Abstract
The yeast interaction trap system was used to identify, NS1-I (for NS1 interactor), which is a human protein that binds to the nonstructural NS1 protein of the influenza A virus. NS1-I is a human homolog of the porcine 17beta-estradiol dehydrogenase precursor protein, to which it is 84% identical. We detected only one NS1-I mRNA species, of about 3.0 kb, in HeLa cells, and the NS1-I cDNA was found to have a coding capacity for a 79.6-kDa protein. However, immunoblot analysis detected predominantly a 55-kDa protein in human cells, suggesting that NS1-I, like the porcine 17beta-estradiol dehydrogenase, is posttranslationally processed. Using an in vitro coprecipitation assay, we showed that NS1-I interacts with NS1 proteins from extracts of cells infected with five different influenza A virus strains as well as with the NS1 of an influenza B virus. The fact that influenza A and influenza B virus NS1 proteins bind to NS1-I suggests that this cellular protein plays a role in the influenza virus life cycle.
Full Text
The Full Text of this article is available as a PDF (536.9 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Adamski J., Husen B., Marks F., Jungblut P. W. Purification and properties of oestradiol 17 beta-dehydrogenase extracted from cytoplasmic vesicles of porcine endometrial cells. Biochem J. 1992 Dec 1;288(Pt 2):375–381. doi: 10.1042/bj2880375. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Adamski J., Normand T., Leenders F., Monté D., Begue A., Stéhelin D., Jungblut P. W., de Launoit Y. Molecular cloning of a novel widely expressed human 80 kDa 17 beta-hydroxysteroid dehydrogenase IV. Biochem J. 1995 Oct 15;311(Pt 2):437–443. doi: 10.1042/bj3110437. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Baez M., Zazra J. J., Elliott R. M., Young J. F., Palese P. Nucleotide sequence of the influenza A/duck/Alberta/60/76 virus NS RNA: conservation of the NS1/NS2 overlapping gene structure in a divergent influenza virus RNA segment. Virology. 1981 Aug;113(1):397–402. doi: 10.1016/0042-6822(81)90166-5. [DOI] [PubMed] [Google Scholar]
- Bartel P., Chien C. T., Sternglanz R., Fields S. Elimination of false positives that arise in using the two-hybrid system. Biotechniques. 1993 Jun;14(6):920–924. [PubMed] [Google Scholar]
- Buonagurio D. A., Nakada S., Parvin J. D., Krystal M., Palese P., Fitch W. M. Evolution of human influenza A viruses over 50 years: rapid, uniform rate of change in NS gene. Science. 1986 May 23;232(4753):980–982. doi: 10.1126/science.2939560. [DOI] [PubMed] [Google Scholar]
- Chen Z., Jiang J. C., Lin Z. G., Lee W. R., Baker M. E., Chang S. H. Site-specific mutagenesis of Drosophila alcohol dehydrogenase: evidence for involvement of tyrosine-152 and lysine-156 in catalysis. Biochemistry. 1993 Apr 6;32(13):3342–3346. doi: 10.1021/bi00064a017. [DOI] [PubMed] [Google Scholar]
- Chien C. T., Bartel P. L., Sternglanz R., Fields S. The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9578–9582. doi: 10.1073/pnas.88.21.9578. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Compans R. W. Influenza virus proteins. II. Association with components of the cytoplasm. Virology. 1973 Jan;51(1):56–70. doi: 10.1016/0042-6822(73)90365-6. [DOI] [PubMed] [Google Scholar]
- Enami K., Sato T. A., Nakada S., Enami M. Influenza virus NS1 protein stimulates translation of the M1 protein. J Virol. 1994 Mar;68(3):1432–1437. doi: 10.1128/jvi.68.3.1432-1437.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fields S., Sternglanz R. The two-hybrid system: an assay for protein-protein interactions. Trends Genet. 1994 Aug;10(8):286–292. doi: 10.1016/0168-9525(90)90012-u. [DOI] [PubMed] [Google Scholar]
- Fortes P., Beloso A., Ortín J. Influenza virus NS1 protein inhibits pre-mRNA splicing and blocks mRNA nucleocytoplasmic transport. EMBO J. 1994 Feb 1;13(3):704–712. doi: 10.1002/j.1460-2075.1994.tb06310.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Frohman M. A., Dush M. K., Martin G. R. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8998–9002. doi: 10.1073/pnas.85.23.8998. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Garfinkel M. S., Katze M. G. How does influenza virus regulate gene expression at the level of mRNA translation? Let us count the ways. Gene Expr. 1993;3(2):109–118. [PMC free article] [PubMed] [Google Scholar]
- Goebel S. J., Johnson G. P., Perkus M. E., Davis S. W., Winslow J. P., Paoletti E. The complete DNA sequence of vaccinia virus. Virology. 1990 Nov;179(1):247-66, 517-63. doi: 10.1016/0042-6822(90)90294-2. [DOI] [PubMed] [Google Scholar]
- Greenspan D., Palese P., Krystal M. Two nuclear location signals in the influenza virus NS1 nonstructural protein. J Virol. 1988 Aug;62(8):3020–3026. doi: 10.1128/jvi.62.8.3020-3026.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gyuris J., Golemis E., Chertkov H., Brent R. Cdi1, a human G1 and S phase protein phosphatase that associates with Cdk2. Cell. 1993 Nov 19;75(4):791–803. doi: 10.1016/0092-8674(93)90498-f. [DOI] [PubMed] [Google Scholar]
- Hatada E., Fukuda R. Binding of influenza A virus NS1 protein to dsRNA in vitro. J Gen Virol. 1992 Dec;73(Pt 12):3325–3329. doi: 10.1099/0022-1317-73-12-3325. [DOI] [PubMed] [Google Scholar]
- Hatada E., Hasegawa M., Shimizu K., Hatanaka M., Fukuda R. Analysis of influenza A virus temperature-sensitive mutants with mutations in RNA segment 8. J Gen Virol. 1990 Jun;71(Pt 6):1283–1292. doi: 10.1099/0022-1317-71-6-1283. [DOI] [PubMed] [Google Scholar]
- Hatada E., Takizawa T., Fukuda R. Specific binding of influenza A virus NS1 protein to the virus minus-sense RNA in vitro. J Gen Virol. 1992 Jan;73(Pt 1):17–25. doi: 10.1099/0022-1317-73-1-17. [DOI] [PubMed] [Google Scholar]
- Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- KILBOURNE E. D., SMART K. M., POKORNY B. A. Inhibition by cortisone of the synthesis and action of interferon. Nature. 1961 May 13;190:650–651. doi: 10.1038/190650a0. [DOI] [PubMed] [Google Scholar]
- Koennecke I., Boschek C. B., Scholtissek C. Isolation and properties of a temperature-sensitive mutant (ts 412) of an influenza A virus recombinant with a ts lesion in the gene coding for the nonstructural protein. Virology. 1981 Apr 15;110(1):16–25. doi: 10.1016/0042-6822(81)90003-9. [DOI] [PubMed] [Google Scholar]
- Kozak M. The scanning model for translation: an update. J Cell Biol. 1989 Feb;108(2):229–241. doi: 10.1083/jcb.108.2.229. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Krug R. M., Etkind P. R. Cytoplasmic and nuclear virus-specific proteins in influenza virus-infected MDCK cells. Virology. 1973 Nov;56(1):334–348. doi: 10.1016/0042-6822(73)90310-3. [DOI] [PubMed] [Google Scholar]
- Leenders F., Adamski J., Husen B., Thole H. H., Jungblut P. W. Molecular cloning and amino acid sequence of the porcine 17 beta-estradiol dehydrogenase. Eur J Biochem. 1994 May 15;222(1):221–227. doi: 10.1111/j.1432-1033.1994.tb18860.x. [DOI] [PubMed] [Google Scholar]
- Leenders F., Husen B., Thole H. H., Adamski J. The sequence of porcine 80 kDa 17 beta-estradiol dehydrogenase reveals similarities to the short chain alcohol dehydrogenase family, to actin binding motifs and to sterol carrier protein 2. Mol Cell Endocrinol. 1994 Sep;104(2):127–131. doi: 10.1016/0303-7207(94)90114-7. [DOI] [PubMed] [Google Scholar]
- Lu Y., Qian X. Y., Krug R. M. The influenza virus NS1 protein: a novel inhibitor of pre-mRNA splicing. Genes Dev. 1994 Aug 1;8(15):1817–1828. doi: 10.1101/gad.8.15.1817. [DOI] [PubMed] [Google Scholar]
- Lu Y., Wambach M., Katze M. G., Krug R. M. Binding of the influenza virus NS1 protein to double-stranded RNA inhibits the activation of the protein kinase that phosphorylates the elF-2 translation initiation factor. Virology. 1995 Dec 1;214(1):222–228. doi: 10.1006/viro.1995.9937. [DOI] [PubMed] [Google Scholar]
- Ludwig S., Schultz U., Mandler J., Fitch W. M., Scholtissek C. Phylogenetic relationship of the nonstructural (NS) genes of influenza A viruses. Virology. 1991 Aug;183(2):566–577. doi: 10.1016/0042-6822(91)90985-k. [DOI] [PubMed] [Google Scholar]
- Ludwig S., Vogel U., Scholtissek C. Amino acid replacements leading to temperature-sensitive defects of the NS1 protein of influenza A virus. Arch Virol. 1995;140(5):945–950. doi: 10.1007/BF01314970. [DOI] [PubMed] [Google Scholar]
- Moore J. B., Smith G. L. Steroid hormone synthesis by a vaccinia enzyme: a new type of virus virulence factor. EMBO J. 1992 May;11(5):1973–1980. doi: 10.1002/j.1460-2075.1992.tb05251.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Norton G. P., Tanaka T., Tobita K., Nakada S., Buonagurio D. A., Greenspan D., Krystal M., Palese P. Infectious influenza A and B virus variants with long carboxyl terminal deletions in the NS1 polypeptides. Virology. 1987 Feb;156(2):204–213. doi: 10.1016/0042-6822(87)90399-0. [DOI] [PubMed] [Google Scholar]
- Nuttley W. M., Aitchison J. D., Rachubinski R. A. cDNA cloning and primary structure determination of the peroxisomal trifunctional enzyme hydratase-dehydrogenase-epimerase from the yeast Candida tropicalis pK233. Gene. 1988 Sep 30;69(2):171–180. doi: 10.1016/0378-1119(88)90428-3. [DOI] [PubMed] [Google Scholar]
- O'Neill R. E., Palese P. NPI-1, the human homolog of SRP-1, interacts with influenza virus nucleoprotein. Virology. 1995 Jan 10;206(1):116–125. doi: 10.1016/s0042-6822(95)80026-3. [DOI] [PubMed] [Google Scholar]
- Persson B., Krook M., Jörnvall H. Characteristics of short-chain alcohol dehydrogenases and related enzymes. Eur J Biochem. 1991 Sep 1;200(2):537–543. doi: 10.1111/j.1432-1033.1991.tb16215.x. [DOI] [PubMed] [Google Scholar]
- Qiu Y., Krug R. M. The influenza virus NS1 protein is a poly(A)-binding protein that inhibits nuclear export of mRNAs containing poly(A). J Virol. 1994 Apr;68(4):2425–2432. doi: 10.1128/jvi.68.4.2425-2432.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Qiu Y., Nemeroff M., Krug R. M. The influenza virus NS1 protein binds to a specific region in human U6 snRNA and inhibits U6-U2 and U6-U4 snRNA interactions during splicing. RNA. 1995 May;1(3):304–316. [PMC free article] [PubMed] [Google Scholar]
- Santos M. J., Imanaka T., Shio H., Small G. M., Lazarow P. B. Peroxisomal membrane ghosts in Zellweger syndrome--aberrant organelle assembly. Science. 1988 Mar 25;239(4847):1536–1538. doi: 10.1126/science.3281254. [DOI] [PubMed] [Google Scholar]
- Shimizu K., Mullinix M. G., Chanock R. M., Murphy B. R. Temperature-sensitive mutants of influenza A/Udorn/72 (H3N2) virus. III. Genetic analysis of temperature-dependent host range mutants. Virology. 1983 Jan 15;124(1):35–44. doi: 10.1016/0042-6822(83)90288-x. [DOI] [PubMed] [Google Scholar]
- Skinner M. A., Moore J. B., Binns M. M., Smith G. L., Boursnell M. E. Deletion of fowlpox virus homologues of vaccinia virus genes between the 3 beta-hydroxysteroid dehydrogenase (A44L) and DNA ligase (A50R) genes. J Gen Virol. 1994 Sep;75(Pt 9):2495–2498. doi: 10.1099/0022-1317-75-9-2495. [DOI] [PubMed] [Google Scholar]
- Skorko R., Summers D. F., Galarza J. M. Influenza A virus in vitro transcription: roles of NS1 and NP proteins in regulating RNA synthesis. Virology. 1991 Feb;180(2):668–677. doi: 10.1016/0042-6822(91)90080-u. [DOI] [PubMed] [Google Scholar]
- Smart K. M., Kilbourne E. D. The influence of cortisone on experimental viral infection. VI. Inhibition by hydrocortisone of interferon synthesis in the chick embryo. J Exp Med. 1966 Feb 1;123(2):299–307. doi: 10.1084/jem.123.2.299. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Smart K. M., Kilbourne E. D. The influence of cortisone on experimental viral infection. VII. Kinetics of interferon formation and its inhibition with hydrocortisone in relation to viral strain and virulence. J Exp Med. 1966 Feb 1;123(2):309–325. doi: 10.1084/jem.123.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
- Snyder M. H., London W. T., Maassab H. F., Chanock R. M., Murphy B. R. A 36 nucleotide deletion mutation in the coding region of the NS1 gene of an influenza A virus RNA segment 8 specifies a temperature-dependent host range phenotype. Virus Res. 1990 Jan;15(1):69–83. doi: 10.1016/0168-1702(90)90014-3. [DOI] [PubMed] [Google Scholar]
- Treanor J. J., Snyder M. H., London W. T., Murphy B. R. The B allele of the NS gene of avian influenza viruses, but not the A allele, attenuates a human influenza A virus for squirrel monkeys. Virology. 1989 Jul;171(1):1–9. doi: 10.1016/0042-6822(89)90504-7. [DOI] [PubMed] [Google Scholar]
- Wolstenholme A. J., Barrett T., Nichol S. T., Mahy B. W. Influenza virus-specific RNA and protein syntheses in cells infected with temperature-sensitive mutants defective in the genome segment encoding nonstructural proteins. J Virol. 1980 Jul;35(1):1–7. doi: 10.1128/jvi.35.1.1-7.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zervos A. S., Gyuris J., Brent R. Mxi1, a protein that specifically interacts with Max to bind Myc-Max recognition sites. Cell. 1993 Jan 29;72(2):223–232. doi: 10.1016/0092-8674(93)90662-a. [DOI] [PubMed] [Google Scholar]
- de Hoop M. J., Ab G. Import of proteins into peroxisomes and other microbodies. Biochem J. 1992 Sep 15;286(Pt 3):657–669. doi: 10.1042/bj2860657. [DOI] [PMC free article] [PubMed] [Google Scholar]
- de la Luna S., Fortes P., Beloso A., Ortín J. Influenza virus NS1 protein enhances the rate of translation initiation of viral mRNAs. J Virol. 1995 Apr;69(4):2427–2433. doi: 10.1128/jvi.69.4.2427-2433.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]