• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of jvirolPermissionsJournals.ASM.orgJournalJV ArticleJournal InfoAuthorsReviewers
J Virol. Oct 1992; 66(10): 6045–6057.
PMCID: PMC241482

cis-acting lesions targeted to the hydrophobic domain of a poliovirus membrane protein involved in RNA replication.

Abstract

The structural requirements of the hydrophobic domain contained in poliovirus polypeptide 3AB were studied by using a molecular genetic approach in combination with an in vitro biochemical analysis. We report here the generation and analysis of deletion, insertion, and amino acid replacement mutations aimed at decreasing the hydrophobic character of the domain. Our results indicated that the hydrophobicity of this region of 3AB is necessary to maintain normal viral RNA synthesis. However, in vitro membrane association assays of the mutated proteins did not establish a direct correlation between 3AB membrane association and viral RNA synthesis. Some of the lethal mutations we engineered produced polyproteins with abnormal P2- and P3-processing capabilities due to an alteration in the normal cleavage order of the polyprotein. A detailed analysis of these mutants suggests that P2 is not the major precursor for polypeptides 2A and 2BC and that P2 protein products are derived from P2-P3-containing precursors (most likely P2-P3 or P2-3AB). Such precursors are likely to result from primary polyprotein cleavage events that initiate a proteolytic cascade not previously documented. Our results also indicated that the function provided by the hydrophobic domain of 3AB cannot be provided in trans. We discuss the implications of these results on the formation of limited-diffusion replication complexes as a means of sequestering P2- and P3-region polypeptides required for RNA synthesis and protein processing.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (3.2M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Anderson DJ, Mostov KE, Blobel G. Mechanisms of integration of de novo-synthesized polypeptides into membranes: signal-recognition particle is required for integration into microsomal membranes of calcium ATPase and of lens MP26 but not of cytochrome b5. Proc Natl Acad Sci U S A. 1983 Dec;80(23):7249–7253. [PMC free article] [PubMed]
  • Andino R, Rieckhof GE, Baltimore D. A functional ribonucleoprotein complex forms around the 5' end of poliovirus RNA. Cell. 1990 Oct 19;63(2):369–380. [PubMed]
  • Baron MH, Baltimore D. Anti-VPg antibody inhibition of the poliovirus replicase reaction and production of covalent complexes of VPg-related proteins and RNA. Cell. 1982 Oct;30(3):745–752. [PubMed]
  • Berstein HD, Baltimore D. Poliovirus mutant that contains a cold-sensitive defect in viral RNA synthesis. J Virol. 1988 Aug;62(8):2922–2928. [PMC free article] [PubMed]
  • Bernstein HD, Sarnow P, Baltimore D. Genetic complementation among poliovirus mutants derived from an infectious cDNA clone. J Virol. 1986 Dec;60(3):1040–1049. [PMC free article] [PubMed]
  • Bienz K, Egger D, Pasamontes L. Association of polioviral proteins of the P2 genomic region with the viral replication complex and virus-induced membrane synthesis as visualized by electron microscopic immunocytochemistry and autoradiography. Virology. 1987 Sep;160(1):220–226. [PubMed]
  • Bienz K, Egger D, Troxler M, Pasamontes L. Structural organization of poliovirus RNA replication is mediated by viral proteins of the P2 genomic region. J Virol. 1990 Mar;64(3):1156–1163. [PMC free article] [PubMed]
  • Blair WS, Semler BL. Role for the P4 amino acid residue in substrate utilization by the poliovirus 3CD proteinase. J Virol. 1991 Nov;65(11):6111–6123. [PMC free article] [PubMed]
  • Butterworth BE, Shimshick EJ, Yin FH. Association of the polioviral RNA polymerase complex with phospholipid membranes. J Virol. 1976 Aug;19(2):457–466. [PMC free article] [PubMed]
  • Caliguiri LA. Analysis of RNA associated with the poliovirus RNA replication complexes. Virology. 1974 Apr;58(2):526–535. [PubMed]
  • Caliguiri LA, Tamm I. The role of cytoplasmic membranes in poliovirus biosynthesis. Virology. 1970 Sep;42(1):100–111. [PubMed]
  • Charini WA, Burns CC, Ehrenfeld E, Semler BL. trans rescue of a mutant poliovirus RNA polymerase function. J Virol. 1991 May;65(5):2655–2665. [PMC free article] [PubMed]
  • Crawford NM, Baltimore D. Genome-linked protein VPg of poliovirus is present as free VPg and VPg-pUpU in poliovirus-infected cells. Proc Natl Acad Sci U S A. 1983 Dec;80(24):7452–7455. [PMC free article] [PubMed]
  • Flanegan JB, Baltimore D. Poliovirus-specific primer-dependent RNA polymerase able to copy poly(A). Proc Natl Acad Sci U S A. 1977 Sep;74(9):3677–3680. [PMC free article] [PubMed]
  • Flanegan JB, Baltimore D. Poliovirus polyuridylic acid polymerase and RNA replicase have the same viral polypeptide. J Virol. 1979 Jan;29(1):352–360. [PMC free article] [PubMed]
  • Giachetti C, Semler BL. Role of a viral membrane polypeptide in strand-specific initiation of poliovirus RNA synthesis. J Virol. 1991 May;65(5):2647–2654. [PMC free article] [PubMed]
  • Gilmore R, Blobel G. Translocation of secretory proteins across the microsomal membrane occurs through an environment accessible to aqueous perturbants. Cell. 1985 Sep;42(2):497–505. [PubMed]
  • Graham FL, van der Eb AJ. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. [PubMed]
  • Jackson RJ. A detailed kinetic analysis of the in vitro synthesis and processing of encephalomyocarditis virus products. Virology. 1986 Feb;149(1):114–127. [PubMed]
  • Jia XY, Ehrenfeld E, Summers DF. Proteolytic activity of hepatitis A virus 3C protein. J Virol. 1991 May;65(5):2595–2600. [PMC free article] [PubMed]
  • Johnson KL, Sarnow P. Three poliovirus 2B mutants exhibit noncomplementable defects in viral RNA amplification and display dosage-dependent dominance over wild-type poliovirus. J Virol. 1991 Aug;65(8):4341–4349. [PMC free article] [PubMed]
  • Johnson VH, Semler BL. Defined recombinants of poliovirus and coxsackievirus: sequence-specific deletions and functional substitutions in the 5'-noncoding regions of viral RNAs. Virology. 1988 Jan;162(1):47–57. [PubMed]
  • Kaplan G, Racaniello VR. Construction and characterization of poliovirus subgenomic replicons. J Virol. 1988 May;62(5):1687–1696. [PMC free article] [PubMed]
  • Klein P, Kanehisa M, DeLisi C. The detection and classification of membrane-spanning proteins. Biochim Biophys Acta. 1985 May 28;815(3):468–476. [PubMed]
  • Kuhn RJ, Tada H, Ypma-Wong MF, Semler BL, Wimmer E. Mutational analysis of the genome-linked protein VPg of poliovirus. J Virol. 1988 Nov;62(11):4207–4215. [PMC free article] [PubMed]
  • Kuhn RJ, Wimmer E, Semler BL. Expression of the poliovirus genome from infectious cDNA is dependent upon arrangements of eukaryotic and prokaryotic sequences in recombinant plasmids. Virology. 1987 Apr;157(2):560–564. [PubMed]
  • Kyte J, Doolittle RF. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • La Monica N, Meriam C, Racaniello VR. Mapping of sequences required for mouse neurovirulence of poliovirus type 2 Lansing. J Virol. 1986 Feb;57(2):515–525. [PMC free article] [PubMed]
  • Lawson MA, Semler BL. Picornavirus protein processing--enzymes, substrates, and genetic regulation. Curr Top Microbiol Immunol. 1990;161:49–87. [PubMed]
  • Li JP, Baltimore D. Isolation of poliovirus 2C mutants defective in viral RNA synthesis. J Virol. 1988 Nov;62(11):4016–4021. [PMC free article] [PubMed]
  • Lundquist RE, Maizel JV., Jr Structural studies of the RNA component of the poliovirus replication complex. I. Purification and biochemical characterization. Virology. 1978 Apr;85(2):434–444. [PubMed]
  • Morrow CD, Dasgupta A. Antibody to a synthetic nonapeptide corresponding to the NH2 terminus of poliovirus genome-linked protein VPg reacts with native VPg and inhibits in vitro replication of poliovirus RNA. J Virol. 1983 Nov;48(2):429–439. [PMC free article] [PubMed]
  • Morrow CD, Hocko J, Navab M, Dasgupta A. ATP is required for initiation of poliovirus RNA synthesis in vitro: demonstration of tyrosine-phosphate linkage between in vitro-synthesized RNA and genome-linked protein. J Virol. 1984 May;50(2):515–523. [PMC free article] [PubMed]
  • Mostov KE, DeFoor P, Fleischer S, Blobel G. Co-translational membrane integration of calcium pump protein without signal sequence cleavage. Nature. 1981 Jul 2;292(5818):87–88. [PubMed]
  • Nomoto A, Detjen B, Pozzatti R, Wimmer E. The location of the polio genome protein in viral RNAs and its implication for RNA synthesis. Nature. 1977 Jul 21;268(5617):208–213. [PubMed]
  • Pallai PV, Burkhardt F, Skoog M, Schreiner K, Bax P, Cohen KA, Hansen G, Palladino DE, Harris KS, Nicklin MJ, et al. Cleavage of synthetic peptides by purified poliovirus 3C proteinase. J Biol Chem. 1989 Jun 15;264(17):9738–9741. [PubMed]
  • Palmenberg AC. Proteolytic processing of picornaviral polyprotein. Annu Rev Microbiol. 1990;44:603–623. [PubMed]
  • Pettersson RF, Ambros V, Baltimore D. Identification of a protein linked to nascent poliovirus RNA and to the polyuridylic acid of negative-strand RNA. J Virol. 1978 Aug;27(2):357–365. [PMC free article] [PubMed]
  • Pincus SE, Wimmer E. Production of guanidine-resistant and -dependent poliovirus mutants from cloned cDNA: mutations in polypeptide 2C are directly responsible for altered guanidine sensitivity. J Virol. 1986 Nov;60(2):793–796. [PMC free article] [PubMed]
  • Reuer Q, Kuhn RJ, Wimmer E. Characterization of poliovirus clones containing lethal and nonlethal mutations in the genome-linked protein VPg. J Virol. 1990 Jun;64(6):2967–2975. [PMC free article] [PubMed]
  • Richards OC, Martin SC, Jense HG, Ehrenfeld E. Structure of poliovirus replicative intermediate RNA. Electron microscope analysis of RNA cross-linked in vivo with psoralen derivative. J Mol Biol. 1984 Mar 5;173(3):325–340. [PubMed]
  • Sample J, Liebowitz D, Kieff E. Two related Epstein-Barr virus membrane proteins are encoded by separate genes. J Virol. 1989 Feb;63(2):933–937. [PMC free article] [PubMed]
  • Sarnow P, Bernstein HD, Baltimore D. A poliovirus temperature-sensitive RNA synthesis mutant located in a noncoding region of the genome. Proc Natl Acad Sci U S A. 1986 Feb;83(3):571–575. [PMC free article] [PubMed]
  • Semler BL, Anderson CW, Hanecak R, Dorner LF, Wimmer E. A membrane-associated precursor to poliovirus VPg identified by immunoprecipitation with antibodies directed against a synthetic heptapeptide. Cell. 1982 Feb;28(2):405–412. [PubMed]
  • Spiess M, Lodish HF. An internal signal sequence: the asialoglycoprotein receptor membrane anchor. Cell. 1986 Jan 17;44(1):177–185. [PubMed]
  • Takeda N, Kuhn RJ, Yang CF, Takegami T, Wimmer E. Initiation of poliovirus plus-strand RNA synthesis in a membrane complex of infected HeLa cells. J Virol. 1986 Oct;60(1):43–53. [PMC free article] [PubMed]
  • Takegami T, Kuhn RJ, Anderson CW, Wimmer E. Membrane-dependent uridylylation of the genome-linked protein VPg of poliovirus. Proc Natl Acad Sci U S A. 1983 Dec;80(24):7447–7451. [PMC free article] [PubMed]
  • Takegami T, Semler BL, Anderson CW, Wimmer E. Membrane fractions active in poliovirus RNA replication contain VPg precursor polypeptides. Virology. 1983 Jul 15;128(1):33–47. [PubMed]
  • Tershak DR. Association of poliovirus proteins with the endoplasmic reticulum. J Virol. 1984 Dec;52(3):777–783. [PMC free article] [PubMed]
  • Toyoda H, Yang CF, Takeda N, Nomoto A, Wimmer E. Analysis of RNA synthesis of type 1 poliovirus by using an in vitro molecular genetic approach. J Virol. 1987 Sep;61(9):2816–2822. [PMC free article] [PubMed]
  • Van Dyke TA, Flanegan JB. Identification of poliovirus polypeptide P63 as a soluble RNA-dependent RNA polymerase. J Virol. 1980 Sep;35(3):732–740. [PMC free article] [PubMed]
  • Wessels HP, Spiess M. Insertion of a multispanning membrane protein occurs sequentially and requires only one signal sequence. Cell. 1988 Oct 7;55(1):61–70. [PubMed]
  • Ypma-Wong MF, Semler BL. In vitro molecular genetics as a tool for determining the differential cleavage specificities of the poliovirus 3C proteinase. Nucleic Acids Res. 1987 Mar 11;15(5):2069–2088. [PMC free article] [PubMed]
  • Ypma-Wong MF, Semler BL. Processing determinants required for in vitro cleavage of the poliovirus P1 precursor to capsid proteins. J Virol. 1987 Oct;61(10):3181–3189. [PMC free article] [PubMed]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • Compound
    Compound
    PubChem Compound links
  • MedGen
    MedGen
    Related information in MedGen
  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...