Replication of Vesicular Stomatitis Virus Facilitated by Shope

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1 INFECTION AND IMMUNITY, July 1979, p /79/ /07 $02.00/0 Vol. 25, No. 1 Replication of Vesicular Stomatitis Virus Facilitated by Shope Fibroma Virus In Vivo NORMAN A. CROUCHt* AND RICHARD L. MITCHELLf Department of Microbiology, College of Medicine, The University of Iowa, Iowa City, Iowa Received for publication 18 April 1979 Previous studies show that Shope fibroma virus facilitates replication of vesicular stomatitis virus (VSV) in some rabbit cells grown in vitro. In the present investigation, the possibility that these two viruses can also interact in vivo was determined. Rabbits inoculated intradermally with both viruses together, or each separately, were examined for the formation of lesions or tumors and for the production of infectious virus. The presence of VSV interfered with tumorigenesis by Shope fibroma virus. In tumors already formed, production of infectious VSV was greater than in normal skin. Hence, each virus affected the other. Sera and tissues of normal rabbits were found to contain a substance which inhibits VSV; this may act to limit replication of VSV in rabbit skin. In addition, cultured rabbit skin cells appeared to adsorb VSV inefficiently. When persistently infected by Shope fibroma virus, however, adsorption of VSV was markedly improved. Our results suggest that in vivo Shope fibroma virus may facilitate adsorption of VSV to reduce the effect of a natural inhibitor and consequently enhance production of infectious virus. Replication of vesicular stomatitis virus (VSV) is facilitated by Shope fibroma virus (SFV) in cultured domestic rabbit kidney (DRK3) cells (3, 7). Despite the broad host range of VSV (10), in the absence of SFV these particular cells are completely nonpermissive when challenged with VSV at a low multiplicity of infection, e.g., 20 infectious particles per cell, apparently because of a defect in the ability of VSV to be adsorbed effectively to the plasma membrane (1, 2). When these cells are challenged at a much higher multiplicity, e.g., 1,000 infectious particles per cell, VSV can enter but the infection is still abortive and no VSV is produced (1). Upon infection with SFV the cells become modified, and these restrictions of VSV adsorption and intracellular replication are eliminated. Even at low input multiplicities, VSV is then able to replicate in a normal manner (1). This facilitation of VSV in certain restrictive cells seems to be a characteristic common to poxviruses. Vaccinia virus, a poxvirus related to SFV, also makes DRK3 cells permissive for VSV (Crouch, unpublished data). Furthermore, vaccinia virus facilitates VSV in cultured rabbit cornea (RC-60) cells (9). Hence this phenomenon is not limited to SFV in DRK3 cells. t Present address: Department of Biomedical Sciences, Rockford School of Medicine, University of Illinois College of Medicine, Rockford, IL t Present address: Department of Microbiology, University of Minnesota Medical School, Minneapolis, MN In the present study, to determine whether poxviruses and VSV can also interact in vivo, rabbits inoculated with SFV and VSV were examined for the development of tumors and the production of infectious virus. MATERIALS AND METHODS Viruses. The Patuxent strain of SFV and the Indiana strain of VSV were grown as previously described (1). Cell cultures. Vero cells were grown in Eagle minimal essential medium with Earle salts supplemented with 5% fetal-bovine serum, 0.23% sodium bicarbonate, 100 U of penicillin per ml, and 100 t&g of streptomycin per ml. Rabbit skin cells were isolated from male New Zealand white rabbits. After chemical depilation with Nair (Carter/Wallace, Inc.) and then disinfection with 70% ethanol, sections of skin approximately 4 by 4 cm were excised from the backs of animals. Dermal tissue was then removed from these fragments with scalpels and placed into a mixture of trypsin-ethylenediaminetetraacetate at 370C. The cells were grown in the same medium used for Vero cells, but with 10% serum. All cells, including Vero, were incubated at 370C in a humidified atmosphere of 5% CO2 in air. Induction of tumors. Weanling male New Zealand white rabbits were injected intradermally along their backs with 0.1-ml suspensions containing 105 infectious SFV particles diluted in tris(hydroxymethyl)aminomethane-buffered saline (TBS; ph 7.4). Each animal was first clipped to remove excess hair and then injected at two sites approximately 6 cm to the left and two sites approximately 6 cm to the right of the 213

2 214 CROUCH AND MITCHELL midline. The two sites on each side were separated by 8 to 10 cm. Tumor size was quantitated at daily intervals by measuring two diameters at right angles to each other. Assay of tissues for infectious virus. To quantitate infectious VSV present in rabbit skin and SFVinduced fibromas, sites inoculated with VSV were excised, homogenized, and then assayed for plaque formation in confluent monolayers of Vero cells. Before excision of the tissues the animals were sacrificed, and hair was removed from external surfaces by chemical treatment. This involved generously coating the area with the cosmetic preparation Nair and allowing it to react for 10 to 15 min at room temperature. After thorough rinsing with water, the skin or tumor sites were bathed in 70% ethanol and allowed to air dry. Circular fragments approximately 3 cm in diameter were removed from each site, placed into 2 ml of TBS, weighed, and then stored at -550C until assayed. All fragments had comparable weights. To assay, each fragment in 2 ml of TBS was minced with scissors and then transferred with an additional 2 ml of TBS to a 5-ml chamber attached to a microhomogenizer (Sorvall). Homogenization was at full speed for 3 min with the chamber suspended in an ice-water bath. Homogenates were clarified by centrifugation at 800 x g for 10 min at room temperature. The supernatant fluids obtained were diluted serially in cold TBS, and 0.1-ml amounts were then inoculated onto Vero monolayers in 35-mm plastic petri dishes. After viral adsorption for 2 h at 370C, 2 ml of minimal essential medium containing 1% methylcellulose (Matheson, Coleman and Bell), 5% heat-inactivated fetal bovine serum, and the usual concentrations of penicillin, streptomycin, and sodium bicarbonate was added to each monolayer. After 24 h at 370C, the monolayers were fixed, stained, and examined for VSV plaques as previously described (1) Ȧssay for VSV inhibitor activity. Sera and supernatant fluids from homogenates of skin and tumors, prepared as described above, were diluted serially in TBS containing 10% heat-inactivated fetal bovine serum. Before dilution, 0.5-ml amounts of these materials were placed into 35-mm plastic petri dishes and exposed to ultraviolet (UV) radiation for 5 min at an intensity of 6 x 103 ergs/s per cm2 for the purpose of inactivating infectious VSV present in the skin and tumor preparations. In addition, all of the materials were also heat inactivated at 560C for 30 min. To each dilution, a constant amount of VSV was added (200 to 300 infectious particles). After incubation in a water bath for 60 min at 37 C, duplicate monolayers of Vero cells were inoculated with 0.1 ml of each dilution to assay for VSV plaques. Assay for infectious centers. The number of rabbit skin cells productively infected by VSV was determined by the method previously described (1). Immunofluorescence. Viral antigen in VSV-infected cells was assayed by indirect immunofluorescence as described previously (1). RESULTS Inhibition of tumorigenesis by VSV. To determine whether VSV affects the ability of SFV to induce fibromas, rabbits were injected intradermally with 0.1 ml of a mixture containing 105 infectious units of SFV and 108 infectious units of VSV. Controls included animals similarly injected with 0.1 ml of each virus separately. At daily intervals the sites of inoculation were examined for tumors or lesions. When VSV was present, tumorigenesis by SFV was completely blocked (Fig. 1). Whereas in controls inoculated with SFV alone tumors rapidly developed over a period of 10 days and then regressed, a characteristic response (8), no tumors were detectable at any time after injection of both viruses simultaneously. The concentration of each virus used in this experiment was found to be optimal for the observed effect. At lower doses of SFV, tumor formation was delayed and variable; at lower doses of VSV, the blockage of tumorigenesis was incomplete. In the animals injected with both viruses at doses in which tumorigenesis was blocked, the only gross evidence of infection was an area of erythema about 2 cm in diameter at each site of inoculation. Similar sites of erythema also developed in the skin of rabbits injected with 108 infectious units of VSV alone. This effect was observable by 24 h and disappeared after several days. To test whether gene functions of VSV were INFECT. IMMUN DAYS AFTER SFV INFECTION FIG. 1. Inhibition of SFV-induced tumor formation by VSV. Separate rabbits were injected intradermally with SFV (-), SFVplus VSV (O), or SFVplus UV-inactivated VSV (A). Other rabbits were injected with SFV alone followed 3 days later by injection of VSV directly into the tumor (0). Each point is the mean size of 12 tumors or sites on 6 different animals.

3 VOL. 25, 1979 necessary for inhibition of SFV tumorigenesis, or induction of localized erythema, VSV was exposed to UV radiation. Suspensions containing 2 x 108 VSV plaque-forming units/0.1 ml were irradiated to abolish all infectivity. The irradiated virus was then diluted with an equal volume of either TBS or a suspension containing 2 x 105 SFV focus-forming units/0.1 ml and injected intradermally as in the previous experiment. This treatment with UV light prevented VSV from blocking the induction of tumors by SFV (Fig. 1). Although the tumors which formed in the presence of UV-irradiated VSV were somewhat smaller, they attained their maximum size and began to regress at the same time as those induced by SFV alone. Irradiation of VSV also abolished its ability to cause erythema; inoculated skin remained completely normal in appearance. Since infectious VSV interfered markedly with tumorigenesis when injected simultaneously with SFV, it was of interest to see whether it could also affect tumors that were already growing. Hence, 0.1 ml of a suspension containing 108 VSV plaque-forming units was injected directly into the centers of tumors which had been induced by intradermal injection of 105 SFV focus-forming units 3 days before. In addition, 0.1-ml amounts containing 109 VSV plaqueforming units were injected intravenously into animals bearing similarly induced tumors. When VSV was injected into the tumors, they remained small and began to regress 4 days earlier than those induced by SFV alone (Fig. 1). Although direct inoculation affected tumor development, when the VSV was inoculated intravenously it had no effect on tumorigenesis (data not shown). It also appeared to have no effect on the health of the inoculated animals over a period of 3 weeks. Enhanced yields of infectious VSV in fibromas. The transient appearance of limited erythema that we observed in the skin of rabbits injected intradermally with VSV alone suggested that replication of VSV in this tissue may be restricted. To examine this possibility, and also determine whether SFV affects replication of VSV in vivo, skin and SFV-induced tumors injected with VSV were homogenized at intervals and assayed for infectious VSV. Results obtained over a period of 3 days after injection of VSV are presented in Fig. 2. In normal skin, production of infectious VSV did appear to be restricted; after an initial decrease in the amount of detectable virus, the mean concentration remained essentially constant. In contrast, there was a progressive increase in the amount of infectious VSV produced in tumor tissue coin- I0 9 -j > 0 -J 7 E,-I GROWTH OF VSV IN FIBROMAS 215 I DAYS AFTER VSV INFECTION FIG. 2. Replication of VSV in rabbit skin and SFV-induced fibromas. VSV was injected intradermally into normal rabbits (A) or into fibromas (0) induced in separate rabbits by intradermal injection of SFV 3 days before. Skin sites or tumors were excised at intervals and homogenized. Homogenates were assayed for infectious VSV by plaque formation in Vero cells. fected with SFV. At 3 days the difference in yields of VSV between skin and tumor was greater than 100-fold. By 4 days after injection of VSV, and later, there was a dramatic decrease in detectable VSV, both in the skin and tumors, presumably from the action of specific neutralizing antibody. Inhibition of VSV by rabbit serum and tissue extracts. The limited amount of infectious VSV found in rabbit skin, compared to fibromas, suggested that in normal skin an inhibitor of VSV may be present, or induced, which is either ineffective or not induced in the tumor tissue. An alternative possibility, that the cells in normal rabbit skin are nonpermissive for VSV, seemed unlikely for two reasons: (i) VSV characteristically has a broad host range, and (ii) erythema plus the amounts of virus present between days 1 and 3 (Fig. 2) indicate that some replication of VSV probably can occur. We therefore examined homogenates of VSV-injected skin and tumors for the presence of such an inhibitor. In addition, we examined homogenates of skin not treated with either virus, as well as serum from noninfected animals. To

4 216 CROUCH AND MITCHELL inactivate virus present in the homogenates of VSV-inoculated tissues, the preparations were exposed to excess UV radiation before being assayed for inhibitor activity. Results of the assays are shown in Fig. 3. An inhibitor of VSV plaque formation in vitro was found in both tumors (Fig. 3A through C) and skin (Fig. 3D through F) injected with VSV. It was not, however, induced by VSV for it was also present in skin not treated with virus (Fig. 3G) and in normal serum (Fig. 3H). Furthermore, in the VSV-inoculated tissues the inhibitor was present already at 6 h after injection of VSV. By 7 days after inoculation with VSV, the inhibitor activity was increased in both tumors (Fig. 3C) and skin (Fig. 3F). This was also observed at 4 days (data not shown), but with considerable variation among different animals. Presumably this increase in inhibition resulted from the production of specific anti-vsv neutralizing antibodies. An A ei Li.. * D 0 E 0100 *6 IJJ-r a. ~50 Cl)o B INFECT. IMMUN. inhibition curve of VSV by serum from an animal hyperimmunized with VSV, shown in Fig. 31, had a similar shape. Since the early inhibitor of VSV was not induced, and since it was present at apparently comparable concentrations in both normal skin and fibromas, it was of interest to determine whether SFV interfered with its inhibitory effect. If so, this might account for the differences we observed in the amounts of infectious VSV present in skin and SFV-containing tumors. For simplification, the effect of SFV was examined in vitro. Cells isolated from rabbit skin were grown as primary cultures. These were then persistently infected with SFV in an attempt to mimic the virus-cell interaction found within fibromas. Infectious VSV was mixed with various dilutions of normal rabbit serum containing inhibitor activity, as described in the previous experiment. Samples of each mixture were then OF,,IIJIII1I1 1J1 I, J I a -I. I,,, DILUTION (LOG2-1) FIG. 3. Detection of an inhibitor of VSV plaque formation. Tissues were homogenized, UV irradiated, heated at 560C for 30 min, and diluted. Each diluted preparation was mixed with a constant amount of infectious VSV, incubated at 370C for 60 min, and then assayed for VSV plaque formation in Vero cells. Controls included VSVplus diluent. Homogenates and preparations assayed were: fibromas-6 h (A), 3 days (B), and 7 days (C) after VSV injection; skin-6 h (D), 3 days (E), and 7 days (F) after VSV injection; skin not injected with either virus (G); normal rabbit serum (H); and rabbit anti- VSV serum (I).

5 VOL. 25, 1979 assayed for the formation of VSV plaques in confluent cultures of SFV-infected skin cells. The presence of SFV did not prevent normal rabbit serum from inhibiting replication of VSV (Fig. 4). At a dilution of 1:2, the serum reduced the number of VSV plaques by greater than 90%, a reduction comparable to that seen in the control cultures not treated with SFV. However, the presence of SFV markedly increased the efficiency of plaque formation. At dilutions of serum containing no inhibitory effect, a maximum number of about 290 plaques formed in the SFVinfected cultures, whereas only about 180 plaques were detected in the controls. Comparable results were obtained in several experiments. Enhancement of VSV adsorption by SFV. The increased efficiency of plaque formation by VSV in SFV-infected skin cells suggested that SFV may affect adsorption of VSV. To test this further, an infectious-center assay was used. Primary cultures of rabbit skin cells, either persistently infected with SFV or noninfected, were challenged with increasing multiplicities of VSV. After allowing 2 h for the VSV to be adsorbed to the plasma membrane, the cells were washed, treated with rabbit anti-vsv immune serum to neutralize unadsorbed virus, washed again, and A DILUTION OF SERUM (LOG2-1) FIG. 4. Inhibition of VSVplaque formation in cultured rabbit skin cells by normal rabbit serum. Heatinactivated rabbit serum was diluted, and each dilution was mixed with a constant amount ofinfectious VSV. After 60 min at 37"C, each mixture was assayed for VSV plaque formation in both monolayers of rabbit skin cells (A) and monolayers of skin cells persistently infected with SFV for 48 h (0). GROWTH OF VSV IN FIBROMAS 217 then seeded onto monolayers of permissive Vero cells. The numbers of infectious centers, i.e., permissive skin cells which had adsorbed input VSV, are presented in Fig. 5. Clearly, the presence of SFV increased the number of cells initially infected by VSV. At a multiplicity of about 20 infectious VSV particles per cell, we observed in these cultures a maximum number of infectious centers; i.e., essentially all of the permissive skin cells were infected. In contrast, at the same multiplicity, 95% fewer infectious centers were found in the controls not infected with SFV. These results were supported by examination of similar cultures by indirect immunofluorescence for the numbers of cells containing VSV-specific antigens (Table 1). At a multiplicity of 20 about 95% more of the cells in SFV-infected cultures contained detectable VSV antigens being synthesized 4 h after viral inoculation. Most of the SFV-infected cells were probably initially infected by the input VSV, for at 8 h essentially all contained viral antigen. In contrast, at 8 h only 35% of the control cells were antigen positive, and these were in clusters, indicating focal spread of the virus. Apparently few cells in these control cultures were infected by input VSV. All of these cells were permissive, however, for by 12 h essentially all contained antigen. (nw z U.- U ' MULTIPLICITY OF VSV FIG. 5. Production of infectious centers by cultured rabbit skin cells challenged with VSV. Monolayers persistently infected with SFV for 48 h (0) or noninfected (A) were inoculated with increasing multiplicities of VSV. After adsorption of VSV at 37rC for 2 h, the cells were trypsinized, washed, diluted in anti-vsv serum, and then seeded onto Vero cells under an overlay of methylcellulose. Productively infected cells produced discrete plaques.

6 218 CROUCH AND MITCHELL TABLE 1. Effect of SFV on the proportion of cultured rabbit skin (RS) cells infected by input VSV Cells Multi- % of VSV antigen-posiplicity of tive' cells at: VSV 4h 8h 12h RS b >95 RS (SFV)C >95 >95 a Assayed by indirect immunofluorescence. b Positive cells were clustered. e Infected with 10 SFV infectious units per cell 48 h before VSV challenge. DISCUSSION In rabbits injected intradermally with both VSV and SFV, we found that each virus can influence the infectious process of the other. The presence of VSV can inhibit growth of tumors induced by SFV, and, in tumors already formed, production of infectious VSV is greater than in comparable normal tissue. This demonstrates that in dual viral infections such as this, modifications can occur which may have important consequences for the host. The mechanism by which VSV interferes with the growth of SFV-induced tumors remains unclear. Since the effect was sensitive to UV irradiation, presumably VSV replication, or at least some transcription of the viral genome, is required. This suggests that VSV-induced cell killing may be involved. Such an effect of VSV on cells can be abolished by UV treatment and is known to require viral transcription (6). Hence, inhibition of tumor growth may result from direct killing of SFV-infected or normal cells at the site of inoculation. As we observed, this should either prevent tumors from developing, i.e., when both viruses are injected simultaneously, or reduce growth and accelerate regression, i.e., when VSV is injected after tumors have begun to form. Although replication of SFV in these tissues was not assayed, in previous studies we found that contemporaneous replication of VSV in cultured cells had no inhibitory effect on replication of SFV (1, 2). It is unlikely, therefore, that inhibition of tumor growth by VSV resulted from direct interference with SFV replication. The greater yields of infectious VSV found in fibromas, compared to those in normal skin, may be the result of two interrelated effects: (i) facilitation of VSV-cell interaction by SFV and (ii) escape of VSV from an inhibitor substance present in rabbit serum and tissue. Possible facilitation of VSV adsorption to SFV-infected tumor cells is based on our in vitro experiments with cultured fibroblasts isolated from rabbit skin. INFECT. IMMUN. These cells were permissive for intracellular replication of VSV in the absence of SFV, for all became infected at an appropriately high multiplicity or after a prolonged period of time. However, adsorption of VSV appeared to be inefficient. Upon infection with SFV, this adsorption was facilitated. Although the relevance of these in vitro experiments to events in vivo must be considered with caution, it is conceivable that in SFV-induced fibromas input VSV attaches to and enters cells more readily than in normal skin. If this is true, more cells may be initially infected by VSV and less of the input virus may be affected by the inhibitory substance we detected in the tissues. Since extracts prepared from VSV-inoculated rabbit skin inhibited plaque formation by VSV in Vero cultures, the possibility that the low yields of infectious VSV observed in this tissue resulted from interference by defective-interfering particles or interferon was considered. The large amounts of VSV injected may have constituted a high multiplicity of infection, which is known to generate defective-interfering particles in vitro (5). It has been suggested that such particles act to restrict viral replication in vivo (4). However, we found the inhibitory effect of these extracts to be unaltered by excessive UV irradiation. Since inhibition caused by VSV defective-interfering particles is sensitive to UV light (5), this indicated that defective-interfering particles were not involved. Interferon also appeared to be not involved. When monolayers of Vero cells were treated for 18 h with the inhibitory extracts from VSV-inoculated skin and then washed thoroughly, VSV formed plaques as well as in nontreated controls (Crouch, unpublished data). If interferon was present, this treatment should have made these cells resistant to VSV. These results suggest that the inhibition factor present in VSV-inoculated skin is probably the same as that present in normal rabbit serum and skin, as well as in SFV-induced fibromas. The inhibitory factor demonstrated in rabbit serum, skin, and SFV-induced fibromas has not yet been characterized. However, in addition to being resistant to UV irradiation and stable at 560C for 30 min, we have found that this inhibitory activity can be removed from rabbit serum by addition of excess UV-inactivated VSV (Crouch, unpublished data). This does not occur with UV-inactivated SFV. These results suggest that the factor is a stable substance widespread in nature which binds to VSV particles. Perhaps it competes with receptors for VSV on the plasma membrane of rabbit skin cells. If so, this may exaggerate the inefficient adsorption of

7 VOL. 25, 1979 VSV suggested by our in vitro experiments. Facilitation of VSV by SFV in vivo, therefore, may involve alteration of the plasma membrane in SFV-infected tumor cells to allow VSV to successfully compete for receptor sites. In this respect, facilitation of VSV in vivo may be related to the facilitation of VSV by SFV in DRK3 cells shown in our previous work. ACKNOWLEDGMENT This work was supported by U.S. Public Health Service General Research Support Grant 5 S01 RR5313 awarded to the University of Iowa from the National Institutes of Health. LITERATURE CITED 1. Chen, C.-Y., and N. A. Crouch Shope fibroma virus-induced facilitation of vesicular stomatitis virus adsorption and replication in nonpermissive cells. Virology 85: Crouch, N. A Replication of vesicular stomatitis virus facilitated in nonpermissive cells by early functions of Shope fibroma virus. Proc. Soc. Exp. Biol. Med. 157: GROWTH OF VSV IN FIBROMAS Crouch, N. A., and H. C. Hinze Modifications of cultured rabbit cells by UV-inactivated noncytocidal Shope fibroma virus. Proc. Soc. Exp. Biol. Med. 155: Huang, A. S., and D. Baltimore Defective viral particles and viral disease processes. Nature (London) 226: Huang, A. S., and R. R. Wagner Defective T particles of vesicular stomatitis virus. II. Biologic role in homologous interference. Virology 30: Marcus, P. I., and M. J. Sekellick Cell killing by viruses. II. Cell killing by vesicular stomatitis virus: a requirement for virion-derived transcription. Virology 63: Padgett, B. L., and D. L. Walker Effect of persistent fibroma virus infection on susceptibility of cells to other viruses. J. Virol. 5: Shope, R. E A transmissible tumor-like condition in rabbits. J. Exp. Med. 56: Thacore, H. R., and J. S. Youngner Abortive infection of a rabbit cornea cell line by vesicular stomatitis virus: conversion to productive infection by superinfection with vaccinia virus. J. Virol. 16: Wagner, R. R Reproduction of rhabdoviruses, p In H. Fraenkel-Conrat and R. R. Wagner (ed.), Comprehensive virology, vol. 4. Plenum Press, New York. Downloaded from on October 30, 2018 by guest