Mechanism of Interferon Action: Inhibition of Vesicular Stomatitis Virus Replication in Human Amnion U Cells by Cloned Human y-Interferon I. EFFECT ON EARLY AND LATE STAGES OF THE VIRAL MULTIPLICATION CYCLE*

The molecular basis of the inhibition of vesicular stomatitis virus (VSV) replication by pure recombinant y-interferon (IFN-y) in human a-nion U cells was examined. A saturating concentration of IFN-y induced, at maximum, about a two log,, reduction in infectious VSV yield. The kinetics of induction of the antiviral activity by IFN-y were first order over the period of about 6-18 h, following a lag of about 3 h, after treatment with a saturating concentration of IFN-y. The relationship of the inhibition in VSV infectivity to the early and late events of the VSV multipli- cation cycle was investigated. IFN-y 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-y-treated U cells, as com- pared to untreated U cells. Progeny virions isolated from IFN-y-treated U cells, although greatly reduced in number, were found to be equally as infectious as those isolated from untreated U cells. Progeny virions from IFN-y-treated cells also poamsed the same composition of viral proteins as was observed for virions from untreated cells. These results suggest that conditions of IFN--y treatment sufficient to

(4, 5). It has recently become possible, by molecular cloning and expression, to obtain pure samples of single species of human IFN proteins absolutely free of any other eukaryotic proteins. Using IFN-(uA expressed in Escherichia coli, we have recently shown that this IFN-a subspecies inhibits VSV replication in human amnion U cells primarily, and probably solely, at the level of translation of viral mRNA into viral polypeptides (6,7). No significant effect of IFN-aA is observed in U cells on the adsorption, penetration, and uncoating of infecting VSV virions, nor is there an inhibition of primary transcription i n vivo or a reduction in the in vitro translational activity of viral mRNA synthesized i n vivo (6)(7)(8). Progeny VSV from IFN-aA-treated U cells, although greatly reduced in number, are equally as infectious as progeny VSV from untreated U cells (8).
Very little is known concerning the nature of the antiviral activities and biochemical changes induced by IFN-7 as compared to those responses induced by IFN-a subspecies. At least two different classes of high-affinity IFN receptors have recently been shown to exist on human lymphoblastoid and f i b r o b l~t cells, one to which IFN-a and IFN-/3 bind and another to which IFN-7 binds (9-11). Recombinant IFN-7, like IFN-aA, induces an antiviral activity against VSV in human amnion U cells (12). However, the antiviral activities of IFN-y and IFN-aA in U cells are synergistic for the inhibition of VSV, suggesting that the molecular mechanism of the antiviral action of IFN-y may be different from that of IFN-aA (12). Synergistic effects of highly purified recombinant IFN-a or IFN-/3 in antiprol~erative as well as antiviral systems have also recently been reported (13).
Using recombinant human IFN-7, we have undertaken a systematic biochemical study of the effect of IFN-7 treatment of human amnion U cells on each stage of the multiplication cycle of VSV. In this paper, we report the effect of IFN-y on the early and the late steps of VSV multiplication; in the following paper (14); the effect of IFN-7 on VSV macromolecular synthesis is examined.

DISCUSSION
We have examined in detail the molecular basis of the antiviral state induced against VSV in human amnion U cells ?-Interferon Inhibition of Vesicular Stomatitis Virus by molecularly cloned human IFN-7. In this paper, we have addressed the question of whether inhibition of VSV replication is manifested to any significant extent at the earliest or the latest steps of the virus multiplication cycle. The results obtained demonstrate that treatment of human U cells with IFN-7 has no detectable effect on the ability of VSV to initiate an infection or on the specific infectivity of the progeny virions produced even though the yield of progeny virions is greatly decreased by IFN-7 treatment.
The kinetics of induction of the antiviral state against VSV in U cells by IFN--y differ from those previously observed for IFN-aA. IFN--/ displayed a single kinetic phase following an initial lag. A maximum of about 2 log,, reduction of VSV yield developed in cells treated for 24 h with saturating concentrations of IFN-7, with the major extent of the reduction occurring after 6-18 h of IFN-./ treatment. By contrast, the antiviral state against VSV induced by IFN-aA in U cells is biphasic with the inflection between the first and second phases occurring after 6-8 h of treatment with saturating concentrations of IFN-aA (8). The first kinetic phase induced by IFN-aA in U cells accounts for the major extent of the total reduction of infectious VSV, typically about 3 log,, (8,15), and displays first-order kinetics at all IFN-aA concentrations examined (8). Our observation that, at saturating IFN concentrations, purified molecularly cloned IFN--y induces an antiviral state more slowly than molecularly cloned IFN-a against VSV in human U cells confirms the original observations reported for the antiviral action of natural IFN preparations in diploid human fibroblast cells measured with Sindbis virus (16).
Although it is generally accepted that IFN treatment has no effect on the adsorption, penetration, or uncoating of parental infecting virions, this notion is generalized from studies with type I ( a , p) IFNs and a very small number of viruses. Recent studies indicating that the replication of SV40 in lytically infected simian cells is inhibited by IFN at a step prior to early transcription (17,18), as well as reports describing effects of type I IFN on cell membrane structure and function (19,20), made it worthwhile to investigate the effect of IFN--/ on the early stages of VSV infection. Under conditions identical to those used to examine the kinetics and concentration dependence of the induction of the antiviral state in U cells by IFN-7, we found no effect by IFN--y on the amount of VSV bound to or internalized by U cells. Furthermore, equal amounts of input parental VSV virions were recovered as uncoated nucleocapsids from untreated cells as compared to IFN-7 treated cells. It has recently been reported that purified natural human a-IFN inhibits fluid-phase pinocytosis as measured by the uptake of horseradish peroxidase in human diploid fibroblasts as well as mouse and chicken cells treated with homologous type I IFNs (21). The lack of a detectable effect of IFN-7 in human U cells on the attachment, penetration, or uncoating of parental input VSV virions indicates that either IFN-7 does not affect pinocytosis in U cells under conditions where viral replication is inhibited, or that the specific aspects of the pinocytotic process affected by type I IFN treatment are not essential for the initiation of VSV infection in U cells.
The latest point in the VSV multiplication cycle at which IFN-y could conceivably act would be virion morphogenesis and release from cells. Our finding that there was no signifi-cant alteration of the specific infectivity or the polypeptide composition of progeny virions produced by IFN-y-treated U cells as compared to untreated U cells suggests that the virions which are produced by IFN-treated cells are normal. However, our method of analysis does not eliminate the possibility that a late step in morphogenesis is indeed affected by IFN-./ treatment, and that the IFN-y alteration results in a greatly reduced efficiency of the assembly and subsequent release by budding of progeny virions which are otherwise normal.
The results described herein that were obtained with IFNy-treated U cell are similar to those recently reported for IFN-aA-treated U cells (8). The single molecularly cloned subspecies of a-IFN, IFN-aA, also did not detectably affect either the early or the late steps of VSV replication in human amnion U cells. Although neither IFN--y nor IFN-aA affects either the early or the late steps of VSV replication, biologic and biochemical studies indicate that the molecular basis of their antiviral activity may be different (9)(10)(11)(12)(13). The fact that the antiviral actions of molecularly cloned human IFN-y and IFN-aA are synergistic with each other for the inhibition of VSV in U cells strongly suggests that the two types of IFNs inhibit VSV replication by different mechanisms in the human amnion cell line (12). Recent

E f f e c t o f Gamma IFN Treatment o n P e n e t r a t i o n o f [3SSjMethionine-labeled VSV into Human Amnion U Cell Monolayers manolayers Of U cells Nhich had been either untreated or treated VSV labeled with [3SS]rnethionine w a s used to infect 21 cm2
with IFN-y. Multiplicity of infection was 10 PFUlcell. After

are thought to pass, via clathrin-coated pits, to endocytic in the extrusion of viral nucleocapsids into the cytoplasm c e l l s a n d u n c a a t i n g w i t h i n c e l l s c o n s t i t u t e s t h e e a r l i e s t s t a g e
( 2 5 , 2 6 ) . (Table I).

Because virion adsorption was the standard conditions under which reduction Of viral yield had been measured (Figs. 1 and 2). t h e r a d i o a c t i v i t y t h a t c o u l d n o t b e w a s h e d a w a y f r o m c e l l m o n o l a y e r s f a l l o w i n g i n f e c t i o n o r d e r t o e s t i m a t e t h e e x t e n t o f v i r i o n p e n e t r a t i o n w a s h e d c e l l ( T a b l e 1) was a measure of virion binding and penetration.
In monolayers were treated with trypsin prior to meas;rement of the cell-associated radioactivity. As shown in Table  2 t h e a m o u n t of trypsin-insensitive cell-associated radioactivit; was not detectably different between untreated U cell monolayers and monolayers treated with IFN-r. After 2 h at 37 about 7.5% o f of both untreated and IFN-y treated cells.

It is conceivable that an 1FN-r, induced alteration in the e f f i c i e n c y b y w h i c h p a r e n t a l v i r i o n s a r e uncoated within U cells to yield nucleocapsids would not be revealed in the experiments polypeptides described above. Therefore, the yield and shown in Tables 1 and 2 or the analysis of parental virion s t r u c t u r e O f U n c o a t e d n~~l e o~d p s l d s d e r i v e d f r o m [3sS]~ethionine-labeled papentdl virions w a s examined. NUCleOCapsidS were separated from intact parental input [3SS]methionine-labeled virions by SUCTOIB gradient fractianauntreated and IFN-Y treated c e l l s ; t h e separated nucleocdpslds t i o n o f c y t o p l a s m i c extyacts prepared at 2 h post-infection from
were then collected by ultracentrifugation and analyzed by NaOodSOb-polyacrylamide gel electrophoresis. As shown in Fig. 3   As can be seen i n F i g .

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l a n e a ) . s u g g e s t i n g