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The effect of interferon on glucocorticoid-stimulated mouse mammary tumor virus expression in a murine mammary tumor cell line, Mm5mt/c 1 , was investigated. Interferon at a concentration of 10–100 IU/ml causes a 5- to 10-fold inhibition of virus production. The effect has the typical time course of interferon action, is dose dependent and reversible, and is not associated with depression of cellular synthesis of macromolecules. The mechanism appears complex. In contrast to the interferon-mediated inhibition of murine leukemia virus, cellular RNA-dependent DNA polymerase (RDDP) is significantly reduced. The ratio of cellular to extracellular RDDP is, however, increased, and expression of mouse mammary tumor virus antigens on the cell membrane is not inhibited. The evidence therefore suggests effects of interferon on translation or transcription of some viral macromolecules as well as on viral assembly and release.

When L y cells were treated with 100 units per milliliter of mouse interferon and then infected with vesicular stomatitis virus (VSV) at a multiplicity of 10–60 PFU/cell, a prolonged infection of cultures ensued, lasting in one case longer than 60 days. After four passages in L y cells at high multiplicities of VSV from a prolonged infection, there was no inhibition of virus growth, and virus from the prolonged infection had the same distribution of intracellular and extracellular RNA forms as was found in wild-type virus passed four times in L y cells. Also, the intracellular and extracellular RNA forms of virus taken directly from a prolonged infection of L y cells were indistinguishable from those of the wild-type virus. A morphological examination of VSV from a prolonged infection did not show a significant number of small defective viral forms after four passages in L y cells at a high multiplicity of infection. There was, however, evidence for the emergence of temperature-sensitive mutants of VSV during the course of the prolonged infection. Changing the incubation temperature of the cultures from 37° to 32° resulted in an increase in virus production and virus-induced cytopathology. The virus produced during prolonged infections grew to higher titers at 32° than at 37° and its plaque size at 39° progressively decreased with the length of time that the infection persisted. Furthermore, interferon production also seemed to have a role in the persistence of infections such as the treatment of cultures with rabbit anti-mouse interferon globulin and resulted in a significant rise in virus production and viral cytopathology in the cell cultures.

We have investigated the sensitivity of a mouse cell line, NIH 3T3 (clone 1), that was found to be resistant to the anticellular and certain antiviral activities of interferon, to 2′,5′-oligoadenylate (2′5′A). The 2′5′A was introduced into the cells by coprecipitation with calcium phosphate and by increasing cell permeability with lysolecithin. In both cases neither cellular protein nor DNA synthesis was inhibited by 2′5′A in the NIH 3T3 cells. In contrast, the synthesis of proteins and DNA in L cells, an IFN sensitive line, was inhibited by low concentrations of 2′5′A. Furthermore, treatment of the infected cell cultures with 2′5′A resulted in the inhibition of vesicular stomatitis virus (VSV) replication in L cells but not in NIH 3T3 cells. The production of MuLV in NIH 3T3 cells was also not affected by 2′5′A. These observations, together with our previous finding that NIH 3T3 (clone 1) cells are deficient in RNase F activity suggest that activation of the RNase F by 2′5′A is responsible for the inhibition of both protein and DNA synthesis in sensitive cells.

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 KBalb 3T3 cells ( Balb 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.

As much as 200-fold inhibition in the titer of infectious vesicular stomatitis virus (VSV) was achieved in cultures of Ly cells treated with 30 reference units/ml of interferon (IF); however, virus particle production, as measured by VSV particle-associated RNA, nucleocapsid (N) protein, or transcriptase, was inhibited a maximum of 10-fold by this concentration of interferon. A significant reduction was seen in the glycoprotein (G) and matrix protein (M) content of VSV from interferon-treated cells. These results indicated that IF-treated cells produce VSV particles with low infectivity. This may be related to the reduced amount of G protein incorporated into such particles. Only 42 S viral RNA was detected in the virus particles produced by interferon-treated cells; these particles did not interfere with the growth of wild-type VSV in BHK or Ly cells. The findings suggested that these particles are not defective-interfering forms of VSV. The results in many respects resemble those previously reported in IF-treated cells infected with murine leukemia viruses.

Interferon treatment of mouse JLS-V9R cells resulted in a 10- to 20-fold increase in the levels of the 2–5A (ppp(A2′p)nA)-dependent RNase. The nuclease was monitored in cell extracts by covalent and noncovalent binding of 32P-labeled 2–5A derivatives to the nuclease and by the appearance of 2–5A-mediated ribosomal RNA cleavage products. Untreated JLS-V9R cells contained very low levels of the nuclease (5–10% of that found in control Ehrlich ascites tumor cells). An increase in the nuclease was first detected between 3 and 6 hr after interferon treatment and was completely inhibited by actinomycin D. Control experiments indicated that the increase in the nuclease was not due to an intracellular redistribution of the enzyme. The results indicate that in JLS-V9R cells, interferon controls the activity of the 2–5A system at two levels: by inducing the synthesis of both the 2–5A-dependent RNase and the 2–5A-synthetase.

Murine leukemia virus (MuLV) ts3 is a temperature-sensitive mutant of Moloney MuLV which harbors a defect in a late stage of virus assembly. Cells chronically infected with ts3 accumulate budding virus particles on the plasma membrane when incubated at the nonpermissive temperature. Interferon has been shown to inhibit the production of oncornaviruses and a similar late stage of virus assembly has been suggested as the site of interferon action. In this study, the temporal relationship between the two inhibitions was examined. It was found that the process of virus assembly affected by the inhibitory action of interferon preceded that affected by the temperature-sensitive defect. Analysis of virus yields by multiple parameters indicated that interferon treatment at the nonpermissive temperature resulted in the release of noninfectious viral particles.