Archives of Virology
Archives of Virology 57, 291--296 (1978)
© by Springer-Verlag 1.978
Actin Filaments in Paramyxovirus-Infected Human Fibroblasts Studied by Indirect Immunofluoreseence By A. FAGRAEUS~D. L. J. TYRRELL1, ~. NORBERG, and E. NoRI~BY Department of Immunology, National Bacteriological Laboratory, Stockholm and Department of Virology, Karolinska Instituter, Stockholm, Sweden With 2 Figures Accepted February 24, 1978
Summary Fibroblasts growing on glass have microfilaments arranged in bundles. These can be demonstrated b y indirect immunofluorcseent technique using human antiactin serum or experimentally produced rabbit anti-actin serum. When monolayer cultures of epithelial cells and fibroblasts are infected with paramyxovirus, such as measles, mumps, Sendal and NI)V, there is a striking decrease of the bundles. Rabies and adenoviruses do not seem to influence the staining of microfilaments. The microfilament decreasing effect in the cells correlates to the finding by SDS-polyacrylamide-gel-electrophoresis of actin within virions of the paramyxoviruses.
Introduction Epithelial and fibroblastic cells grown in tissue culture adhere and flatten out on a plastic or glass surface. Microfilaments found beneath the plasma membrane and running in parallel bundles within the cell can be seen with phase microscopy, electron microscopy or immunofluoreseenee (1, 6). One of the major constituents of the mierofilaments is actin (5, 6). When cells divide or are exposed to trypsin or divalent cation chelating agents, the actin filament bundles assume a random cellular distribution (9). The mechanism of assembly of paramyxoviruses has not been completely elucidated. However, the nucIeoeapsids move to an area of the cell membrane rich in viral matrix and glycoproteins before being enveloped (2). The movement of viral glycoproteins on the cell surface m a y be influenced by actin filaments (3). Actin has been shown to be a major constituent of R N A tumor viruses and the paramyxoviruses (4, 16, 18). Moreover anti-aetin antibodies have been transiently demonstrated in connection with i.a. measles infection. I t was therefore of interest 1 D. G. J. TYR~EL~,is a Centennial Fellow of the 5~[edica]Research Council of Canada.
0304-8608/78/0057/0291/$ 01.20
292
A. FAGRAEUS,
D. L. J. TYI~RELL, i%. NOI~BEI~G, and E. NOaRBY:
to compare the a e t i n f i l a m e n t p a t t e r n in p a r a m y x o v i r u s - i n f e c t e d cells with cells infected with viruses in which a c t i n has n o t been d e m o n s t r a t e d . The d i s t r i b u t i o n of aetin tilaments in cells was d e m o n s t r a t e d b y indirect immunofluoreseenee using h u m a n a n t i a e t i n antibodies.
Materials and Methods I-Iuman diploid lung fibrobIasts were grown on glass slides in Bellco tubes containing E @ e s minimM essential medium (MEM) supplemented with 5 per cent. recta,1 calf serum. VV*henthe cultures were 5 0 8 0 per eent confluent the cells were infected with either measles, mumps, Sendai, Newcastle disease (NDV), adenovirus type 2 or rabies at multiplicity resulting in infection in 20--30 per cent of the cells. After about 40 hours the slides were washed twice in PBS, dried and fixed in acetone at --20 ° C. Adeno, Sendai and NDV virus inclusions were stained by virus specific rabbit antisera. A h u m a n serum from a patient vaccinated against rabies was used to stain those inclusions. A h u m a n anti-aetin serum, containing antibodies against measles and mumps was used to visualize these inclusions as well as the mierofilament bundles. The characteristics of the h u m a n anti-aetin serum, as well as the technique for absorption of serum with aetin have been described elsewhere (t0). FITC conjugates used were sheep anSi-human immunoglobulin (SBL 073910) and anti-rabbit immunoglobulin (SBL 647111). Both conjugates were obserbed with immunoglobulin of the other species involved. The antisera were mixed before being added, so were the conjugates. Staining and reading of the preparations were otherwise performed as earlier described (10).
Results I n all p r e p a r a t i o n s we restricted our observations to cells t h a t were still a t t a c h e d to the glass surface. The actin mierofilament p a t t e r n s could n o t be compared in cells t h a t showed a d v a n c e d eytopathie effects. F o r example, r o u n d e d or detached cells were n o t studied i n this comparison. Uninfected cells in the infected cell preparations showed the normM p a t t e r n with parallel mierofilament b u n d l e s often proceeding u n i n t e r r u p t e d for the total length of the cell. Cells infected with p a r a m y x o v i r u s e s indicated b y the presence of inclusions, f r e q u e n t l y d e m o n s t r a t e d a decrease i n the n u m b e r of a e t i n f i l a m e n t bundles, or a n i n t e r r u p t i o n of the mierofilaments near the region of viral inclusions. This effec~ was seen with measles, m u m p s , N D V a n d Sendai virus, b u t was most p r o n o u n c e d with measles a n d m u m p s infected cells (Figs. 1 a arid b). L u n g fibroblasts infected w i t h adenovirus type 2 showed n u c l e a r inclusions, b u t the a e t i n f i l a m e n t b u n d l e s r e m a i n e d n n a l t e r e d (Fig. i e). The B H K cells infected with rabies virus d e m o n s t r a t e d cytoplasmic inclusions, b u t no or m u c h less a l t e r a t i o n in the microfilament p a t t e r n (Fig. 1 d) as compared to the p a r a m y x o virus infected ceils. Fig. 1 a - - d . Lung fibroblasts infected with measles (Fig. t a), mumps (Fig. l b) and adenovirus type 2 (Fig. lc) and B H K cells infected with rabies (Fig. ld). The cells were stained by indirect immnnofluorescence using a h u m a n anti-actin serum which also stained the viral inclusions in Figures 1 a, b and d. R a b b i t antibodies stained the viral inclusions in Figure 1 c. Note that very few microfilament bundles are seen in the measles (Fig. 1 a) and mumps (Fig. l b) infected cells, especially in the vicinity of the virus inclusions, whereas an ample amount of microfilament bundles are stained in the uninfected cells. Seemlingly uninterrupted bundles of filaments are shown in adenovirus (Fig. 1 e) and rabies virus (Fig. t d) infected cells. Magnification × 500 (Figs. 1 a--e) and × 800 (Fig. 1 d)
A e t i n F i l a m e n t s in V i r u s I n f e e t e d F i b r o b l a s t s
Fig. 1 a - d
293
294
A. FAGnA~US,
D. L. J. TY;aRELL, R. NORBERG,
and E. NORRBY:
In. all eases absorption of the anti-aetin serum with actin totally abolished the mierofilament staining but left the staining of viral inclusions unaltered (Fig. 2).
Fig. 2. Measles infected cells stained by the humarl anti-aetin serum absorbed with actin. The measles inclusions are still stained but the staining of microfilaments is abolished
Diseussion Indirect iramunofluorescent staining of lung fibroblasts infected with paramyxoviruses suggested t h a t there were fewer mierofitament bundles t h a n in uninfected cells. This decrease in actin filament bundles was not seen in cells infected with rabies or adenovirus. The mechanism b y which the aetin filament bundles were decreased is unknown. However, it has been shown t h a t the total a m o u n t of aetin decreases in cells infected with Sendal virus (8). The aetin present in measles virions has been shown to be inside the virion. The a m o u n t of aetin found in the virus is unaltered b y t r e a t m e n t with nonionie detergents used to remove envelope proteins. Furthermore, t r e a t m e n t of purified virions with high concentrations of trypsin, does not affect the viral associated aetin (16), but does remove aetin adsorbed to the surface of the virions (Tyrrell, personal observations). Evidence has also been presented t h a t an intact eytoskeleton is a prerequisite for m o v e m e n t of measles nueleoeapsids to the site of budding on. the cell m e m b r a n e in connection with capping (t5). The failure of rabies virus to alter significantly the aetin filament bundles is of interest since rhabdoviruses are assembled in the cytoplasms a n d bud from the cell
Actin Filaments in Virus Infected Fibroblasts
295
m e m b r a n e i~ f i b r o b l a s t s (7). I n n e u r a l tissue, rabies virus m a y acquire its m e m b r a n e i n t r a e y t o p l a s m i e a l l y (11). H o w e v e r , t h e failure to define a e t i n as a c o m p o n e n t in r h a b d o v i r u s e s (17), suggested t h a t t h e r e m a y be significant differences in the a s s e m b l y of r h a b d o v i r u s e s a n d p a r a m y x o v i r u s e s . These differences m a y e x p l a i n t h e different effects of these viruses on t h e a e t i n f i l a m e n t bundles in infected cells. The failure of a d e n o v i r u s infection to a l t e r t h e ac~in f i l a m e n t b u n d l e s was n o t surprising as ~his virus is a s s e m b l e d in t h e nucleus a n d does n o t c o n t a i n actiu. I n a p a r a l l e l s t u d y (14) changes in t h e d i s t r i b u t i o n of a c t i n in cells infected w i t h N D V a n d Vesicular s t o m a t i t i s virus (VSV) were described. H o w e v e r , in this s t u d y t h e p a r a m y x o v i r u s caused a n increase in a e t i n containing f i l a m e n t s s u b j a c e n t to t h e cell surface whereas V S V infection caused a decrease in the corresponding filaments. Differences in e x p e r i m e n t a l conditions such as t y p e of cells a n d t i m e for h a r v e s t i n g of cultures m a y e x p l a i n t h e differences in our findings. I l l other studies t h e v i r a l - i n d u c e d changes of a c t i n m i c r o f i l a m e n t p a t t e r n s h a v e been a s s o c i a t e d w i t h cell t r a n s f o r m a t i o n . The long a e t i n f i l a m e n t b u n d l e s f o u n d in n o r m a l c u l t u r e d 3 T 3 cells c o n v e r t e d to a diffuse a e t i n m a t r i x on t r a n s f o r m a t i o n of t h e cells w i t h SV40 virus (12, 13).
Acknowledgments The skillfull technical assistance of Miss A n i t a 0 s t b o r n and Mr. G6ran Utter, M. Sci., is gratefully acknowledged. This work was supported by a grant from The Swedish Medical I~eseareh Council B 77-16 X-04976-01.
References 1. BUCKLEY, I., PO~TER, K. : Cytoplasmic fibrils in living culmred cells. A light and electron microscopic study. Protoplasma 64, 349--356 (1967). 2. C~oPPIN, P. W., CONPANS, tl,. ~hT.: Reproduction of paramyxoviruses. I n : F~AENXE]5-CoNRAT, I:I., WAGNER, R. I~. (eds.), Comprehensive Virology, Vol. 4, 95---125. New York: Plenum Press 1975. 3. E~]~srsT, A., SuSrDQVlS% K.-G. : Polar appearance and nonligand induced spreading of measles virus hemaggtutinin at the surface of chronically infected cells. Ceil 5, 351--359 (1975). 4. FLEISSNEg, E., TRESS, E. : Chromatographic and electrophoretie analysis of viral proteins from hamster and chicken cells transformed by g o u s sarcoma virus. J. Virol. 11, 250--262 (1973). 5. GOLDMAN, K., KNIPE, D. : Functions of cytoplasmic fibers in non-muscle cell motility. Cold Spring H a r b o r Symp. quant. Biol. 31, 529--535 (1973). 6. GOLDS~A~, t~., LAZARmES, E., POLLACK, g . , %VEBER, K . : The distribution of actin in non-muscle cells: The use of ac~in antibody in the localization of aetin within microfilament bundles of mouse 3 T 3 cells. Exp. CetI l~es. 99, 333~344 (1975). 7. I~[ow~Tso~, A. F., W~IT~ORE, G. F. : The development and structure of vesicular stomatitis virus. Virology I6, 466--478 (1962). 8. L~)~B, R. A., M A ~ ¥ , B. W. T., CnoI'PI~, P. W. : The synthesis of Sendal virus potypeptides in infected cells. Virology 69, 116-- 13 t ( 1976). 9. LAZAlClDES,E. : Aspects of the structural organization of actin filaments in tissue culture cells. I n : GOJ,D~A~r, t~., POLLe~D, T., ROSEN~ALrM, J. (eds.), Cell Motility. Cold Spring H a r b o r Conferences on Cell Proliferation 3, 347--360 (1976).
296
A. FAGRAEUSet al. : Actin Filaments in Virus Infected Fibroblasts
10. LID:M&N, K., BIBERFELD, (~., FAGRAEUS, A., NORBERG, R., TORSTENSSON, 1~., UTTER, G., CARLSSON, L., LUCA, J., LINDBERG, U. : Antiactin specificity of human smooth muscle antibodies in chronic active hepatitis. Clin. exp. Immunot. 24, 266--272 (1976). i l . MATSU~ATO, S., KAWAI, A.: Comparative studies on development of rabies in different host cells. Virology 39, 449--459 (1969). 12. McN~TT, N., CuLP, N., BLACK, P. : R e v e r t a n t cell lines isolated from 8V 40 transformed cells. IV. Mierofilament disruption and cell shape in untransformed, transformed and revertant cells. J. Ceil Biol. 50, 412---418 (1973). 13. POL]~ACK, R., OSBORN, M., WEBER, K . : Patterns of organization of aetin and myosin in normal and transformed cultured cells. Proc. Nat. Acad. Sci. U.S.A. 72, 994--998 (1975). 14. RUTTER, G., MANNWEILER, K . : Alterations of aetin-contalning structures in B H K 21 cells infected with Newcastle disease virus and vesicular stomatitis virus. J. ten. Virol. 37, 233--242 (1977). 15. TYRRELL, D. L. J., Ea~NST, A. : Transmembranal communication of measles viral proteins. A possible role for aetin in viral assembly. Submitt.ed for publication. 16. Tx~aRELL, D. L. J., No~aR]3,z, ]9]. : Structural polypeptides of measles virus. J. gen. Virol. (in press, 1978). 17. ~¥A~GER, R. R., PREVEO, L., BRO~.VN,F., SLTM]M[ERS,D. F., SoKo;r~,F., MACLEOD, I~. : Classification of rhabdovirus proteins: a proposal. J. Virol. 10, 1228--1230 (1972). 18. WANe, E., WOLF, B. A., L A ~ , K. A., CHOP]'I>r, 1~. W., GOLDBERa, A. g . : The presence of actin in enveloped viruses. In : GOLD~AN, R., POLLARD, 1~., ]~OSENBAUM, J. (eds.), Cell Motility. Cold Spring Harbor Conferences on Cell Proliferation 3, 589--599 (1976). Authors' address: Dr. A. FAGRAEUS, The National Bacteriological Laborato W, Lundagatan 2, S-105 21 Stockholm, Sweden. Received
January 4, 1978
Archives of Virology 57, 291--296 (1978)
© by Springer-Verlag 1.978
Actin Filaments in Paramyxovirus-Infected Human Fibroblasts Studied by Indirect Immunofluoreseence By A. FAGRAEUS~D. L. J. TYRRELL1, ~. NORBERG, and E. NoRI~BY Department of Immunology, National Bacteriological Laboratory, Stockholm and Department of Virology, Karolinska Instituter, Stockholm, Sweden With 2 Figures Accepted February 24, 1978
Summary Fibroblasts growing on glass have microfilaments arranged in bundles. These can be demonstrated b y indirect immunofluorcseent technique using human antiactin serum or experimentally produced rabbit anti-actin serum. When monolayer cultures of epithelial cells and fibroblasts are infected with paramyxovirus, such as measles, mumps, Sendal and NI)V, there is a striking decrease of the bundles. Rabies and adenoviruses do not seem to influence the staining of microfilaments. The microfilament decreasing effect in the cells correlates to the finding by SDS-polyacrylamide-gel-electrophoresis of actin within virions of the paramyxoviruses.
Introduction Epithelial and fibroblastic cells grown in tissue culture adhere and flatten out on a plastic or glass surface. Microfilaments found beneath the plasma membrane and running in parallel bundles within the cell can be seen with phase microscopy, electron microscopy or immunofluoreseenee (1, 6). One of the major constituents of the mierofilaments is actin (5, 6). When cells divide or are exposed to trypsin or divalent cation chelating agents, the actin filament bundles assume a random cellular distribution (9). The mechanism of assembly of paramyxoviruses has not been completely elucidated. However, the nucIeoeapsids move to an area of the cell membrane rich in viral matrix and glycoproteins before being enveloped (2). The movement of viral glycoproteins on the cell surface m a y be influenced by actin filaments (3). Actin has been shown to be a major constituent of R N A tumor viruses and the paramyxoviruses (4, 16, 18). Moreover anti-aetin antibodies have been transiently demonstrated in connection with i.a. measles infection. I t was therefore of interest 1 D. G. J. TYR~EL~,is a Centennial Fellow of the 5~[edica]Research Council of Canada.
0304-8608/78/0057/0291/$ 01.20
292
A. FAGRAEUS,
D. L. J. TYI~RELL, i%. NOI~BEI~G, and E. NOaRBY:
to compare the a e t i n f i l a m e n t p a t t e r n in p a r a m y x o v i r u s - i n f e c t e d cells with cells infected with viruses in which a c t i n has n o t been d e m o n s t r a t e d . The d i s t r i b u t i o n of aetin tilaments in cells was d e m o n s t r a t e d b y indirect immunofluoreseenee using h u m a n a n t i a e t i n antibodies.
Materials and Methods I-Iuman diploid lung fibrobIasts were grown on glass slides in Bellco tubes containing E @ e s minimM essential medium (MEM) supplemented with 5 per cent. recta,1 calf serum. VV*henthe cultures were 5 0 8 0 per eent confluent the cells were infected with either measles, mumps, Sendai, Newcastle disease (NDV), adenovirus type 2 or rabies at multiplicity resulting in infection in 20--30 per cent of the cells. After about 40 hours the slides were washed twice in PBS, dried and fixed in acetone at --20 ° C. Adeno, Sendai and NDV virus inclusions were stained by virus specific rabbit antisera. A h u m a n serum from a patient vaccinated against rabies was used to stain those inclusions. A h u m a n anti-aetin serum, containing antibodies against measles and mumps was used to visualize these inclusions as well as the mierofilament bundles. The characteristics of the h u m a n anti-aetin serum, as well as the technique for absorption of serum with aetin have been described elsewhere (t0). FITC conjugates used were sheep anSi-human immunoglobulin (SBL 073910) and anti-rabbit immunoglobulin (SBL 647111). Both conjugates were obserbed with immunoglobulin of the other species involved. The antisera were mixed before being added, so were the conjugates. Staining and reading of the preparations were otherwise performed as earlier described (10).
Results I n all p r e p a r a t i o n s we restricted our observations to cells t h a t were still a t t a c h e d to the glass surface. The actin mierofilament p a t t e r n s could n o t be compared in cells t h a t showed a d v a n c e d eytopathie effects. F o r example, r o u n d e d or detached cells were n o t studied i n this comparison. Uninfected cells in the infected cell preparations showed the normM p a t t e r n with parallel mierofilament b u n d l e s often proceeding u n i n t e r r u p t e d for the total length of the cell. Cells infected with p a r a m y x o v i r u s e s indicated b y the presence of inclusions, f r e q u e n t l y d e m o n s t r a t e d a decrease i n the n u m b e r of a e t i n f i l a m e n t bundles, or a n i n t e r r u p t i o n of the mierofilaments near the region of viral inclusions. This effec~ was seen with measles, m u m p s , N D V a n d Sendai virus, b u t was most p r o n o u n c e d with measles a n d m u m p s infected cells (Figs. 1 a arid b). L u n g fibroblasts infected w i t h adenovirus type 2 showed n u c l e a r inclusions, b u t the a e t i n f i l a m e n t b u n d l e s r e m a i n e d n n a l t e r e d (Fig. i e). The B H K cells infected with rabies virus d e m o n s t r a t e d cytoplasmic inclusions, b u t no or m u c h less a l t e r a t i o n in the microfilament p a t t e r n (Fig. 1 d) as compared to the p a r a m y x o virus infected ceils. Fig. 1 a - - d . Lung fibroblasts infected with measles (Fig. t a), mumps (Fig. l b) and adenovirus type 2 (Fig. lc) and B H K cells infected with rabies (Fig. ld). The cells were stained by indirect immnnofluorescence using a h u m a n anti-actin serum which also stained the viral inclusions in Figures 1 a, b and d. R a b b i t antibodies stained the viral inclusions in Figure 1 c. Note that very few microfilament bundles are seen in the measles (Fig. 1 a) and mumps (Fig. l b) infected cells, especially in the vicinity of the virus inclusions, whereas an ample amount of microfilament bundles are stained in the uninfected cells. Seemlingly uninterrupted bundles of filaments are shown in adenovirus (Fig. 1 e) and rabies virus (Fig. t d) infected cells. Magnification × 500 (Figs. 1 a--e) and × 800 (Fig. 1 d)
A e t i n F i l a m e n t s in V i r u s I n f e e t e d F i b r o b l a s t s
Fig. 1 a - d
293
294
A. FAGnA~US,
D. L. J. TY;aRELL, R. NORBERG,
and E. NORRBY:
In. all eases absorption of the anti-aetin serum with actin totally abolished the mierofilament staining but left the staining of viral inclusions unaltered (Fig. 2).
Fig. 2. Measles infected cells stained by the humarl anti-aetin serum absorbed with actin. The measles inclusions are still stained but the staining of microfilaments is abolished
Diseussion Indirect iramunofluorescent staining of lung fibroblasts infected with paramyxoviruses suggested t h a t there were fewer mierofitament bundles t h a n in uninfected cells. This decrease in actin filament bundles was not seen in cells infected with rabies or adenovirus. The mechanism b y which the aetin filament bundles were decreased is unknown. However, it has been shown t h a t the total a m o u n t of aetin decreases in cells infected with Sendal virus (8). The aetin present in measles virions has been shown to be inside the virion. The a m o u n t of aetin found in the virus is unaltered b y t r e a t m e n t with nonionie detergents used to remove envelope proteins. Furthermore, t r e a t m e n t of purified virions with high concentrations of trypsin, does not affect the viral associated aetin (16), but does remove aetin adsorbed to the surface of the virions (Tyrrell, personal observations). Evidence has also been presented t h a t an intact eytoskeleton is a prerequisite for m o v e m e n t of measles nueleoeapsids to the site of budding on. the cell m e m b r a n e in connection with capping (t5). The failure of rabies virus to alter significantly the aetin filament bundles is of interest since rhabdoviruses are assembled in the cytoplasms a n d bud from the cell
Actin Filaments in Virus Infected Fibroblasts
295
m e m b r a n e i~ f i b r o b l a s t s (7). I n n e u r a l tissue, rabies virus m a y acquire its m e m b r a n e i n t r a e y t o p l a s m i e a l l y (11). H o w e v e r , t h e failure to define a e t i n as a c o m p o n e n t in r h a b d o v i r u s e s (17), suggested t h a t t h e r e m a y be significant differences in the a s s e m b l y of r h a b d o v i r u s e s a n d p a r a m y x o v i r u s e s . These differences m a y e x p l a i n t h e different effects of these viruses on t h e a e t i n f i l a m e n t bundles in infected cells. The failure of a d e n o v i r u s infection to a l t e r t h e ac~in f i l a m e n t b u n d l e s was n o t surprising as ~his virus is a s s e m b l e d in t h e nucleus a n d does n o t c o n t a i n actiu. I n a p a r a l l e l s t u d y (14) changes in t h e d i s t r i b u t i o n of a c t i n in cells infected w i t h N D V a n d Vesicular s t o m a t i t i s virus (VSV) were described. H o w e v e r , in this s t u d y t h e p a r a m y x o v i r u s caused a n increase in a e t i n containing f i l a m e n t s s u b j a c e n t to t h e cell surface whereas V S V infection caused a decrease in the corresponding filaments. Differences in e x p e r i m e n t a l conditions such as t y p e of cells a n d t i m e for h a r v e s t i n g of cultures m a y e x p l a i n t h e differences in our findings. I l l other studies t h e v i r a l - i n d u c e d changes of a c t i n m i c r o f i l a m e n t p a t t e r n s h a v e been a s s o c i a t e d w i t h cell t r a n s f o r m a t i o n . The long a e t i n f i l a m e n t b u n d l e s f o u n d in n o r m a l c u l t u r e d 3 T 3 cells c o n v e r t e d to a diffuse a e t i n m a t r i x on t r a n s f o r m a t i o n of t h e cells w i t h SV40 virus (12, 13).
Acknowledgments The skillfull technical assistance of Miss A n i t a 0 s t b o r n and Mr. G6ran Utter, M. Sci., is gratefully acknowledged. This work was supported by a grant from The Swedish Medical I~eseareh Council B 77-16 X-04976-01.
References 1. BUCKLEY, I., PO~TER, K. : Cytoplasmic fibrils in living culmred cells. A light and electron microscopic study. Protoplasma 64, 349--356 (1967). 2. C~oPPIN, P. W., CONPANS, tl,. ~hT.: Reproduction of paramyxoviruses. I n : F~AENXE]5-CoNRAT, I:I., WAGNER, R. I~. (eds.), Comprehensive Virology, Vol. 4, 95---125. New York: Plenum Press 1975. 3. E~]~srsT, A., SuSrDQVlS% K.-G. : Polar appearance and nonligand induced spreading of measles virus hemaggtutinin at the surface of chronically infected cells. Ceil 5, 351--359 (1975). 4. FLEISSNEg, E., TRESS, E. : Chromatographic and electrophoretie analysis of viral proteins from hamster and chicken cells transformed by g o u s sarcoma virus. J. Virol. 11, 250--262 (1973). 5. GOLDMAN, K., KNIPE, D. : Functions of cytoplasmic fibers in non-muscle cell motility. Cold Spring H a r b o r Symp. quant. Biol. 31, 529--535 (1973). 6. GOLDS~A~, t~., LAZARmES, E., POLLACK, g . , %VEBER, K . : The distribution of actin in non-muscle cells: The use of ac~in antibody in the localization of aetin within microfilament bundles of mouse 3 T 3 cells. Exp. CetI l~es. 99, 333~344 (1975). 7. I~[ow~Tso~, A. F., W~IT~ORE, G. F. : The development and structure of vesicular stomatitis virus. Virology I6, 466--478 (1962). 8. L~)~B, R. A., M A ~ ¥ , B. W. T., CnoI'PI~, P. W. : The synthesis of Sendal virus potypeptides in infected cells. Virology 69, 116-- 13 t ( 1976). 9. LAZAlClDES,E. : Aspects of the structural organization of actin filaments in tissue culture cells. I n : GOJ,D~A~r, t~., POLLe~D, T., ROSEN~ALrM, J. (eds.), Cell Motility. Cold Spring H a r b o r Conferences on Cell Proliferation 3, 347--360 (1976).
296
A. FAGRAEUSet al. : Actin Filaments in Virus Infected Fibroblasts
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