(~) INSTITUT PASTEUR/ELSEVIER Paris 1992
Res. ViroL 1992, 143, 401-406
Role of acylation of viral haemagglutinin during the influenza virus infectious cycle P. Portincasa, G. Conti t°) and C. Chezzi lstituto di Microbiologia, Universita" degfi Studi di Parma, Facolta" di Medicina e Chirurgia, Parma (Italy)
SUMMARY We investigated fatty acid residues bound to the haemagglutinin (HAl of type A influenza viruses by growing the viruses in permissive chick embryo fibroblasts (CEF) and in non-permissive HeLa-229 cells using a maintenance medium containing 3H-palmitic acid. Our results suggest that fatty acid acylation of the major viral glycoprotain may be an important prerequisite for the production of mature viral particles. Indeed, palmitoylation is found in infected CEF, but is completely lacking in non-permissive HeLa-229 cells infected by the same virus strains. We conclude that this type of post-translational modification of virus HA glycoprotein could be a general phenomenon regulating the maturation and budding of influenza virus. Key-words: Influenza virus, Acylation, Palmitic acid, Haemagglutinin; Glycoprotein, Viral maturation.
INTRODUCTION
Glycoproteins present in a number of enveloped viruses have been shown to contain palmitic acid residues covalently bound to polypeptides (Schmidt, 1982; Schmidt et al., 1979; Schmidt and Schlesinger, 1979; Klochmann and Deppert, 1985). This modification has also been described for viral proteins of influenza A viruses, such as the membrane M2 protein (Sugrue et al., 1990) and the precursor of the major structural protein of HIV1 (Bryant and Ratner, 1990). There is some evidence that acylation of virusencoded polypeptides and glycoproteins may be essential for the assembly and budding of the virus from the host cell and may play a possible
Submitted May 14, 1992, accepted October 26, 1992. (*) To whom reprint requests should be addressed.
role in intracellular protein transport (Cross et al., 1984; Moreman and Touster, 1985; Olson et al., 1985; Pellman et al., 1985; Schlesinger and Malfer, 1982 ; Schultz et al., 1985 ; Zilberstein et al., 1980). However, the necessity for this kind of post-translational modification and its significance, are not yet clear, since it does not occur in polypeptides of certain virus strains which are able to undergo productive multiplication in the cellular host (Sugrue et al., 1990; Girish and Ghosh, 1984). We recently reported that HeLa-229 cells do not permit the multiplication of type A influenza viruses (Portincasa et al., 1990) and that the non-permissive condition could be due to the inability of the haemagglutinin (HA) polypeptide
402
P. P O R TINCA SA E T A L .
to be correctly inserted into the plasma membrane (Portincasa et al., 1991). Since one of the most important events in virus assembly and propagation concerns protein modification, we investigated post-translational changes in the virus-encoded H A polypeptide by ascertaining the presence or absence of bound fatty acids in HeLa-229 cells infected by various type A influenza viruses.
MATERIALS AND METHODS
Fowl plague virus, Ulster strain (A, FPV, Ulster 73, H7N,), fowl plague virus, Rostock strain (A, FPV, Rostock, HTNI) and the Texas strain (A,H3N2) were grown in the allantoic cavity of 10-day-old embryonated hen eggs. After a 28-h incubation period at 37°C for avian Ulster and Rostock strains, and after 42-h incubation for the human Texas strain, the allantoic fluid was harvested, centrifuged at 2,000 rpm to separate cellular debris and titrated by haemagglutination with chicken red blood cells (Borland and Mahy, 1968) and the plaque assay. Monolayer cultures of mammalian HeLa-229 cells and chick embryo fibroblasts (CEF) were grown in Dulbecco's medium and minimal essential medium (Earle's MEM) respectively, both supplemented with 10 % foetal calf serum as described elsewhere (Choppin, 1969). To verify that viral HA correctly performed its cytoplasmic migration during influenza virus infection, experiments using 35S-methionine labelling were carried out in permissive CEF and nonpermissive HeLa-229 cells. Both cellular monolayers were infected with avian Rostock and Ulster strains as well as with the human Texas strain. Six hours post-infection (p.i.), infected monolayers were pulselabelled with 3SS-methionine (20 izCi/ml) and then processed by 15 % SDS-slab gel electrophoresis according to standard procedure (Inglis, 1978). In order to investigate the presence of covalently bound fatty acids to virus-encoded structural HA polypeptide, experiments with 3H-palmitic acid labelling were performed in non-permissive HeLa-229 and permissive CEF cells. Monolayers of confluent cells infected by type A influenza viruses were labelled with 50 }~Ci/ml of JH-palmitic acid (10-30 Ci/mmol)
CEF FPV HA
= = =
chick embryo fibroblast. fowl plague virus. haemasglutinin.
starting at 3 h after infection. Labelling of infected and non-infected HeLa-229 and CEF cells was halted by placing the Petri dishes in an ice-cold water bath; the infected cellular monolayers were then scraped off with a rubber teflon policeman and pelleted by centrifugation at 3,000 rpm for 5 min. Cellular pellets were then resuspended in a small volume of sterile water, sonicated 3 times for 30 s in an icecold water bath and centrifuged at 43,500 rpm in an "SW50L Beckman" rotor to separate cellular membranes. The supernatant fraction was discarded and the pellet was made soluble by adding lysis buffer (Schlesinger et al., 1980). Samples were loaded onto a 12.5 % SDS-polyacrylamide slab gel and electrophoresis was performed according to standard procedures (Inglis, 1978). The gel was then processed by fluorography with "En3Hance '' (NEN, DuPont) and a fluorogram was used to visualize labelled protein bands by exposure on X-ray "Omat" film autoradiography.
RESULTS Figure 1 shows the autoradiogram of 3Hpalmitic-acid-labelled cellular extracts of Ulsterinfected and mock-infected CEF and HeLa-229 cells. Comparison of virus-encoded proteins was made with 35S-methiordne-labelled polypeptides o f infected CEF cells. Only the cellular extracts of infected CEF cells evidenced comigration of 3H-labelled material with viral H A polypeptide. Comigration did not take place in uninfected CEF, in infected and mock-infected HeLa-229 cells. Data obtained from infection of such cellular hosts with Rostock and Texas strains were strictly comparable (data not shown). We performed an immunoprecipitation test using hyperimmune polyclonal antiserum to Ulster strain and virus-encoded H A glycoprotein (Madeley et al., 1971) to confirm the effective correspondence of 3H-labelled material w i t h viral HA, and to assess the absence of paimitoylation in infected HeLa 229 cells. Cellular extracts of infected C E F and H e L a 229 cells labelled with 3 H - p a l m i t i c acid were ira-
MEM = minimalessential p.i. = post-infection.
medium.
I N F L U E N Z A VIRUS C Y C L E A N D H A F A T T Y A C I D A C Y L A T I O N
munoprecipitated according to Schlesinger and Malfer (1982), and analysed by SDS-slab gel electrophoresis. As shown in figure 2, a 3H-labelled protein was recovered in infected CEF by immuno-
~"
"~~IC CEF
•'
403
precipitation assay; it corresponded to the HA polypeptide. In this case as well, 3H-labelled material was not present in infected HeLa 229 cells. It is reasonable to conclude that virusencoded HA of influenza viruses underwent
t
IC
II
I
m
'
" m
Fig. 2. Immunoprecipitation of labelled 3H-palmitic-acid Fig. I. SDS-polyacrylamide
slab gel electrophoresis (12.5 %) of 3H-palmitic-acid-labelled chick embryo fibroblasts (CEF) and HeLa-229 cells uninfected and infected by type A influenza virus F P V Ulster 73. Pulse labelling was performed from 3 to 8 h p.i. U C = uninfected cells; IC = infected cells. PB2, PBI and P A are subunits of virus-encoded RNA-dependent R N A polymerase; H A = viral haemagglutinin; N P represents viral nucleoprotein; M l is viralmembrane component and N S I is a virus-induced non-structural polypeptide.
HA glycoprotein from CEF and HeLa-229 cells unninfected and infected with influenza A virus FPV Ulster 73. Cell lysates were immunoprecipitated with anti-HA polyclonal serum and applied to a 12.5 % SDSpolyacrylamide slab gel electrophoresis. UC = uninfected cells; IC = infected cells. PB2, PBI and PA are subunits of virus-encoded RNA-dependent RNA polymerase; HA = viral haemagglutinin; NP represents viral nucleoprotein; MI is viral membrane component and NS1 is a virus-induced non-structural polypeptide.
404
P. P O R T I N C A S A E T A L .
post-translational modification by palmitoylation only during the productive infectious cycle in the permissive cellular host.
this cellular host, all virus-induced proteins were synthesized and, in addition, that migration o f virus-encoded H A was effective.
Experiments using 35S-methionine labelling p e r f o r m e d at a later time during infection were carried out in HeLa-229 cells to ensure that, in
As indicated in figure 3, the presence o f the HA1 glycoprotein in lane R corresponding to the polypeptides o f F P V Rostock, in both C E F and
...
R.U.T.
R.U.T.
PB|.. PBI,PA ~ ' " HA
, PA
. . . . .
NP HA1
N S'
.! X
NS2 III
II
i.c
• CEF
i
i
uc.
ic.
" HeL
a229
Fig. 3. SDS-polyacrylamide slab gel electrophoresis (17.5 %) of CEF and HeLa-229 cells infected by influenza A viruses FPV Rostock (R), FPV Ulster 73 (U) and Texas (T) strains, and pulse-labelled at 6 h p.i. using 3~S-methionine. PB2, PBl and PA are subunits of virus-coded RNA-dependent RNA polymerase; HA = viral haemagglutinin; HAl is the cleavage product of the FPV Rostock HA; NP represents viral nucleoprotein; Ml is viral membrane component; NS! and NS2 are virus-induced non-structural polypeptide.
INFLUENZA VIRUS C Y C L E A N D HA F A T T Y ACID A C Y L A T I O N
HeLa-229 infected cells, demonstrated that HeLa-229 cells permitted the migration of HA protein during virus strain multiplication.
DISCUSSION
Our results provide evidence that HA of the three strains of type A influenza virus is not acylated during the infectious cycle in HeLa-229 cells, while CEF infected by the same avian and human strains of influenza virus show acylation of viral HA. The infectious cycle in HeLa-229 is completely non-permissive, whereas CEF cells produce a great number of mature viral particles (Portincasa et al., 1990). We have recently established that HeLa-229 cells do not permit the multiplication of type A influenza viruses, and that the reason for this behaviour seems to be the result of incorrect insertion of viral HA in the cellular membrane during the final stages of viral infection. It is well known that the budding process requires the establishment of interactions between viral and cellular proteins, and that the plasma membrane of the infected cell must interact with influenza components during the maturation of viral particles (Zabedee and Lamb, 1989; Hay et al., 1986). Specific interactions are necessary for several steps during the late stages of infection, such as the insertion of viral HA into the cellular membrane and migration of this viral component through the cytoplasmic compartment. In fact, it is well known that viral HA is synthesized on the rough endoplasmic reticulum and then migrates to the plasma membrane via the smooth endoplasmic reticulum. During this migration, viral HA is cleaved by cellular proteolytic enzymes. In the present case, however, there is no evidence that inhibition of migration of HA is the defect shown by infected HeLa-229 cells, inasmuch as HA1, which represents the cleavage product of Rostock HA, is strongly present in the autoradiogram shown in figure 3. In the same electrophoretic pattern, the other two lines corresponding to FPV Ulster 73 03) and Texas
405
(T) strains do not show any HA1 product, since they do not possess cleaved HA (Conti et al., 1985). Moreover, the fact that HA1 of FPV Rostock is present in infected HeLa-229 cells clearly demonstrates that these cells permit the migration of this structural component through the cytoplasm and to the cellular surface. Despite this, since palmitoylation of viral HA is completely lacking in these cells and seems to be the unique defect (Portincasa et al., 1990), we hypothesize that the absence of this modification in viral glycoprotein might explain the non-permissive infection we observed. Palmitoylation could act as an anchor for the cellular membrane or allow HA insertion into the plasma membrane. In fact, results of haemadsorption obtained in HeLa-229 infected by these influenza virus strains suggest that the lack of mature viral particles could be due to abnormal insertion of HA into the cellular membrane (Portincasa et al., 1991). The inability to cross the lipid bilayer of the infected cells could be explained by the failure of post-translational modification of this virus-coded glycoprotein. In this respect, fatty acid acylation of HA protein might be the pre-requisite for this specific step during maturation and budding of influenza virus from HeLa-229, even if' another possible function of the fatty acid residue must be taken into account: palmitoylation might, in fact, provide a signal for the interaction of the HA glycoprotein with the viral nucleocapsid at specific sites on the plasma membrane. Although the function of acylated components is intriguing, it is reasonable to conclude that acylation of viral glycoprotein may play an important role in the assembly and maturation of influenza viral particles. Experiments involving the use of specific inhibitors of protein acylation are presently being conducted on the permissive cellular system to better understand the role of palmitoylation of viral HA during the influenza virus multiplication cycle.
Acknowledgements This studywas supportedby a grant fromthe Ministero Pubblica Istruzione(60 %) of the ItalianGovernment1990.
406
P. P O R T I N C A S A
R61e de racylation de I'h~magglutinine virale dans le cycle infectieux du virus grippal Dans le present travail effectu6 sur des fibroblastes d'embryon de poulet et sur des cellules H e L a (non permissives) dans un milieu contenant de l'acide palmitique, nous avons observ6 que l'acylation de cet acide gras li~/L la prot6ine de l'h~magglutinine du virus grippal peut apporter une modification d6terminante pour la maturation et la production des particules virales. Mots-cl~s: Virus grippal, Acylation, Acide palmitique, H~magglutinine; Glycoprot~ine, Maturation virale.
References Borland, R. & Mahy, B.W.J. (1968), Deoxyribonucleic acid-dependent RNA polymerase activity in cells infected with influenza virus. J. Virol., 2, 33-39. Bryant, M. & Ratner, L. (1990), Myristoylation-dependent replication and assembly of human immunodeficiency virus-l. Proc. nat. Acad. Sci. (Wash.), 87, 523-527. Choppin, P.W. (1969), Replication of influenza virus in a continuous cell line: high yield of infective virus from cells inoculated at high multiplicity. Virology, 39, 130-134. Conti, G., Portincasa, P., Pesce, A. & Chezzi, C. (1985), Biological characterization of FPV, Ulster 73, replicatire cycle. Microbiologica, 8, 151-164. Cross, F.R., Garber, E.A., Pellman, D. & Hanafusa, H. (1984), A short sequence in the p60 src N-terminus is required for p60 src myristylation and membrane association and for cell transformation. Mol. Cell. Biol., 4, 1834-1842. Girish, J.K. & Ghosh, H.P. (1984), Role of fatty acid acylation of membrane glycoproteins. J. biol. Chem., 259, 4699-4701. Hay, A.J., Zambon, M.C., Wolstenholme, A.J., Skehel, J.J. & Smith, M.H. (1986), Molecular basis of resistence of influenza A viruses to amantadine. J. Antimicrob. Chemoter. 18 (suppl. B), 19-29. Inglis, S.C. (1978), The synthesis of influenza virus proteins. PhD Thesis, University of Cambridge, UK. Klockmann, U. & Deppert, W. (1985), Evidence for transmembrane orientation of acylated simian virus 40 large T antigen. J. Virol., 56, 541-548.
ET AL.
Madeley, C.R., Allan, W.H. & Kendall, A.P. (1971), Studies with influenza A viruses: serological relations of the haemagglutinin and neuraminidase antigens of ten viruse isolates. J. gen. Virol., 12, 69-78. Moreman, K.W. & Touster, O. (1985), Biosynthesis and modification of Golgi mannosidase-2 in HeLa and 3T3 cells. J. biol. Chem., 260, 6654-6662. Oison, E.N., Towler, D.A. & Glaser, L. (1985), Specificity of fatty acid acylation of cellular proteins. J. biol. Chem., 260, 3784-3790. Pellman, D., Garber, E.A., Cross, F.R. & Hanafusa, H. (1985), Fine structural mapping of a critical NH2-terminal region of p60 src. Proc. nat. Acad. Sci. (Wash.), 82, 1623-1627. Portincasa, P., Conti, G. & Chezzi, C. (1990), Abortive replication of influenza A viruses in HeLa 229 cells. Virus Res., 18, 29-40. Portincasa, P., Conti, G. & Chezzi, C. (1991), Defective insertion of HA polypeptide as a cause of abortivity of influenza A viruses in HeLa 229 cells. Microbiologica, 14, 351-356. Schlesinger, M.J., Magee, A.I. & Schmidt, M.F.G. (1980), Fatty acid acylation of proteins in cultured cells. J. biol. Chem., 225, 10021-10024. Schlesinger, M.J. & Malfer, C. (1982), Cerulenin blocks fatty acid acylation of glycoproteins and inhibits vesicular stomatitis and Sindbis virus particle formation. J. biol. Chem., 257, 9887-9890. Schmidt, M.F.G., Bracha, M. & Schlesinger, M.J. (1979), Evidence for covalent attachment of fatty acid to Sindbis virus glycoproteins. Proc. nat. Acad. Sci. (Wash.), 76, 1687-1691. Schmidt, M.F.G. & Schlesinger, M.J. (1979), Fatty acid binding to vesicular stomatitis virus glycoprotein: a new type of post translational modification of the viral glycoprotein. Cell, 17, 813-819. Schmidt, M.F.G. (1982), Acylation of viral spike glycoproteins: a feature of enveloped RNA viruses. Virology, 116, 327-338. Schultz, A.M., Henderson, L.E., Oroszlan, S., Garber, E.A. & Hanafusa, H. (1985), Amino terminal myristoylation of the protein kinase p60 src, a retroviral transforming protein. Science, 227, 427429. Sugrue, R.J., Belshe, R.D. & Hay, A.J. (1990), Palmitoylation of the influenza A virus M2 protein. Virology, 179, 51-56. Zabedee, S.L. & Lamb, R.A. (1989), Growth restriction of influenza A virus by M2 protein antibody is genetically linked to the M 1 protein. Proc. nat. Acad. Sci. (Wash.), 86, 1061-1065. Zilberstein, A., Smider, M.D., Porter, M. & Lodish, M.F. (1980), Mutants o f vesicular stomatitis virus blocked at different stages in maturation of the viral glycoprotein. Cell, 21,417-427.
Res. ViroL 1992, 143, 401-406
Role of acylation of viral haemagglutinin during the influenza virus infectious cycle P. Portincasa, G. Conti t°) and C. Chezzi lstituto di Microbiologia, Universita" degfi Studi di Parma, Facolta" di Medicina e Chirurgia, Parma (Italy)
SUMMARY We investigated fatty acid residues bound to the haemagglutinin (HAl of type A influenza viruses by growing the viruses in permissive chick embryo fibroblasts (CEF) and in non-permissive HeLa-229 cells using a maintenance medium containing 3H-palmitic acid. Our results suggest that fatty acid acylation of the major viral glycoprotain may be an important prerequisite for the production of mature viral particles. Indeed, palmitoylation is found in infected CEF, but is completely lacking in non-permissive HeLa-229 cells infected by the same virus strains. We conclude that this type of post-translational modification of virus HA glycoprotein could be a general phenomenon regulating the maturation and budding of influenza virus. Key-words: Influenza virus, Acylation, Palmitic acid, Haemagglutinin; Glycoprotein, Viral maturation.
INTRODUCTION
Glycoproteins present in a number of enveloped viruses have been shown to contain palmitic acid residues covalently bound to polypeptides (Schmidt, 1982; Schmidt et al., 1979; Schmidt and Schlesinger, 1979; Klochmann and Deppert, 1985). This modification has also been described for viral proteins of influenza A viruses, such as the membrane M2 protein (Sugrue et al., 1990) and the precursor of the major structural protein of HIV1 (Bryant and Ratner, 1990). There is some evidence that acylation of virusencoded polypeptides and glycoproteins may be essential for the assembly and budding of the virus from the host cell and may play a possible
Submitted May 14, 1992, accepted October 26, 1992. (*) To whom reprint requests should be addressed.
role in intracellular protein transport (Cross et al., 1984; Moreman and Touster, 1985; Olson et al., 1985; Pellman et al., 1985; Schlesinger and Malfer, 1982 ; Schultz et al., 1985 ; Zilberstein et al., 1980). However, the necessity for this kind of post-translational modification and its significance, are not yet clear, since it does not occur in polypeptides of certain virus strains which are able to undergo productive multiplication in the cellular host (Sugrue et al., 1990; Girish and Ghosh, 1984). We recently reported that HeLa-229 cells do not permit the multiplication of type A influenza viruses (Portincasa et al., 1990) and that the non-permissive condition could be due to the inability of the haemagglutinin (HA) polypeptide
402
P. P O R TINCA SA E T A L .
to be correctly inserted into the plasma membrane (Portincasa et al., 1991). Since one of the most important events in virus assembly and propagation concerns protein modification, we investigated post-translational changes in the virus-encoded H A polypeptide by ascertaining the presence or absence of bound fatty acids in HeLa-229 cells infected by various type A influenza viruses.
MATERIALS AND METHODS
Fowl plague virus, Ulster strain (A, FPV, Ulster 73, H7N,), fowl plague virus, Rostock strain (A, FPV, Rostock, HTNI) and the Texas strain (A,H3N2) were grown in the allantoic cavity of 10-day-old embryonated hen eggs. After a 28-h incubation period at 37°C for avian Ulster and Rostock strains, and after 42-h incubation for the human Texas strain, the allantoic fluid was harvested, centrifuged at 2,000 rpm to separate cellular debris and titrated by haemagglutination with chicken red blood cells (Borland and Mahy, 1968) and the plaque assay. Monolayer cultures of mammalian HeLa-229 cells and chick embryo fibroblasts (CEF) were grown in Dulbecco's medium and minimal essential medium (Earle's MEM) respectively, both supplemented with 10 % foetal calf serum as described elsewhere (Choppin, 1969). To verify that viral HA correctly performed its cytoplasmic migration during influenza virus infection, experiments using 35S-methionine labelling were carried out in permissive CEF and nonpermissive HeLa-229 cells. Both cellular monolayers were infected with avian Rostock and Ulster strains as well as with the human Texas strain. Six hours post-infection (p.i.), infected monolayers were pulselabelled with 3SS-methionine (20 izCi/ml) and then processed by 15 % SDS-slab gel electrophoresis according to standard procedure (Inglis, 1978). In order to investigate the presence of covalently bound fatty acids to virus-encoded structural HA polypeptide, experiments with 3H-palmitic acid labelling were performed in non-permissive HeLa-229 and permissive CEF cells. Monolayers of confluent cells infected by type A influenza viruses were labelled with 50 }~Ci/ml of JH-palmitic acid (10-30 Ci/mmol)
CEF FPV HA
= = =
chick embryo fibroblast. fowl plague virus. haemasglutinin.
starting at 3 h after infection. Labelling of infected and non-infected HeLa-229 and CEF cells was halted by placing the Petri dishes in an ice-cold water bath; the infected cellular monolayers were then scraped off with a rubber teflon policeman and pelleted by centrifugation at 3,000 rpm for 5 min. Cellular pellets were then resuspended in a small volume of sterile water, sonicated 3 times for 30 s in an icecold water bath and centrifuged at 43,500 rpm in an "SW50L Beckman" rotor to separate cellular membranes. The supernatant fraction was discarded and the pellet was made soluble by adding lysis buffer (Schlesinger et al., 1980). Samples were loaded onto a 12.5 % SDS-polyacrylamide slab gel and electrophoresis was performed according to standard procedures (Inglis, 1978). The gel was then processed by fluorography with "En3Hance '' (NEN, DuPont) and a fluorogram was used to visualize labelled protein bands by exposure on X-ray "Omat" film autoradiography.
RESULTS Figure 1 shows the autoradiogram of 3Hpalmitic-acid-labelled cellular extracts of Ulsterinfected and mock-infected CEF and HeLa-229 cells. Comparison of virus-encoded proteins was made with 35S-methiordne-labelled polypeptides o f infected CEF cells. Only the cellular extracts of infected CEF cells evidenced comigration of 3H-labelled material with viral H A polypeptide. Comigration did not take place in uninfected CEF, in infected and mock-infected HeLa-229 cells. Data obtained from infection of such cellular hosts with Rostock and Texas strains were strictly comparable (data not shown). We performed an immunoprecipitation test using hyperimmune polyclonal antiserum to Ulster strain and virus-encoded H A glycoprotein (Madeley et al., 1971) to confirm the effective correspondence of 3H-labelled material w i t h viral HA, and to assess the absence of paimitoylation in infected HeLa 229 cells. Cellular extracts of infected C E F and H e L a 229 cells labelled with 3 H - p a l m i t i c acid were ira-
MEM = minimalessential p.i. = post-infection.
medium.
I N F L U E N Z A VIRUS C Y C L E A N D H A F A T T Y A C I D A C Y L A T I O N
munoprecipitated according to Schlesinger and Malfer (1982), and analysed by SDS-slab gel electrophoresis. As shown in figure 2, a 3H-labelled protein was recovered in infected CEF by immuno-
~"
"~~IC CEF
•'
403
precipitation assay; it corresponded to the HA polypeptide. In this case as well, 3H-labelled material was not present in infected HeLa 229 cells. It is reasonable to conclude that virusencoded HA of influenza viruses underwent
t
IC
II
I
m
'
" m
Fig. 2. Immunoprecipitation of labelled 3H-palmitic-acid Fig. I. SDS-polyacrylamide
slab gel electrophoresis (12.5 %) of 3H-palmitic-acid-labelled chick embryo fibroblasts (CEF) and HeLa-229 cells uninfected and infected by type A influenza virus F P V Ulster 73. Pulse labelling was performed from 3 to 8 h p.i. U C = uninfected cells; IC = infected cells. PB2, PBI and P A are subunits of virus-encoded RNA-dependent R N A polymerase; H A = viral haemagglutinin; N P represents viral nucleoprotein; M l is viralmembrane component and N S I is a virus-induced non-structural polypeptide.
HA glycoprotein from CEF and HeLa-229 cells unninfected and infected with influenza A virus FPV Ulster 73. Cell lysates were immunoprecipitated with anti-HA polyclonal serum and applied to a 12.5 % SDSpolyacrylamide slab gel electrophoresis. UC = uninfected cells; IC = infected cells. PB2, PBI and PA are subunits of virus-encoded RNA-dependent RNA polymerase; HA = viral haemagglutinin; NP represents viral nucleoprotein; MI is viral membrane component and NS1 is a virus-induced non-structural polypeptide.
404
P. P O R T I N C A S A E T A L .
post-translational modification by palmitoylation only during the productive infectious cycle in the permissive cellular host.
this cellular host, all virus-induced proteins were synthesized and, in addition, that migration o f virus-encoded H A was effective.
Experiments using 35S-methionine labelling p e r f o r m e d at a later time during infection were carried out in HeLa-229 cells to ensure that, in
As indicated in figure 3, the presence o f the HA1 glycoprotein in lane R corresponding to the polypeptides o f F P V Rostock, in both C E F and
...
R.U.T.
R.U.T.
PB|.. PBI,PA ~ ' " HA
, PA
. . . . .
NP HA1
N S'
.! X
NS2 III
II
i.c
• CEF
i
i
uc.
ic.
" HeL
a229
Fig. 3. SDS-polyacrylamide slab gel electrophoresis (17.5 %) of CEF and HeLa-229 cells infected by influenza A viruses FPV Rostock (R), FPV Ulster 73 (U) and Texas (T) strains, and pulse-labelled at 6 h p.i. using 3~S-methionine. PB2, PBl and PA are subunits of virus-coded RNA-dependent RNA polymerase; HA = viral haemagglutinin; HAl is the cleavage product of the FPV Rostock HA; NP represents viral nucleoprotein; Ml is viral membrane component; NS! and NS2 are virus-induced non-structural polypeptide.
INFLUENZA VIRUS C Y C L E A N D HA F A T T Y ACID A C Y L A T I O N
HeLa-229 infected cells, demonstrated that HeLa-229 cells permitted the migration of HA protein during virus strain multiplication.
DISCUSSION
Our results provide evidence that HA of the three strains of type A influenza virus is not acylated during the infectious cycle in HeLa-229 cells, while CEF infected by the same avian and human strains of influenza virus show acylation of viral HA. The infectious cycle in HeLa-229 is completely non-permissive, whereas CEF cells produce a great number of mature viral particles (Portincasa et al., 1990). We have recently established that HeLa-229 cells do not permit the multiplication of type A influenza viruses, and that the reason for this behaviour seems to be the result of incorrect insertion of viral HA in the cellular membrane during the final stages of viral infection. It is well known that the budding process requires the establishment of interactions between viral and cellular proteins, and that the plasma membrane of the infected cell must interact with influenza components during the maturation of viral particles (Zabedee and Lamb, 1989; Hay et al., 1986). Specific interactions are necessary for several steps during the late stages of infection, such as the insertion of viral HA into the cellular membrane and migration of this viral component through the cytoplasmic compartment. In fact, it is well known that viral HA is synthesized on the rough endoplasmic reticulum and then migrates to the plasma membrane via the smooth endoplasmic reticulum. During this migration, viral HA is cleaved by cellular proteolytic enzymes. In the present case, however, there is no evidence that inhibition of migration of HA is the defect shown by infected HeLa-229 cells, inasmuch as HA1, which represents the cleavage product of Rostock HA, is strongly present in the autoradiogram shown in figure 3. In the same electrophoretic pattern, the other two lines corresponding to FPV Ulster 73 03) and Texas
405
(T) strains do not show any HA1 product, since they do not possess cleaved HA (Conti et al., 1985). Moreover, the fact that HA1 of FPV Rostock is present in infected HeLa-229 cells clearly demonstrates that these cells permit the migration of this structural component through the cytoplasm and to the cellular surface. Despite this, since palmitoylation of viral HA is completely lacking in these cells and seems to be the unique defect (Portincasa et al., 1990), we hypothesize that the absence of this modification in viral glycoprotein might explain the non-permissive infection we observed. Palmitoylation could act as an anchor for the cellular membrane or allow HA insertion into the plasma membrane. In fact, results of haemadsorption obtained in HeLa-229 infected by these influenza virus strains suggest that the lack of mature viral particles could be due to abnormal insertion of HA into the cellular membrane (Portincasa et al., 1991). The inability to cross the lipid bilayer of the infected cells could be explained by the failure of post-translational modification of this virus-coded glycoprotein. In this respect, fatty acid acylation of HA protein might be the pre-requisite for this specific step during maturation and budding of influenza virus from HeLa-229, even if' another possible function of the fatty acid residue must be taken into account: palmitoylation might, in fact, provide a signal for the interaction of the HA glycoprotein with the viral nucleocapsid at specific sites on the plasma membrane. Although the function of acylated components is intriguing, it is reasonable to conclude that acylation of viral glycoprotein may play an important role in the assembly and maturation of influenza viral particles. Experiments involving the use of specific inhibitors of protein acylation are presently being conducted on the permissive cellular system to better understand the role of palmitoylation of viral HA during the influenza virus multiplication cycle.
Acknowledgements This studywas supportedby a grant fromthe Ministero Pubblica Istruzione(60 %) of the ItalianGovernment1990.
406
P. P O R T I N C A S A
R61e de racylation de I'h~magglutinine virale dans le cycle infectieux du virus grippal Dans le present travail effectu6 sur des fibroblastes d'embryon de poulet et sur des cellules H e L a (non permissives) dans un milieu contenant de l'acide palmitique, nous avons observ6 que l'acylation de cet acide gras li~/L la prot6ine de l'h~magglutinine du virus grippal peut apporter une modification d6terminante pour la maturation et la production des particules virales. Mots-cl~s: Virus grippal, Acylation, Acide palmitique, H~magglutinine; Glycoprot~ine, Maturation virale.
References Borland, R. & Mahy, B.W.J. (1968), Deoxyribonucleic acid-dependent RNA polymerase activity in cells infected with influenza virus. J. Virol., 2, 33-39. Bryant, M. & Ratner, L. (1990), Myristoylation-dependent replication and assembly of human immunodeficiency virus-l. Proc. nat. Acad. Sci. (Wash.), 87, 523-527. Choppin, P.W. (1969), Replication of influenza virus in a continuous cell line: high yield of infective virus from cells inoculated at high multiplicity. Virology, 39, 130-134. Conti, G., Portincasa, P., Pesce, A. & Chezzi, C. (1985), Biological characterization of FPV, Ulster 73, replicatire cycle. Microbiologica, 8, 151-164. Cross, F.R., Garber, E.A., Pellman, D. & Hanafusa, H. (1984), A short sequence in the p60 src N-terminus is required for p60 src myristylation and membrane association and for cell transformation. Mol. Cell. Biol., 4, 1834-1842. Girish, J.K. & Ghosh, H.P. (1984), Role of fatty acid acylation of membrane glycoproteins. J. biol. Chem., 259, 4699-4701. Hay, A.J., Zambon, M.C., Wolstenholme, A.J., Skehel, J.J. & Smith, M.H. (1986), Molecular basis of resistence of influenza A viruses to amantadine. J. Antimicrob. Chemoter. 18 (suppl. B), 19-29. Inglis, S.C. (1978), The synthesis of influenza virus proteins. PhD Thesis, University of Cambridge, UK. Klockmann, U. & Deppert, W. (1985), Evidence for transmembrane orientation of acylated simian virus 40 large T antigen. J. Virol., 56, 541-548.
ET AL.
Madeley, C.R., Allan, W.H. & Kendall, A.P. (1971), Studies with influenza A viruses: serological relations of the haemagglutinin and neuraminidase antigens of ten viruse isolates. J. gen. Virol., 12, 69-78. Moreman, K.W. & Touster, O. (1985), Biosynthesis and modification of Golgi mannosidase-2 in HeLa and 3T3 cells. J. biol. Chem., 260, 6654-6662. Oison, E.N., Towler, D.A. & Glaser, L. (1985), Specificity of fatty acid acylation of cellular proteins. J. biol. Chem., 260, 3784-3790. Pellman, D., Garber, E.A., Cross, F.R. & Hanafusa, H. (1985), Fine structural mapping of a critical NH2-terminal region of p60 src. Proc. nat. Acad. Sci. (Wash.), 82, 1623-1627. Portincasa, P., Conti, G. & Chezzi, C. (1990), Abortive replication of influenza A viruses in HeLa 229 cells. Virus Res., 18, 29-40. Portincasa, P., Conti, G. & Chezzi, C. (1991), Defective insertion of HA polypeptide as a cause of abortivity of influenza A viruses in HeLa 229 cells. Microbiologica, 14, 351-356. Schlesinger, M.J., Magee, A.I. & Schmidt, M.F.G. (1980), Fatty acid acylation of proteins in cultured cells. J. biol. Chem., 225, 10021-10024. Schlesinger, M.J. & Malfer, C. (1982), Cerulenin blocks fatty acid acylation of glycoproteins and inhibits vesicular stomatitis and Sindbis virus particle formation. J. biol. Chem., 257, 9887-9890. Schmidt, M.F.G., Bracha, M. & Schlesinger, M.J. (1979), Evidence for covalent attachment of fatty acid to Sindbis virus glycoproteins. Proc. nat. Acad. Sci. (Wash.), 76, 1687-1691. Schmidt, M.F.G. & Schlesinger, M.J. (1979), Fatty acid binding to vesicular stomatitis virus glycoprotein: a new type of post translational modification of the viral glycoprotein. Cell, 17, 813-819. Schmidt, M.F.G. (1982), Acylation of viral spike glycoproteins: a feature of enveloped RNA viruses. Virology, 116, 327-338. Schultz, A.M., Henderson, L.E., Oroszlan, S., Garber, E.A. & Hanafusa, H. (1985), Amino terminal myristoylation of the protein kinase p60 src, a retroviral transforming protein. Science, 227, 427429. Sugrue, R.J., Belshe, R.D. & Hay, A.J. (1990), Palmitoylation of the influenza A virus M2 protein. Virology, 179, 51-56. Zabedee, S.L. & Lamb, R.A. (1989), Growth restriction of influenza A virus by M2 protein antibody is genetically linked to the M 1 protein. Proc. nat. Acad. Sci. (Wash.), 86, 1061-1065. Zilberstein, A., Smider, M.D., Porter, M. & Lodish, M.F. (1980), Mutants o f vesicular stomatitis virus blocked at different stages in maturation of the viral glycoprotein. Cell, 21,417-427.
