Mechanism of interferon action: inhibition of vesicular stomatitis virus replication in human amnion U cells by cloned human leukocyte interferon. I. Effect on early and late stages of the viral multiplication cycle.

The kinetics of induction in human amnion U cells of the antiviral activity against vesicular stomatitis virus (VSV) produced by a single molecularly cloned subspecies of human leukocyte interferon (IFN-alpha A) were examined. IFN-alpha A-induced inhibition was found to be biphasic over a period of 24 h with the major extent of VSV inhibition occurring within the first 6 h of IFN treatment. The relationship of this major phase of inhibition to the early and late events of the VSV multiplication cycle was investigated. IFN-alpha A treatment had no detectable effect on the adsorption and penetration of VSV virions or on their uncoating to yield viral nucleocapsids. The polypeptides of adsorbed or uncoated VSV particles were neither preferentially degraded nor detectably altered in IFN-treated cells, as compared to untreated cells. Progeny virions released from IFN-treated cells, although greatly reduced in number, were found to be equally as infectious as those released from untreated cells. Progeny virions from IFN-treated cells also had a normal complement of VSV proteins in the same ratios as were seen in virions from untreated cells; specifically, IFN treatment produced no reduction in the incorporation of G or M protein into assembled virions. These results suggest that conditions of IFN treatment sufficient to reduce the yield of infectious VSV progeny greater than 99% do not detectably affect either the early or the late stages of the VSV multiplication cycle.


I "
The effects of recombinant human y-interferon (IFNy) on vesicular stomatitis virus (VSV) macromolecular synthesis in human amnion U cells were examined. Saturating concentrations of IFN-y caused only a 3 to &fold reduction of viral protein synthesis in wild-type VSV-infected cells, an extent insufficient to account for the 100-fold inhibition of viral infectivity. By use of the VSV mutant tsG41, which is competent in RNA transcription but defective in RNA replication at 4OoC, it was shown that the apparent IFN-induced inhibition of viral protein synthesis was likely due to a reduction in the synthesis of primary transcripts in IFN-y-treated U cells. Dot blot hybridization analysis revealed that saturating concentrations of IFN-y reduced both primary (measured with mutant tsG4linfected U cells) and total (measured with wild-typeinfected U cells) viral RNA synthesis by about 4-fold, an extent of inhibition comparable to the observed reduction in viral protein synthesis. Analysis of RNA, fractionated by agarose gel electrophoresis after denaturation with glyoxal, with cDNA probes to individu d VSV mRNAs did not reveal any detectable difference in the structural integrity of VSV mRNA isolated from IFN-y treated as compared to untreated U cells.
These results suggest that IFN-y treatment causes a small reduction in the efficiency of transcript formation catalyzed by input parental virions. However, the results also indicate that the principal cause of the IFNy-induced inhibition of VSV replication in U cells is the alteration of a step in replication other than viral macromolecular synthesis. This implies that the moleeular mechanism of viral inhibition by IFN-y i s fundamentally different from that of IFN-a in human amnion U cells. Vesicular stomatitis virus (VSV') has been widely utilized as a challenge virus, both for the assay of interferons (IFN) and for the study of the molecular mechanisms of interferons * This work was supported in part by Research Grants AI-12520 and AI-20611 from the National Institutes of Health, United States Public Health Service. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore he hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. interferon; IFN-y, y-interferon (type II interferon, immune interferon); NaDodS04, sodium dodecyl sulfate; pfu, plaque-forming unit; eIF, eukaryotic inkiation factor. antiviral actions (1, 2). The suitability of VSV for IFN research derives from its acute sensitivity to the antiviral effects of IFN, its broad host range, its relatively rapid multiplication cycle, and the extensive information available concerning the molecular biology of the virus (1,3). Numerous studies have contributed to our understanding of the molecular mechanisms by which type I (a and p) IFNs inhibit the replication of VSV (2). A recent systematic biochemical study using a mole~ularly cloned subspecies of human leukocyte IFN (IFN-aA) revealed that, in human amnion U cells, the major and possibly only effect of this type I IFN on VSV replication in U cells is the discriminatory inhibition of viral protein synthesis (4). The IFN-a-induced inhibition of VSV protein synthesis and viral infectivity in U cells appears to be due to the alteration of a component o f the translational machinery other than the viral mRNA template (4,5), and correlates with the induction of a "double-stranded RNA-dependent', protein kinase which catalyzes the phosphorylation of protein synthesis initiation factor eIF-2a (6). Phosphorylation of the a subunit of eIF-2 is believed to play an important role in the regulation of translation in eukaryotic systems (7), including type I IFN-treated animal celis (2,8).
In contrast to our understanding of the molecular mechanism by which type I IFNs inhibit the replication of VSV, very little is known concerning the molecular basis of the type I1 (y) IFN-induced in~ibition of VSV replicat~on. Recent studies using highly purified molecularly cloned IFN-aA and IFN-y revealed that the antiviral actions of these IFNs are synergistic in various human cells (6,9), including VSVinfected human amnion U cells (61, confirming earlier observations from studies with less pure natural type I and type I1 IFN preparations (IO). The synergistic antiviral actions of IFN-cuA and IFN-7 against VSV in U cells suggest that these human IFNs induce mechanistically distinct antiviral states in U cells. Therefore, using purified molecularly cloned human immune IFN (IFN-y), we have systematically studied the effect of IFN-y action in human amnion U cells on each stage of the VSV multiplication cycle in order to determine the significant point or points acted upon by the IFN-y-~nduced antiviral state. In the preceding paper (Il), we examined the dose dependence of the induction of antiviral activity against VSV by IFN-7, and showed that IFN-y treatment of U cells has no detectable effect on the adsorption, penetration, and uncoating of infecting VSV virions, nor does IFN-y treatment reduce the specific infectivity of progeny virions. In this paper, we report the effect of IFN-y treatment on VSV macromolecular synthesis in human amnion U cells.
~-I~~e r f e r o n ~n~~~i t i o n of Vesicular S t o~a t i~i s V i r~s 4325 EXPERIMENTAL PRO~EDURES'

Viral Protein Synthesis in Wild-type VSV-infected Cells-
The effect of IFN-7 on the synthesis of VSV polypeptides was examined under conditions identical to those used to study the dose response of the induction of antiviral activity by IFN-y against wild-type VSV (11). Human amnion U cell monolayers, which had been treated with varying concentrations of IFN-7 for 24 h, were infected with wild-type VSV at a multiplicity of 10 pfulcell. Cell monolayers were then labeled with [35S]methionine from 4 to 4.5 h post-infection and analyzed by N a D~S O~-p o l y a c~l a m i d e gel electrophoresis. In order to clearly resolve the viral polypeptides, especially L, G, and N, from protein bands of cellular origin, samples were quantitatively immunoprecipitated with anti-VSV antiserum before electrophoresis (4). As shown in Fig. 1 in the Miniprint, viral protein synthesis was reduced as a function of increasing concentration of IFN-y treatment. In order to quantitate the extent of reduction of viral protein synthesis, viral protein hands were excised from gels and counted for radioactivity. At IFN-y concentrations which were saturating for both the inhibition of viral protein synthesis and the inhibition of infectious virus yield, viral protein synthesis was reduced only 3-5-fold in IFN-y treated as compared to untreated U cells, whereas infectious virus yield was reduced nearly lOO-fold (Fig. 2). The relatively small extent of inhibition of viral protein synthesis caused by IFN-y treatment was uniform for the synthesis of the five VSV polypeptides; no single polypeptide differed significantly from the others in its sensitivity to IFN-y-induced inhibition of synthesis (Fig. 2).
We do not believe that the 3-&fold inhibition of total (primary plus secondary) viral protein synthesis is sufficient to account for the overall reduction of infectious virus yield ( Fig. 2; see "Discussion"). However, the inhibition of VSV protein synthesis would be expected to contribute to the overall inhibition of viral infectivity observed in IFN-ytreated cells. For this reason, we attempted to determine the molecular basis of the inhibition. Viral RNA S y~t~e s i s in W i~d -~y~ VSV-~nfec~ed Cells-In order to determine whether the reduction in viral protein synthesis observed in IFN-y-treated U cells infected with wild-type VSV was possibly due to a reduction in the amount of viral mRNA available for translation, the effect of IFN-y treatment on the amount and the structural integrity of viral mRNA was analyzed by dot blot and Northern gel hybridization procedures. Whole cytoplasmic RNA was isolated at 4 h post-infection from IFN-y-treated, wild-type VSV-infected cell cultures parallel to those in which protein synthesis and viral infectivity had been quantitated (Fig. 2). Because we wanted to accurately quantitate the amount of viral mRNA, RNA samples were not selected for polyadenylated fractions since such selection often gives variable yields; furthermore, we could not preclude the possibility that viral message polyadenylation could be affected by IFN-7. As can be seen from Fig. 3, as the concentration of IFN-y used to treat U Portions of this paper (including "Experimental Procedures" and Figs. I, 6, and 8) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 84M-3250, cite the authors, and include a check or money order for $2.40 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press. The highest IFN concentration examined, 300 ng/ml, caused about a 4-fold reduction in total (primary plus secondary) viral transcripts (Fig. 3, lanes e ) as compared to the amount of transcripts in untreated cells (Fig. 3, lanes b). For the four VSV mRNAs analyzed, N, NS, M, and G, the IFN-7 mediated reduction in the amount of the viral transcripts was uniform; no single mRNA differed significantly from the others in its sensitivity to IFN-7 (Fig. 3).
In order to examine the structural integrity of viral RNAs from IFN-y-treated U cells as compared to untreated cells, glyoxylated RNA samples were fractionated by agarose gel electrophoresis prior to being blotted onto nitrocellulose and probed with 32P-labeled VSV cDNA. Because dot blot hybridizations had shown that 300 ng/ml of IFN-y caused about a 4-fold reduction in the amount of each of the VSV mRNAs ( Fig. 3), 8-fold more cytoplasmic RNA isolated from IFN-ytreated cells (4.0 pg) as compared to untreated cells (0.5 pg) was fractionated electrophoretically in order to insure that the hybridization signals detected for the monocistronic mRNAs isolated from the IFN-7-treated cells would be at least as strong as the signals for the mRNAs isolated from untreated cells if their respective structural integrities were indeed comparable. As can be seen in Fig. 4, there were no significant differences in the size of either the viral N or M monocistronic message species from IFN-y treated as compared to untreated U cells. Similar results were obtained when hybridizations were carried out with the NS and G cDNA probes (data not shown). The N and M hybridization signals were slightly stronger for the RNA samples isolated from IFN-y-treated cells as compared to the diluted samples from untreated cells. From the dot blot concentration analysis (Fig.  3), a 2-fold greater amount of VSV-specific RNA from IFNy treated as compared to untreated cells had been fractionated on the agarose gels used for the Fig. 4 blots. The slightly stronger hybridization signals observed in the Northern gel analysis (Fig. 4) for IFN-y-treated samples as compared to untreated samples therefore further suggests that the amount of VSV-specific RNA is indeed reduced about 4-fold by IFN- y treatment, but that the size of the VSV mRNAs are comparable in IFN-y treated as compared to untreated cells.
Also evident in the autoradiograms shown in Fig. 4 are a number of hybridizing bands intermediate to the bands of message and genome length (42 S) material. These intermediate molecules are the polycistronic mRNAs that have previously been detected in vitro by heteroduplex analysis (12) and in vivo by Northern gel analysis (13). IFN-y treatment did not affect the size of the N or M hybridizing polycistronic VSV RNA species; the steady-state levels of the polycistronic VSV RNAs appeared to vary in a manner similar to the monocistronic VSV RNAs, suggesting that their biogenesis was not selectively altered by IFN-y treatment (Fig. 4). translation, or to a combination of these processes, since the overwhelming majority of VSV protein synthesis results from the translation of viral mRNA synthesized by progeny nucleocapsids (4, 14) and VSV genome replication has an absolute requirement for viral protein synthesis (15). In order to distinguish a possible direct effect by IFN-y on VSV transcription from a direct effect on the translation of VSV mRNA, it was necessary to examine only primary transcription and translation, i.e. viral mRNA synthesis and translation occurring in the absence of amplification by parental genome replication. Primary viral processes were studied by the use of mutant tsG41, a complementation group IV mutant which, at the nonpermissive temperature of 40 "C, carried out transcription but no detectable synthesis of genome length 42 S (+)-or (-)-strand species of viral RNA (4,5,16,17).

Viral Protein and RNA Synthesis in Mutant tsG41-infected
U cell monolayers, treated with varying concentrations of IFN-y for 24 h at 37 "C, were infected with mutant VSV tsG41 at 40 "C, and proteins were labeled with [35S]methionine from 3.5 to 4 h post-infection. Viral proteins were immunoprecipitated with anti-VSV antiserum and analyzed by NaDodS04-polyacrylamide gel electrophoresis (Fig. 5). Primary viral protein synthesis was seen to decrease as a function of concentration of IFN-7 treatment (Fig. 5) the same gel to provide relative concentration standards. By comparison of band intensities, the maximum reduction in synthesis of primary VSV polypeptides appeared to be about 4-fold by saturating concentrations of IFN-y. It can be seen in Fig. 6 in the Miniprint that without immune precipitation, primary viral protein synthesis was not apparent against the background of cellular protein synthesis. VSV tsG41 infection of U cells at the nonpermissive temperature did not produce any detectable shutoff of host protein synthesis, nor did IFNy treatment detectably inhibit overall cellular protein synthesis. The only significant effect of IFN-y on cellular protein synthesis in U cells was the IFN-y concentration-dependent increase in synthesis of a cellular protein (Fig. 6 ) of apparent M, 54,000 as estimated by electrophoresis in denaturing gels with known molecular weight standard^.^ The striking increase in synthesis of the 54,000 protein by IFN-y was comparable in uninfected U cells and in U cells infected with either mutant VSV tsG41 or mutant reovirus tsC447 (data not shown).
The effect of IFN-y treatment on the amount and size of primary viral RNA transcripts was examined by dot blot (Fig.  7) and Northern gel blot (Fig. 8) Fig. 7, IFN-y treatment caused a small reduction in the steady state amount of primary VSV transcripts. The maximum reduction observed with a saturating concentration of IFN-y, 300 or 3000 ng/ml, was about 4-fold in the case of each of the four VSV-specific RNAs analyzed, N, NS, M, and G (Fig. 7).

tsG41. As shown by the dot blots in
To examine the structural integrity of the primary viral RNA transcripts from IFN-y-treated cells, RNA samples were fractionated by agarose gel electrophoresis, blotted onto nitrocellulose, and probed with the 32P-labeled N, NS, M, or G cDNAs. As can be seen from Fig. 8 in the Miniprint, there were no significant differences in the sizes of the primary message species as a function of concentration of IFN-7 treatment. The hybridizations of the Northern gel blots shown in Fig. 8 did suggest that the amount of the VSV monocistronic sized mRNAs decreased with increasing concentration of IFN-y, as predicted by the dot blot hybridization results which showed a 4-fold reduction in virus-specific RNA sequences (Fig. 7).

DISCUSSION
We have examined in detail the effect of molecularly cloned human IFN-y on vesicular stomatitis virus macromolecular synthesis in human amnion U cells. Treatment of U cells with IFN-y caused a decrease in total (primary plus secondary) viral protein and RNA syntheses in U cells infected with wildtype VSV. At each concentration of IFN-y which was examined, including low subsaturating concentrations and high saturating concentrations, the synthesis of each of the five VSV polypeptides was inhibited to the same extent; at saturating IFN-y concentrations, the inhibition was about 4-fold. There was not, however, a detectable change in the mobility of any of the individual viral proteins, including the glycoprotein G, synthesized in IFN-y treated as compared to untreated U cells. This suggests that IFN-y treatment does not cause an impairment of post-translational protein modification processes such as protein glycosylation which have been described in other systems (18), because unglycosylated G has a decreased mobility as compared to glycosylated G in Na-DodS04-polyacrylamide gels (19). Like the viral polypeptides synthesized in IFN-y-treated U cells, each of the VSV mRNAs were also reduced by the same relative amount, about 4-fold, by treatment with saturating concentrations of IFN-y. Our finding that there was no detectable alteration in the size or structural integrity of either primary or total VSV RNA as a function of IFN-y treatment suggests that viral RNA degradation by the 2'3'-oligoadenylate synthetase-nuclease system (20) probably does not play an important role in the antiviral mechanism of IFN--y in U cells.
The fact that primary and total (primary plus secondary) viral macromolecular synthesis are all reduced by a comparable albeit small amount (about 4-fold) by saturating concentrations of IFN-y suggests that the principal cause of this inhibition resides at a step or steps prior to or including primary transcription. The IFN--y sensitive step that affects the efficiency of formation of primary viral transcripts presumably is after virion attachment and penetration, because direct analysis did not reveal a reduction in either of these processes (11). Although the yield of uncoated nucleocapsids derived from input parental virions likewise was not detectably reduced, it is conceivable that subtle differences do exist in either the intracellular environment or the structural form of uncoated nucleocapsids produced in IFN-y-treated U cells (11) as compared to either untreated U cells (4, 11) or IFN-aA-treated U cells (4). Such putative differences might reduce the efficiency of primary transcription in IFN-y-treated cells by a mechanism that does not result in an alteration of the structural integrity of the primary transcripts.
Consistent with the interpretation that IFN-y does not directly inhibit the translation of VSV mRNA in U cells is the observation that 1FN"y also does not induce the protein PJeIF-2a protein kinase in U cells (6). By contrast, IFN-aA induces an antiviral activity in U cells that inhibits the translation of VSV mRNA by alteration of a component of the translational machinery other than the amount or the integrity of the viral mRNA template (4). IFN-aA, unlike IFN-7, also induces the protein PJeIF-a kinase in U cells in a manner that correlates with the antiviral activity of IFN-aA against VSV (6). These observations therefore appear to provide a molecular explanation for the synergistic actions observed between IFN-y and IFN-aA in the inhibition of VSV replication in IFN-treated U cells (6).
Natural human 1FN"y is a glycoprotein (21); the IFN-y that we have used in our studies was highly purified recombinant IFN-y synthesized in E. coli and therefore is not glycosylated. Although the carbohydrate modification of IFN-y may affect some of its biologic properties, particularly in intact animals, presently available information suggests that the degree of IFN-y glycosylation does not affect either the antiviral activity or the target cell specificity in cell culture systems (22). The 4-fold reduction in the amount of primary VSV RNA transcripts observed in U cells treated with saturating concentrations of IFN-y does not appear to cause a cascade effect on viral macromolecular synthesis sufficient to account for the nearly 100-fold reduction in infectious virus progeny. Primary VSV protein synthesis, and the subsequent secondary synthesis of VSV transcripts and polypeptides which represents greater than 95% of the viral macromolecular synthesis that occurs in infected cells (4,14) and which is absolutely dependent upon primary macromolecular synthesis ( E ) , are all inhibited to a comparable extent by IFN-y treatment, about 4-fold. In contrast to IFN-y, IFN-aA does not cause a detectable reduction in the amount of primary RNA synthesis in VSV-infected U cells (4). Based on previous studies with IFN-aA and U cells in which the reduction in infectious virus yield was correlated with the inhibition of VSV protein synthesis (4), we do not believe that the extent of overall viral protein synthesis inhibition observed in IFNy-treated U cells is sufficient to account for the overall reduction of infectious virus yield mediated by IFN-y treatment. Investigators have attempted to use, without much success in the case of VSV, the protein synthesis inhibitor cycloheximide to examine the relationship between viral macromolecular synthesis and infectious virus yield (23,24). Surprisingly, even though VSV protein synthesis and infectious virus yield are reduced by cycloheximide, treatment with cycloheximide appears to cause an enhancement both of primary VSV mRNA synthesis (23) and of the overall rate of protein synthesis in VSV-infected cells (24) by mechanisms that are not understood. These responses thereby complicate interpretations relating to the level of viral protein synthesis and infectious progeny because of unknown effects of the drug on other functions that may affect the efficiency of the replication process.
Although both IFN-y and IFN-a do possess antiviral activity, the primary function of IFN--y may be related to its properties as an immunoregulatory agent (25)(26)(27)(28). Conceivably the molecular basis of the immunoregulatory activity of IFN-y resides, in part, at the level of alteration of the cellular plasma membrane or cytoplasmic structure by the cellular gene products induced by IFN-y. Both the early and late steps in the replication of VSV (29), as well as perhaps steps associated with viral macromolecular synthesis and maturation (30)(31)(32), are dependent upon the nature of the cell ultrastructure. Thus, the inhibition of VSV replication observed as the result of IFN-y treatment may possibly be principally due to the alteration of cellular membrane or cytoskeletal structures normally associated with immunoregulatory activities of IFN-y rather than with an IFN-y antiviral activity per se. Although the 4-fold reduction in viral macromolecular synthesis undoubtedly has some impact on the yield of infectious progeny, the major cause of the reduction in infectious VSV yield by IFN-y may relate to alterations in the cellular cytoplasmic or membrane structures and organization that affect the efficiency of virion morphogenesis.