J. gen. Virol. 0978), 4x, 315-323

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Printed in Great Britain

Mastomys natalensis Papilloma Virus (MnPV), the Causative Agent of Epithelial Proliferations: Characterization of the Virus Particle By H E R M A N N

M I ~ L L E R AYD L U T Z G I S S M A N N

Institut fiir Virologie, Justus-Liebig-Universitiit Giessen, Frankfurter Strasse IO7, 63oo Lahn-Giessen and Institut fiir Virologie, Zentrum fiir Hygiene, Hermann-Herder-Strasse I I, 78oo Freiburg, Federal Republic of Germany (Aecepted 3I May I978) SUMMARY

A virus (MnPV) with the structural characteristics of papilloma viruses was isolated from benign and malignant proliferations of adult animals of the inbred line ' G R A Giessen' of Mastomys natalensis. The particles can be banded in CsC1 gradients at densities of 1-34 g/ml (full particles) and 1.29 g/ml (empty particles). The virus D N A has a buoyant density of 1.7IO 4 g/ml and can exist in three different conformations (supercoiled circular, nicked circular and linear), the sedimentation values of which have been determined as 23 to 24S, 16 to I7S and 14 to I5S, respectively. Although the mol. wt. of MnPV D N A is similar to that of HPV I DNA, the size of the fragments obtained after cleavage of MnPV D N A with the restriction endonuclease Hae III is quite different from the pattern seen with human papilloma virus. The virion contains I2 different polypeptides; the major structural protein has a mol. wt. of 56ooo. MnPV is shown to be the causative agent of the skin proliferations, because tumours can be induced by inoculation of purified virus, whereas no cutaneous alterations are observed when the particles are inoculated in the presence of anti-MnPV serum. MnPV can be re-isolated from the experimentally induced tumours. INTRODUCTION

Warts or papillomas are frequently encountered in man and many animal species. Papilloma viruses, a subgroup of the Papovaviridae (Melnick et al. I974) have been shown to be the causative agents of these epithelial and sometimes fibro-epithelial proliferations (for review see zur Hausen, 1977). In this communication we describe the causative agent of epithelial proliferations occurring in about 3 % of adult animals of the inbred line ' G R A Giessen' of Mastomys natalensis (also called Praomys natalensis or 'multimammate mouse'). These epithelial proliferations can be classified as exophytic keratotic papillomas, as keratinizing squamous epithelial carcinomas and, in the majority of cases, as a type which resembles, macroscopically and histologically, keratoacanthoma in man (Burtscher et al. I973; Rudolph & Thiel, I976). Whereas the aetiology of spontaneous keratoacanthomas of man is unknown, the virus aetiology of keratoacanthomas of Mastomys natalensis could be verified by transmitting the tumours with cell-free extracts and by demonstrating virus-like particles in ultrathin sections

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of turnout cells (Rudolph & Miiller, I976). In this paper we present some physico-chemical and morphological characteristics of this Mastomys natalensis papilloma virus (MnPV) in greater detail. METHODS

Animals and transmission of tumours. Animals of the inbred line ' G R A Giessen' of Mastomys natalensis (Texdorf, 1967) with and without skin tumours were obtained from the Institute of Parasitology, University of Giessen. The animals' origin and age as well as the techniques of tumour transmission have been described previously (Rudolph & Miiller, I976). Purification of virus and isolation of virus DNA. Human papilloma virus (HPV) was purified from plantar warts as described by Gissmann & zur Hausen (1976). MnPV was purified from tumours according to Lancaster et al. (I976). The yield from a typical keratoakanthoma weighing 9"5 g was approx, too #g of virus protein. Electron micrographs of such a purified virus preparation did not show any major contaminants (Fig. I). For the extraction of virus D N A as well as the purification of the supercoiled form (co I) the procedure outlined by Tai et al. (1972) was followed. B K virus D N A was a gift from H. J. Rziha, Erlangen. Determination of sedimentation coefficients. To determine the s20,, value of MnPV, purified virions were sedimented through I M-NaC1 for 24 min at 15000 rev/min and 2o °C in an analytical ultracentrifuge (Beckman Model E, An-H-Ti rotor with a bandforming cell type II). To estimate the sedimentation coefficient of the virus D N A the preparations were centrifuged through a neutral CsC1 solution with a density of 1.5o g/ml for 6"5 h at I Ioooo g and 25 °C in a SW 56 rotor (Gissmann, 1977). The localization of the three components was determined by continuous measurements of the extinction (260 nm) during fractionation in a Gilford spectrophotometer. Determination of buoyant densities in CsCI. Virions were banded by i sopycnic centrifugation as described above. The gradient was fractionated and the density of individual fractions was determined by measuring the refractive index (Weigle et al. I959). The buoyant density of virus D N A was calculated by equilibrium density centrifugation in an analytical ultracentrifuge (see above) for 55 h at 44ooo rev/min and 25 °C. D N A from Micrococcus lyscdeicticus (I"731 g/ml) or from HPV 1 with a density of 1"7oo g/ml (Gissmann, I977) were used as density markers. Polyacrylamide gel electrophoresis. Purified MnPV was dissociated by boiling for 2 min in the presence of 2 M-urea, 1% SDS and o.2 % 2-mercaptoethanol and electrophoresed on cylindrical IO% polyacrylamide gels according to Maizel 0971) at lOO V for 4 h. After staining with Coomassie brilliant blue R 25o, the gels were scanned with a Gilford spectrophotometer at 6oo nm. Cleavage of MnP V DNA with the Hae III restriction endonuclease and estimation of the tool wt. of MnPV DNA. Purified virus D N A (co I) was treated with Hae III restriction endonuclease (purchased from Miles, Great Britain) as described (Gissmann & zur Hausen, 1976). Five hundred ng of the digested D N A were run on a 4 % polyacrylamide slab gel (5 % cross-linking) at IOO V for 4 h at IO °C, stained in a o'5 #g/ml solution of ethidium bromide and visualized under u.v. light. Photographs were takenwith a Polaroid Land camera. To estimate the tool. wt. of MnPV DNA, the migration distances of the segments obtained after cleavage with Hae III restriction endonuclease were compared with those of HPV I with known tool. wt. (Gissmann et aL I977) obtained after the same treatment and run on

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the same gel. The mol. wt. of individual segments were summed up to give the mol. wt. of the total DNA. Electron microscopy. Concentrated MnPV in STE buffer (IOO mM-NaC1, 5o mM-tris, o'5 mM-EDTA, p H 7"4) was applied to Formvar (Wacker, Miinchen, W. Germany) -coated copper grids. Excess virus was removed after 3o s and a drop of z % phosphotungstic acid, p H 7"z, was applied to the grids. This drop was removed immediately and the specimens were examined with a Siemens Elmiskop IOI operated at 80 kV.

Preparation of rabbit hyperimmune serum, complement fixation tests and neutralization of virus. Hyperimmune serum against purified MnPV was prepared in rabbits by subcutaneous inoculation of approx. I5O pg of a purified virus preparation incorporated into Freund's incomplete adjuvant. The same dose was given 3 weeks later and blood was drawn 2 weeks thereafter. For the removal of antibodies directed against Mastomys natalensis normal cellular components, serum was passed through a Sepharose 4 B / C L (Pharmacia, Sweden) column to which an extract from skins of tumour-free animals had been conjugated according to Porath et al. (I967) and Porath & Sundberg (1972). The complement fixing activity of the antiserum was determined by the microtitre method after heating it at 6o °C for 2o rain. For neutralization tests the serum was diluted I : 4 with PBS, mixed with an equal volume of MnPV containing suspension and kept at room temperature for 6o min. As a control the virus suspension was mixed with normal rabbit serum. The mixtures were stored at o °C until they were applied to the scarified skins of Mastomys natalensis.

RESULTS

Morphology of purified virus Virus particles could be purified from keratinizing cells of keratoacanthomas as well as papillomas and keratinizing squamous cell carcinomas of Mastomys natalensis ' G R A Giessen'. Virus yields were about the same when spontaneously occurring tumours or artificially induced tumours were used as virus source. Electron micrographs of a purified virus preparation (Fig. 1) negatively stained with phosphotungstic acid showed nonenveloped particles with an average diam. of 52 nm. The capsomeres appear to be hollow cylinders with a mean outer diam. of about 7 nm. This morphology corresponds well with main morphological characteristics of papilloma viruses of other species.

Buoyant density and sedimentation behaviour of purified virus When MnPV was centrifuged to equilibrium in CsC1 gradients a distinct main band formed at the density of 1-34 g/rot. Sometimes a faint band consisting of empty shells was visible at a density of I-29 g/ml. Tubular forms could never be observed in these fractions. In the analytical ultracentrifuge particles with a density of 1-34 g/ml sedimented as a homogeneous sharp band with a sedimentation coefficient (s2o,~) of 290 to 295S. Before the run neither broken nor aggregated particles could be seen in the electron microscope.

Buoyant density and sedimentation behaviour of virus DNA Isolated virus D N A was centrifuged to equilibrium in CsC1 in the analytical ultracentrifuge and compared with the density ofMicroeoccus lysodeicticus as shown in Fig. 2. The resulting value was I"7I o4 g/ml which corresponds to a guanine + cytosine content of5o % (Schildkraut et al. 1962 ). The sedimentation pattern of MnPV D N A is shown in Fig. 3. The s20,,, values of the three forms of D N A were determined as 23 to 248, 16 to I7S and 14 to I 5 8 , respectively. 2I

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Fig. I. Electron micrograph of purified Mastomys natalensis papilloma virus (MnPV) after negative staining with phosphotungstic acid. The arrow marks a particle with clearly visible pentons and hexons. The bar represents 6o nm. I

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Buoyant density (g/ml) Fig. 2. Determination of buoyant density of MnPV DNA. Virus D N A (I) and Microeoceus lysodeietieus D N A (II) were diluted in a neutral CsCl-solution (initial density 1.7i 3 g/ml) and then centrifuged as described in Methods. The buoyant density was calculated as described byIfft etal. (I960.

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Determination of the restriction enzyme cleavage pattern and estimation of the tool. wt. of MnPV DNA The restriction enzyme cleavage pattern of MnPV D N A after Hae III endonuclease treatment was completely different from the pattern of HPV [ D N A as documented in Fig. 4. Eighteen different fragments could be detected, the mol. wt. of which are listed in the legend to Fig. 5. The sum of these values corresponds to a total tool. wt. of 4"9 × [o6-

Polyaerylamide gel electrophoresis of MnPV polypeptides Polyacrylamide gel electrophoresis of the denatured virus proteins indicated the existence of at least I2 different components as shown in Fig. 6. The mol. wt. of these polypeptides are also shown in the figure. Component V was estimated to have mol. wt. of about 56ooo and represents about 75 % of the total protein.

Induction of tumour growth in Mastomys natalensis with purified MnPV and neutralization with hyperimmune serum As shown in Table I, in 36 % of the animals infected with purified MnPV (containing approx. 15o/~g/ml of virus protein), malignancies developed which were histologically indistinguishable from those induced by crude or cell-free tumour extracts. Again, virus particles with the same morphology as the virus described could be purified from these neoplasias. On the other hand, no tumour growth developed during an observation period of ~2 months if purified MnPV virions were incubated with hyperimmune serum prior to transmission. 2I-2

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Fig. 4. Separation of HPV 1 D N A (left slot) and MnPV D N A (right slot) after cleavage with Hae III endonuclease and electrophoresis on a 4 % polyacrylamide gel after staining with ethidium bromide. Conditions are described in Methods. The fragment indicated by arrow might be due to partial digestion.

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Migration distance (cm) Fig. 5. U s i n g the Hae III f r a g m e n t s of H P V I D N A (11) as m a r k e r s ( G i s s m a n n et al. I977), t h e m o l . wt. c f t h e Hae II1 M n P V f r a g m e n t s ((3) were d e t e r m i n e d as I ' 2 5 x lo 6, o 9 5 x 1o 6, o ' 4 o x Io 6, o . 2 5 x io 6, o . 2 2 x 1o 6, 0 . 2 0 × IO n, o ' I 9 x IO e, o . I 7 X lO 6, o ' I 7 × IO 6, o ' I 4 × IO 6, o-I4 x IO ~, o ' I o × IO 6, o . I o x Io 6, 0'095 x IO 6, 0"08 x 1o 6, 0'07 x io n, 0"065 x io e a n d 0'065 x 1o 6, respectively. DISCUSSION

The aetiological agent (MnPV) of the different types of skin turnouts which occur in the inbred line ' G R A Giessen' of Mastomys natalensis could be characterized as a typical representative of papilloma viruses. Its size, density and morphology is consistent with this class of viruses. Its DNA could be isolated in the usual three forms (supercoiled circular, nicked circular and linear) when examined in the electron microscope (data not shown here). One can note, however, an exceptionally high guanine + cytosine content of 50 % in MnPV DNA in comparison with 4I % in HPV I (Gissmann, I977), 43 % in papilloma virus of dogs, 45 % in bovine papilloma virus (Crawford & Crawford, I963) and 48 to 49"5 % in rabbit papilloma virus (Watson & Littlefield, I96o). The high guanine + cytosine content is consistent with the greater number of Hae III sites in comparison to HPV I (Fig. 4) since Hae I22 recognizes GGCC as its specific cleavage site (Yang et al. I976). The tool. wt. of 4"9 x io 6 for both HPV I and MnPV as estimated by summing up the tool. wt. of the individual bands is in good agreement with the molecular weight determined for HPV I DNA determined by electron microscopic measurements (Gissmann & zur Hausen, I976). There is no indication of any differences in virus structure when virus preparations which had been isolated from keratinizing squamous epithelial carcinomas, true papillomas or keratoacanthomas were examined. Isolates from all these sources had identical physico-chemical characteristics and were uniform in their antigenic structure, because antiviral serum prepared in rabbits suppressed any type of tumour.

H. M U L L E R A N D L. G I S S M A N N

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Distance migrated (ram) Fig. 6. Polypeptide pattern of MnPV. Gel electrophoresis was performed as described in Methods. The tool. wt. of the polypeptides were estimated as outlined by Shapiro et al. (1967) using bovine serum albumin, ovalbumin, chymotrypsinogen and horse-myoglobin as markers. T a b l e I. Neutralization o f skin tumour induction by antiviral serum* Number of animals infected

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* Mastomys natalensis ' G R A Giessen' were inoculated with purified MnPV (protein content about 15o #g/ml) pretreated with rabbit hyperimmune serum or with normal rabbit serum. The titre of complement fixing antibodies of the hyperimmune serum used in this experiment was I : lO24. The observation period was I 2 months. So far all attempts have failed to p r o p a g a t e M n P V in cell culture. Nevertheless, this system appears to be very suitable for studying the m o l e c u l a r b i o l o g y or the biological b e h a v i o u r o f an o n c o g e n i c agent in m o r e detail because this virus is available in sufficient a m o u n t s a n d induces a kind o f t u m o u r w h i c h is n o t f o r m e d by the o t h e r k n o w n p a p i l l o m a viruses.

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We thank H. Becht, R. Rott and H. zur Hausen for critical suggestions. This work was supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 47 and Ha 449/12). REFERENCES BURTSCHER,H., GR/.JNBERG,W. &MEINGASSNER,G. (1973). Infekti6se Keratoakanthome by Praomys (Mastomys) natalensis. Naturwissenschaften 6o, 2o9-2IO. CRAWFORD, L. V. & CRAWFORD,E. M. (1963). A comparative study of polyoma and papilloma viruses. Virology 2x, 258-263,

GlSSMANN,L. (1977)- Vergleichende Charakterisierung der Desoxyribonucleins~iuren menschlicher Papillomviren. Doctoral thesis, University of Erlangen-Niirnberg. GlSSMANN, L. & ZUR HAUSEN, H. (1976). Human papilloma viruses: physical mapping and genetic heterogeneity. Proceedings of the National Academy of Sciences of the United States of America 73, 131 o--I 313. GlSSMANN, L., PFISTER, H. & ZUR HAUSEN, H. (1977). Human papilloma viruses (HPV): characterization o f four different isolates. Virology 76, 569-58o. 1EFT, J. B., VOET,D. H. & VINOGRAD,J. (196I). The determination of density distributions and density gradients in binary solutions at equilibrium in the ultracentrifuge. Journal of Physical Chemistry 65, I 138-1145. LANCASTER, W. D., OLSON, C. & MEINKE, W. (1976). Quantitation of bovine papilloma viral D N A in viralinduced tumors. Journal of Virology 17, 824-831. MAIZEL, JUN. J. V. 0971). In Methods in Virology, vol. 5, PP. 179-246. Edited by K. Maramorosch and H. Koprowsky. London and New York: Academic Press. MELNICK, J. D., ALLISON~A. C.~ BUTEL, J. S.~ ECKHART, W., EDDY, B. E., KIT, S., LEVINE, A. J.~ MILES~J. A. R., PAGANO, J. S., SACHS,L. & VONKA,V. (I974). Papovaviridae. Intervirology 3, IO6-I2O. PORATH, J. & SUNDBERG, L. (1972). High capacity chemiesorbents for protein immobilization. Nature New Biology 238, 261-262. PORATH, J., AXEN,R. & ERNBACK, S. (I967). Chemical coupling of proteins to agarose. Nature, London 215, I491-I492. RUDOLPH, R. & M/.JLLER,H. (1976). Induktion von epidermalem Tumorwachstum in der Haut yon Mastomys natalensis dutch O bertragung virushaltigen Tumorgewebes eines Platten-Epithelkarzinoms. Zentralblatt far Veteriniirmedizin B23, 143-I5O. RUDOLPH, R. & THIEL, W. 0976). Pathologische Anatomic und Histologie yon spontanen, epithelialen Hauttumoren bei Mastomys natalensis. Zentralblatt far Veteriniirmedizin A23, 429-4.4I. SCHILDKRAUT, C. L., MARMUR,J. & DOTY, P. (I962). Determination of the base composition of desoxyribonucleic acids from its buoyant density in CsC1. Journal of Molecular Biology 4, 43o-443. SEEHAFER, J. A., SALMI, A., SCRABA,D. G. & COLTER, J. S. (1975). A comparative study of BK and polyoma viruses. Virology 66, I92-2o 5. SHAPIRO, g. L., VINUELA,E. & MAIZEL,JUN. J. V. (1967). Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochemical and Biophysical Research Communications 28, 815-82o. TA1, U. R., SMITH, C. g., SHARP,V. A. & VINOGRAD,S. (I972). Sequence heterogeneity in closed simian virus 40 desoxyribonudeic acid. Journal of Virology 9, 317-325. TEXDORF, 1. (1967). Mastomys natalensis (SMITH, 1843) und ihre Eignurtg als Endwirt ffir Trematoden im Vergleich zu anderen Laboratoriumstieren. Doctoral thesis, University of Giessen. WATSON, J.D. & LtTTLEFIELD, J. W. 0960). Some properties of D N A of Shope papilloma virus. Journal of

Molecular Biology % 161-I72. WEIGLE, J. M., MESELSON,M. & PAIGEN, K. (1959). Density alteration associated with transducing ability in the phage Lambda. Journal of Molecular Biology x, 379-386. YANG, R. C. A., VAN DE VOORDE, A. & FIERS, W. (1976). Cleavage map of the simian-virus-4o genome by the restriction endonuclease iII of Haemophilus aegyptius. European Journal of Biochemistry 6I, lOI- 118. ZUR HAUSEN,H. (1977) Human papilloma viruses and their possible role in squamous cell carcinomas. Current Topics in Microbiology and Immunology 78, I-3O.

(Received 2I December 1977)