropic murine leukemia virus.     

Effect of interferon on exogenous murine leukemia virus infection.

When interferon (IF)-treated NIH/3T3 cells were exogenously infected with the Moloney strain of murine leukemia virus (M-MLV), no viral progeny release was detected as long as IF remained in the medium. This was evidently an inhibition of the virus replication and not a consequence of interfering with the establishment of the infection since, when IF was removed either before infection or later after infection, virus release gradually approached the rate of untreated control after a temporary delay. Furthermore, a direct examination revealed that IF treatment had no effect on the formation of infectious centers. IF treatment led to a reduced viral RNA synthesis. A similar inhibition of viral RNA synthesis was observed when the potent protein synthesis inhibitor cycloheximide (CH) was added early after infection. However, when added at a late stage, the drug had no effect on viral RNA synthesis. It appears, therefore, that IF-induced inhibition of viral RNA synthesis is not a feedback consequence of an inhibition of a later step but, rather, an inhibition of some early step. This conclusion was substantiated by the finding that when IF was eliminated from pretreated cultures up to 10 hr before infection, progeny release was still affected.     

A short-term quantitative XC assay for murine leukemia virus.

A syncytial-plaque (SXC) assay for murine leukemia virus is described which utilizes a single cycle of infection, thus allowing quantitation of virus-producing mouse cells in 3 to 4 days. Depending upon the Fv-1 genotype of infected mouse cells, the assay will reflect single or multiple “hitness” after infection by N- or B-tropic MuLV. The test is equally sensitive to the uv-XC plaque assay for tissue culture adapted MuLV, but is less sensitive to in vivo derived MuLV from an AKR/J mouse. Newly isolated AKR MuLV requires a longer latent period than the in vitro adapted Moloney strain of MuLV.     

Efficient release of murine xenotropic oncornavirus after murine leukemia virus infection of mouse cells.

A simple and a most efficient method of eliciting murine xenotropic type oncornavirus from virus-free mouse cells is an infection of such cells with murine ecotropic oncornavirus. Xenotropic virus appeared after a single passage of Moloney leukemia virus not only from 3T3 type cells of inbred mice but also from 3T3 cells of outbred Swiss mice and from wild mouse SC-1 cells which were not inducible by halogenated pyrimidines. Thus unexpected contamination of mouse oncornaviruses with related viruses of a broader host range is possible.     

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.     

An improved murine leukemia virus immunofluorescence assay

The use of DEAE-dextran and polybrene during infection of mouse embryo fibroblast cell lines of Fv-1^bb origin with the radiation leukemia virus strikingly increased the proportion of cells in which virus-induced cytoplasmic antigens were detectable by indirect immunofluorescence and significantly accelerated the time course of their detection, thus making possible the development of a rapid, sensitive, and practical new assay based upon the percentage of immunofluorescence-positive cells. Direct comparisons with several other in vitro assays are presented. The assay was also tested successfully with Gross leukemia virus grown on cells of Fv-1ⁿⁿ origin, and should be applicable to other murine leukemia viruses and, perhaps with minor modifications, to other mammalian leukemia viruses as well.     

The GIX antigen of murine leukemia virus: an analysis with monoclonal antibodies

The G_IX antigen of murine retrovirus gp70 is a marker for virus replication in various cell types, as well as for T-cell differentiation and leukemogenesis in particular mouse strains. The serological definition of G_IX depends on the cytotoxicity of a particular rat antiserum for thymocytes from strain 129 mice. We have found that two rat monoclonal antibodies, elicited by immunization with AKR ecotropic virus or AKR leukemia cells, co-type closely with G_IX in several assay systems. Like G_IX, the epitopes identified by the monoclonal antibodies are amplified on thymocytes during leukemogenesis in AKR mice. These antibodies are cytotoxic for G_IX⁺ leukemia cells and for fibroblasts infected with G_IX⁺ viral serotypes. Though absorbed by G_IX⁺ thymocytes of various strains, the antibodies are not cytotoxic for these cells. We ascribe the latter result to lower representation of antigenic sites on normal thymocytes, and postulate that more than one epitope may participate in classical G_IX-mediated cytotoxicity. The monoclonal antibodies do not react with viral env products synthesized in the presence of the inhibitor of glycosylation, tunicamycin. Reactivity with the antibodies appears to require a stable configurational change in the env precursor protein coinciding with glycosylation, rather than direct participation of carbohydrate in the antigenic site. Thus subsequent enzymatic removal of carbohydrate chains in vitro does not alter reactivity with the antibodies.     

Isolation of temperature-sensitive mutants of murine leukemia virus

A microtiter technique was developed which allowed screening of large numbers of clones of murine leukemia virus for the presence of temperature-sensitive mutants. Potential mutants were detected by their inability to induce XC syncytium formation at the nonpermissive temperature. A total of 9 mutant viruses which have a markedly reduced infectivity at the nonpermissive temperature have been isolated. The spontaneous rate of reversion of each mutant was found to be very low.     

Amino-terminal amino acid sequence and carboxyl-terminal analysis of Rauscher murine leukemia virus glycoproteins

The two glycoproteins of Rauscher murine leukemia virus, gp70 and gp45, were found to have the following identical NH₂-terminal amino acid sequences: Ala-Ala-Pro-Gly-Ser-Pro-His-Gln-Val-Tyr-X-Ile-Thr-X-Glu-Val-(X - unidentified). The COOH-terminal amino acid of gp70 is tyrosine and that of gp45 is leucine. Computer-analyzed amino acid compositional data indicated that the protein moiety of gp45 is smaller than that of gp70. These results are compatible with the suggestion that gp45 is derived from gp70 by proteolytic cleavage.     

Characterization of temperature-sensitive mutants of murine leukemia virus

The isolation of temperature-sensitive (ts) mutants of the Rauscher strain of murine leukemia virus (MuLV) is described. These and a number of previously isolated ts mutants of the Kirsten strain of MuLV have been partially characterized and have been separated into three distinct classes based on the stage at which viral replication is blocked at the nonpermissive temperature.     

T and B lymphocyte susceptibility to murine leukemia virus moloney.

The susceptibility of T and B lymphocytes to productive infection and transformation by murine leukemia virus Moloney was determined by enumeration of cells producing infectious virus after in vitro infection of mitogen-stimulated, isolated cell populations and by in vivo infection of euthymic BALB/c and thymus-deficient (nude) mice. Our in vitro results demonstrated that the majority of splenic T cells and thymocytes are resistant to productive infection in vitro; a specific subpopulation of susceptible nylon-adherent splenic T cells was identified, however. Similarly, surface immunoglobulin-positive B cells also represent susceptible targets in vitro; mature B cells, however, did not represent the principal target for transformation in the in vivo experiments. Infected euthymic mice expressed increasing titers of murine leukemia virus and uniformly developed fatal T-cell lymphomas at 10 to 12 weeks postinfection; nude mice, in contrast, maintained high, stable levels of viremia throughout the 28 weeks of observation. Infected nude mice remained free of malignancy or developed either granulocytic leukemias or, in one case, reticulum cell sarcoma. Collectively, the results indicate that while the majority of T cells are resistant to productive infection, they represent the principle targets for transformation; B cells, however, represent permissive targets for virus replication, but are resistant to transformation.     

PG-4 cell plaque assay for xenotropic murine leukemia virus.

Xenotropic murine leukemia virus (X-MuLV) is often used in retrovirus elimination studies involving rodent cells. Currently, X-MuLV is measured using a focus-forming assay on mink (MiCl1 S+L-) or cat (PG-4 S+L-) cell lines. An easier and quicker PG-4 cell plaque assay, which retains the statistical reproducibility of the focus-forming assay, was developed and evaluated in this study. The PG-4 plaque assay is more sensitive than the MiCl1 focus assay for titering X-MuLV. The best results were achieved by passaging PG-4 cells at a seeding density of 4x10(6) cells/T185 flask twice a week, inoculating 3x10(5) cells/well on six-well plates and performing the assay at 35 degrees C. The overall variability of the assay was 0.30 log10 titer unit. A linear response to dilution was observed for wells containing 5-70 plaques. The limit of quantitation was 10 PFU/ml. Using six wells per dilution, the 95% confidence limit of the grand mean titer was within +/-0.5 log10.     

A plaque assay for murine leukemia virus using enzyme-coupled antibodies.

Cells infected with murine leukemia virus can be selectively stained with peroxidase-coupled antibodies. When stained 3–5 days after infection, monolayer cultures exhibit plaques which are easily seen at a low magnification. The assay can be used for titration of xenotropic as well as ecotropic murine leukemia viruses.