Infection of chick-embryo fibroblasts and other ceils by certain animal viruses results in alterations in the intracellular concentrations of Na + and K +. Dramatic alterations in monovalent-cation concentrations of lytic-virus-infected ceils may favor the synthesis of viral proteins over cellular proteins. More subtle alterations in retrovirus-transformed cells may result in the expression of many morphological and biochemical changes associated with the transformed phenotype.Public Health Service Grant CA 26169, CA 27003National Cancer Institute CA 06593Integrative Biolog

Bose, Henry R.

Garry, Robert F.

Ulug, Emin T.

Bioscience Reports

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Bioscience Reports 2, 617-62Z (1982) 617 Printed in Great Britain Membrane-mediated  a l te ra t ions  of i n t race l lu la r  Na + and K + in l y t i c -v i rus - in fec ted  and re t rov i rus - t rans formed cel ls  Robert F. GARRY, Emin T. ULUG, and Henry R. BOSE, Jr. The University of Texas at Austin, Austin, Texas 78712-1095, U.S.A. (Received 12 July 1982) Infection of chick-embryo fibroblasts and other ceils by cer ta in  animal viruses results in alterations in the intracellular concentrations of Na + and K +. Dramatic a l te ra t ions  in monova lent -cat ion  concentrat ions of ly t i c -v i rus - in fected  ceils may favor the synthesis of viral  proteins over cel lu lar  proteins. More subtle alterations in retrovirus-transformed cells may result in the expression of many morphological and biochemical changes associated with the transformed phenotype. E luc idat ion of the mechanisms by which viruses influence cell- spec i f ied macromolecu lar  synthesis is one of the most intensely pursued goals of molecular biology. Lytic viruses which induce the death of the host cell generally do so by subordination of host- spec i f ied macromolecu lar  synthesis. Lytic viruses may have two effects on protein synthesis in the infected cell (192) which may not occur simultaneously. One effect is a reduction in the overall rate of protein synthesis, which may be nonselective. Virus- and cell-specified proteins are synthesized at a reduced rate in the infected cell. A second ef fect  is a selective inhibition of cellular protein synthesis and a switchover from cell- to virus-specified protein synthesis. Both the decl ine in the overall rate of protein synthesis and the selective inhibit ion of host-specified protein synthesis appear to involve the initiation step of protein synthesis. Host mRNAs are not degraded in the lytic-virus-infected cell and are functional in in vitro protein- synthes iz ing systems (2-5).  Host and viral mRNAs must have a distinguishing feature to account for the selective translation of viral mRNA in the infected cell. Discrimination between host and virus mRNA translation may involve an alteration in a component(s) of the initiation complex. Transforming viruses, such as retroviruses, generally do not inhibit host macromolecular synthesis, but they induce the expression of a new set  of phenotyp ic  character i s t i cs  te rmed t rans format ion  parameters. Retroviruses contain genes, oncogenes, which are believed to encode proteins responsible for phenotypically altering the trans- formed cell (6). The mechanism by which these proteins induce cell transformation is unknown. Results summarized here suggest that both lyt ic  and t rans forming viruses may employ a common strategy, alteration of the intracellular Na + and K + concentrations, to affect cellular gene expression. 01982 The Biochemical Society 618 GARRY ET AL. Methods  Methods  for measurement  of ce l l -  and virus-specif ied macro- mo lecu lar  synthesis9 determinat ion  of in t race l lu la r  Na + and K + concent ra t ions9  and determinat ion  of host po lysome profi les in chick-embryo fibroblasts and BHK ceils have been described previously (7 -9) .  Interferon assays and assays of the levels of antiviral proteins have also been described (g). The evaluation of retrovirus- induced transformat ion parameters was performed as detai led in Garry et al. (IO). Resu l ts  and D iscuss ion  Effect of altered Na + media on cell- and virus-specified protein synthesis The intracel lular concentrat ion of Na + is low and the intracel lular concent ra t ion  of K + is high relat ive to the concentrat ions in the extracel lu lar  medium ( i f ) .  Koch  and colleagues (12) demonstrated that  incubat ion  of uninfected ceils in medium containing elevated concentrat ions of Na + (among other t reatments)  inhibits the initiation 9 of protein synthesis. Initiation of translation of cellular proteins is also inh ib i ted  by incubat ing  cel ls  in media containing decreased concent ra t ions  of Na + (798). Initiation of translation of cellular mRNAs is inhibited because incubation in high-Na § medium results in an increase in intracel lular Na + and incubation in low-Na + medium resu l ts  in a decrease  in intracel lular K + (7).  Protein synthesis specif ied by a var iety of d i f ferent viruses is much more resistant than ce l l - spec i f ied  prote in  synthesis to the initiation block induced by a l tered Na + medium (7-15) .  Virus-specified mRNAs are translated more  e f f i c ient ly  than ce l lu la r  mRNAs across a wider range of monovalent-cat ion concentrat ion in vitro. Na + is increased and the concent ra t ion  of K + decreased  by t reatment  with ouabain9 which inhibits the membrane-associated (Na+/K+)ATPase (9).  Consequently9 ouaba in  t reatment  inhib i ts  ce l lu la r  protein synthesis, but is less inhibitory to  protein synthesis specif ied by certain viruses (9916). Not all cell mRNAs are inhibited equally by lyt ic virus infection (Tab le  1). Nuss and Koch (17) showed that compared to other cellular proteins the synthesis of IgG in myeloma ceils was much more res i s tant  to the se lec t ive  inhibi t ion of cellular protein synthesis induced by vesicular stomatit is  virus (VSV) infection. Interferon and certa in heat-shock proteins are induced during lyt ic virus infections (891g; Garry9 Niesel9 and Bose~ submitted).  An excel lent correlat ion ex is ts  between the ability of a cellular protein to be synthesized dur ing ly t i c  virus infection and in cells with altered intracel lular concentrat ions of Na + and K +. The synthesis of IgG is much more resistant to hypertonic medium than the synthesis of most cellular proteins (19).  Fibroblast interferon synthesi% but not synthesis of the interferon- induced antiviral protein% is as resistant as the synthesis of v i rus -spec i f ied  proteins to altered Na + media (8) and to ouabain (20921). The synthesis of heat-shock proteins is also resistant to a l te red  Na § media  (22; Gar ry  9 Niesel9 and Bose9 submi t ted) .  Dif ferent ial  sensit ivity of the translation of certain lens proteins may play an important role in the formation of cataracts  (23).  MEMBRANE-MEDIATED ALTERATIONS OF Na + AND K + 619 Table i. Synthesis of cellular proteins under conditions which alter intracellular Na + and K + Numbers in parentheses are reference citations. Lytic virus Altered Na+ Protein infection medium Ouabain IgG resistant (17) Interferon resistant (8) Antiviral protein Heat-shock proteins High-mol.-wt. 6-crystalin not tested Low-mol.-wt. ~-crystalin not tested resistant (19) resistant (8) not tested resistant (20,21) sensitive (8) sensitive (8) sensitive (20,21) resistant (22;*) resistant (22;*) not tested not tested not tested resistant (23) sensitive (23) *Garry et al., submitted. Effect  of lyt ic viruses on intracel lu lar  Na + and K + concentrat ions In cells lyt ical ly infected with Sindbis virus (SB), an alphavirus, or VSV, a rhabdovirus, the intracel lular Na + concentrat ion increases ( from 20 mM to over 60 mM) while the intracel lular concentrat ion of K + decreases  ( f rom 150 mM to under  60 mM) (Tab le  2) (7 ,g ) .  Alterat ions of intracellular ion concentrat ions also develop in cells infected by encephalomyocardit is  virus (EMC),  a picornavirus (Table 2) (24). The changes in intracel lular ion concentrat ions found in these lyt ic-v irus- infected cells are of suff ic ient magnitude to inhibit the translat ion of most host mRNAs. Translation of virus-specif ied mRNAs is unaffected or even st imulated by these changes. In SB- and VSV-infected cells the alterat ion in intracel lular ion concentrat ion,  the reduction in the overall rate of protein synthesis, and the select ive inhibition of host protein synthesis occur simultaneously. In EMC- in fec ted  ce l ls  an alterat ion in intracel lular ion concentrat ion also occurs simultaneously with the overall reduction in the rate of protein synthes is .  The se lec t ive  inh ib i t ion of host protein synthesis in EMC- infected cells, however, occurs later in infect ion at a t ime when viral mRNAs accumulate.  Manipulating the concentrat ion of salts in the medium, which presumably restores the intracel lular concentrat ion of Na + to the levels found in uninfected cells, reverses the select ive inh ib i t ion  of ce l l -speci f ied protein synthesis in EMC-infected cells (25). These results suggest that the reduction in overall rate of protein synthesis observed in cells infected with SB, VSV, and EMC may be the result of the alterat ion in the intracel lular ion concen- trations. Alterations in the intracel lular ion concentrat ions may also be required to select ively inhibit host-specif ied protein synthesis. This e f fec t  requires the accumulation of viral mRNAs which may compete 620 GARRY ET AL. Table 2. Effects of lytic RNA-virus infections on intracellular Na + and K + levels and protein synthesis Numbers in parentheses are reference citations. Virus/cell Alteration in Decline in Selective intracellular overall rate inhibition of Na + and/or K + of protein host protein levels synthesis synthesis Sindbis virus/ CE cells (9) 2 h Vesicular stomatitis virus/CE cells (8) 3 h Encephalomyocarditis virus/L cells (24) 3 h 2h  2h  3h  3h  3h  4h  with the host mRNAs for a protein synthesis factor that is sensitive to the altered ionic environment. Alterations in the intracellular concentrations of Na + and K + have also been documented in cells infected with SV40 (26), adenovirus (5), and, at a late stage of in fect ion ,  with pol iovirus (27; A. Schaefer, R. Zibirre, P. Kabus, 3. Kuhne, and G. Koch, personal communication, and ref. 2g), but may not occur during infections by other viruses (29). Effect of Sindbis virus infection on monovalent cation transport Carrasco (30) proposed that an increase in the permeability of lyric-virus-infected cells to Na + results in an increase in intracellular Na + and a se lec t ive  te rminat ion  of host prote in synthesis.  Intracellular concentrations of Na + and K + are maintained by a number of act ive  and passive ion- t ransport  systems (11,31). We have examined ion transport and permeability in cells infected by Sindbis virus (Ulug and Bose, in preparation). Sindbis virus infection results in a 40% inhibit ion of the membrane-associated (Na+/K+)ATPase (sodium pump) act iv i ty  as determined by ouabain-sensitive influx experiments utilizing the K + tracer 86Rb. The furosemide-sensitive Na+/K+/CI - cotransport system (31) is not inhibited during Sindbis virus in fect ion.  Fur ther  ev idence that  the (Na+/K+)ATPase is inhibited is provided by a paral lel  decrease  in the binding of [3H]ouabain to SB-infected cells. Likewise, there is a substantial decrease  in the (Na+/K+)ATPase act iv i ty  of plasma membrane f ract ions  obtained from SB-infected ceils. Moreover, we find no evidence for increased permeability or leakage of monovalent ions. The changes in (Na+/K+)ATPase activity are temporally correlated with the selective inhibition of cellular protein synthesis. Thus, while the intracellular concentration of Na + increases (and the concentration of K + decreases) during Sindbis virus infection these changes are due to an inhibit ion o f  the (Na+/K+)ATPase activity and not to an increase in nonspecific membrane permeability. MEMBRANE-MEDIATED ALTERATIONS OF Na + AND K + 621 Na + and K + retrov i rus  t ransformat ion One of the earliest events after stimulation of lymphocytes by mitogens is the activation of the sodium pump (32). Nerve growth factor also activates the sodium pump (S. Varon, personal communica- t ion),  and increased fluxes of monovalent ions are an early event in rowth stimulation induced in cultured cells by the addition of serum 3,34). Transformation by retroviruses also results in alterations in the growth propert ies of animal cells. Chick-embryo (CE) cells transformed by the Bryan (B) and Schmidt-Ruppin (SR) strains of Rous sarcoma virus (RSV) exhibit strain-specific changes in their intracellular Na + and K + concentrations (10,35,36). The magnitudes of these changes are less or the directions are different from the changes in ion concentrations in lyt ic-virus-infected cells. Lowering the Na + concentration of the medium in which normal chick-cell cultures are incubated causes them to enlarge, vacuolate, and appear morpho- logically similar to cells transformed by B-RSV ( I0 ) .  Raising the Na + concent rat ion  of the medium causes cel ls to become round or spindle-shaped and to appear morphologically similar to cells trans- formed by SR-RSV. Uninfected chick-cell cultures incubated in either low-  or h igh-Na + medium also express many other phenotypic characteristics of transformed cells. They lose contact inhibition of growth and movement, grow to higher saturation densities, synthesize reduced amounts of fibronectin, exhibit increased lectin agglutinability, transport increased amounts of hexoses, have reduced levels of succinic dehydrogenase act iv i ty ,  produce increased amounts of lactate and pyruvate, and can be serially passaged in Culture signif icantly longer than normal chick fibroblasts. Chick cells incubated in altered Na media do not exh ib i t  all transformation parameters expressed by RSV-transformed ceils. The ceils did not grow in soft agar or in low-serum medium.  These results identi fy a possible new trans-  fo rmat ion  parameter ,  a l te ra t ion  of the intracel lular Na + and K + concent ra t ions ,  and suggest that many, but not all, t ransformat ion parameters  may be consequences  of the  a l te red  in t race l lu la r  concentrat ions of monovalent cations. The al terat ions in intracel lular Na + and K + induced by RSV are dependent  on a funct ional  t ransforming gene: src. The src gene product,  pp60 src, possesses a protein kinase act iv i ty  with the unusual spec i f i c i ty  for  ty rosy l  res idues  (6) .  The mechanism uti l ized by pp60 src to alter intracel lular ion concentrat ions is unknown. Several ion-transport  systems,  such as the (Na+/K+)ATPase,  are known to be regulated by phosphorylation (5).  In addition, Baner jee and coworkers (37) reported that the level of [3H]ouabain-binding sites decreases in cells t ransformed by SR-RSV. Bader et al. (36) reported that  ceils in fec ted  by B-RSV have the same number of [3H]ouabain-binding s i tes /mg protein as control  ceils. Since B-RSV-transformed CE cells are larger and contain more protein than untransformed cells (38),  the number of [3H]ouabain-binding sites per cell is higher in the B-RSV- t ransformed CE cells. Acknowledgements This work was supported by Public Health Serv ice Grants CA 26169 622 GARRY ET AL. and CA 27003. R.F.G. was a postdoctoral  fel low of the National Cancer  Inst i tute (CA 06593). Re ferences  i. Wengler G (1980) in The Togaviruses (Schlesinger RW, ed), pp 459-472. 2. Jaye MC 9 Godchaux WI I I  & Lucas-Lenard J (1982) Virology 116, 142-162. 3. Carrasco L & Smith AE (1976) Nature (London) 264, 807-809. 4. Lodish HF & Porter M (1980) J. Virol. 369 719-733. 5. Cherney CS & Wilhelm JM (1979) J. Virol. 309 533-542. 6. Bishop JM (1982) Scientific American 2469 80-92. 7. Garry RF, Bishop JM, Parker S 9 Westbrook K, Lewis G & Waite MRF (1979) Virology 969 108-120. 8. Garry RF & Waite MRF (1979) Virology 96, 121-128. 9. Garry RF, Westbrook K & Waite MRF (1979) Virology 99~ 108-120. i0. Garry RF9 Moyer MP 9 Bishop JM, Moyer RC & Waite MRF (1981) Virology III, 427-439. ii. Dahl JL & Hokin LE (1974) Ann. Rev. Bioehem 439 327-356. 12. Saborio JL, Pong SS & Koch G (1974) J. Mol. Biol. 859 195-211. 13. Waite MRF & Pfefferkorn ER (1968) J. Virol. 29 759-760. 14. Oppermann H & Koch G (1976) Arch. Virol. 529 123-134. 15. Madore HP & England JM (1975) J. Virol. I09 1351-1354. 16. Nagai Y, Macho K, Linuma M, Yoshida T & Natsumoto T (1972) Virology 9, 234-243. 17. Nuss DL & Koch G (1976) J. Virol. 19, 572-578. 18. Peluso R, Lamb RA & Choppin PW (1978) Proc. Natl. Acad. Sci. U.S.A. 759 6120-6124. 19. Nuss DL & Koch G (1976) J. Mol. Biol. 102, 601-612. 20. Lebon P, Moreau MC, Cohen L & Chany C (1975) Proc. Soc. Exp. Biol. Med. 149, 108-112. 21. Kuwata T, Fuse A & Moringaga N (1977) J. Gen. Virol. 34, 537-540. 22. Hickey ED & Weber LA (1982) Biochemistry 219 1515-1521. 23. Piatigorsky J (1980) Curr. Top. in Lens Res. 3, 2-39. 24. Rhamabhadran TV & Thach RE (1981) J. Virol. 39, 573-583. 25. Alonso M & Carrasco A (1981) J. Virol. 379 535-540. 26. Eggleton KH & Norkin LC (1981) Virology II0, 73-86. 27. Nair CN (1981) J. Virol. 379 268-273. 28. Schaefer A, Zibirre R, Kabus P, Kuhne J & Koch G (1982) Biosci. Rep. 2, 613-615. 29. Pasternak CA, Gray MA & Micklem KJ (1982) Biosci. Rep. 2, 609-612. 30. Carrasco L (1977) FEBS Lett. 769 11-15. 31. Geck P, Pietrzyk C, Burckhardt B-C 9 Pfeiffer B & Heinz E (1980) Biochim. Biophys. Acta 600, 432-447. 32. Kaplan JG & Owens T (1982) Biosci. Rep. 2, 577-581. MEMBRANE-MEDIATED ALTERATIONS OF Na + AND K + 623 33. Rozengurt E & Heppel LA (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 4492-4495. 34. Koch KS & Leffert HL (1979) Cell 18~ 153-163. 35. Johnson MA & Weber MJ (1979) J. Cell Phys. i01~ 89-100. 36. Bader JP9 Okazaki T & Brown NR (1981) J. Cell Phys. I06~ 235-242. 37. Banerjee SP, Bosmann HB & Morgan HR (1977) Exp. Cell Res. 104, 111-117. 38. Bader JP~ Ray DA & Brown NR (1974) Cel l  3, 307-313. 

Membrane-Mediated Alterations of Intracellular Na+ and K+ in Lytic-Virus-Infected and Retrovirus-Transformed Cells

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Membrane-Mediated Alterations of Intracellular Na+ and K+ in Lytic-Virus-Infected and Retrovirus-Transformed Cells

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