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. 1991 Jan;65(1):112–122. doi: 10.1128/jvi.65.1.112-122.1991

In vitro mRNA degradation system to study the virion host shutoff function of herpes simplex virus.

C R Krikorian 1, G S Read 1
PMCID: PMC240495  PMID: 1845879

Abstract

The virion host shutoff (vhs) gene of herpes simplex virus encodes a virion polypeptide that induces degradation of host mRNAs at early times and rapid turnover of viral mRNAs throughout infection. To better investigate the vhs function, an in vitro mRNA degradation system was developed, consisting of cytoplasmic extracts from HeLa cells infected with wild-type herpes simplex virus type 1 or a mutant encoding a defective vhs polypeptide. Host and viral mRNAs were degraded rapidly in extracts from cells productively infected with wild-type herpes simplex virus type 1 but not in extracts from mock-infected cells or cells infected with the mutant vhs1. In contrast, 28S rRNA was stable in all three kinds of extract. Accelerated turnover of host mRNAs was also observed in extracts from cells infected with wild-type virus in the presence of dactinomycin, indicating that the activity was induced by a structural component of the infecting virions. The in vitro vhs activity was inactivated by heat or proteinase K digestion but was insensitive to brief treatment of the extracts with micrococcal nuclease. It was not inhibited by placental RNase inhibitor, it exhibited a strong dependence upon added Mg2+, it was active at concentrations of K+ up to 200 mM, and it did not require the components of an energy-generating system. In summary, the in vitro mRNA degradation system appears to accurately reproduce the vhs-mediated decay of host and viral mRNAs and should be useful for studies of the mechanism of vhs action.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adams J. M., Harris A. W., Pinkert C. A., Corcoran L. M., Alexander W. S., Cory S., Palmiter R. D., Brinster R. L. The c-myc oncogene driven by immunoglobulin enhancers induces lymphoid malignancy in transgenic mice. Nature. 1985 Dec 12;318(6046):533–538. doi: 10.1038/318533a0. [DOI] [PubMed] [Google Scholar]
  2. Bandyopadhyay R., Coutts M., Krowczynska A., Brawerman G. Nuclease activity associated with mammalian mRNA in its native state: possible basis for selectivity in mRNA decay. Mol Cell Biol. 1990 May;10(5):2060–2069. doi: 10.1128/mcb.10.5.2060. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bernstein P., Peltz S. W., Ross J. The poly(A)-poly(A)-binding protein complex is a major determinant of mRNA stability in vitro. Mol Cell Biol. 1989 Feb;9(2):659–670. doi: 10.1128/mcb.9.2.659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bernstein P., Ross J. Poly(A), poly(A) binding protein and the regulation of mRNA stability. Trends Biochem Sci. 1989 Sep;14(9):373–377. doi: 10.1016/0968-0004(89)90011-x. [DOI] [PubMed] [Google Scholar]
  5. Brawerman G. mRNA decay: finding the right targets. Cell. 1989 Apr 7;57(1):9–10. doi: 10.1016/0092-8674(89)90166-9. [DOI] [PubMed] [Google Scholar]
  6. Brewer G., Ross J. Poly(A) shortening and degradation of the 3' A+U-rich sequences of human c-myc mRNA in a cell-free system. Mol Cell Biol. 1988 Apr;8(4):1697–1708. doi: 10.1128/mcb.8.4.1697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brewer G., Ross J. Regulation of c-myc mRNA stability in vitro by a labile destabilizer with an essential nucleic acid component. Mol Cell Biol. 1989 May;9(5):1996–2006. doi: 10.1128/mcb.9.5.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brock M. L., Shapiro D. J. Estrogen stabilizes vitellogenin mRNA against cytoplasmic degradation. Cell. 1983 Aug;34(1):207–214. doi: 10.1016/0092-8674(83)90151-4. [DOI] [PubMed] [Google Scholar]
  9. Brown G. D., Peluso R. W., Moyer S. A., Moyer R. W. A simple method for the preparation of extracts from animal cells which catalyze efficient in vitro protein synthesis. J Biol Chem. 1983 Dec 10;258(23):14309–14314. [PubMed] [Google Scholar]
  10. Campisi J., Gray H. E., Pardee A. B., Dean M., Sonenshein G. E. Cell-cycle control of c-myc but not c-ras expression is lost following chemical transformation. Cell. 1984 Feb;36(2):241–247. doi: 10.1016/0092-8674(84)90217-4. [DOI] [PubMed] [Google Scholar]
  11. Caput D., Beutler B., Hartog K., Thayer R., Brown-Shimer S., Cerami A. Identification of a common nucleotide sequence in the 3'-untranslated region of mRNA molecules specifying inflammatory mediators. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1670–1674. doi: 10.1073/pnas.83.6.1670. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Coppola J. A., Cole M. D. Constitutive c-myc oncogene expression blocks mouse erythroleukaemia cell differentiation but not commitment. Nature. 1986 Apr 24;320(6064):760–763. doi: 10.1038/320760a0. [DOI] [PubMed] [Google Scholar]
  13. Dani C., Blanchard J. M., Piechaczyk M., El Sabouty S., Marty L., Jeanteur P. Extreme instability of myc mRNA in normal and transformed human cells. Proc Natl Acad Sci U S A. 1984 Nov;81(22):7046–7050. doi: 10.1073/pnas.81.22.7046. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dawid I. B., Wellauer P. K. A reinvestigation of 5' leads to 3' polarity in 40S ribosomal RNA precursor of Xenopus laevis. Cell. 1976 Jul;8(3):443–448. doi: 10.1016/0092-8674(76)90157-4. [DOI] [PubMed] [Google Scholar]
  15. Fenwick M. L., Clark J. Early and delayed shut-off of host protein synthesis in cells infected with herpes simplex virus. J Gen Virol. 1982 Jul;61(Pt 50):121–125. doi: 10.1099/0022-1317-61-1-121. [DOI] [PubMed] [Google Scholar]
  16. Fenwick M. L., Clark J. The effect of cycloheximide on the accumulation and stability of functional alpha-mRNA in cells infected with herpes simplex virus. J Gen Virol. 1983 Sep;64(Pt 9):1955–1963. doi: 10.1099/0022-1317-64-9-1955. [DOI] [PubMed] [Google Scholar]
  17. Fenwick M. L., McMenamin M. M. Early virion-associated suppression of cellular protein synthesis by herpes simplex virus is accompanied by inactivation of mRNA. J Gen Virol. 1984 Jul;65(Pt 7):1225–1228. doi: 10.1099/0022-1317-65-7-1225. [DOI] [PubMed] [Google Scholar]
  18. Fenwick M. L., Owen S. A. On the control of immediate early (alpha) mRNA survival in cells infected with herpes simplex virus. J Gen Virol. 1988 Nov;69(Pt 11):2869–2877. doi: 10.1099/0022-1317-69-11-2869. [DOI] [PubMed] [Google Scholar]
  19. Fenwick M. L., Walker M. J. Suppression of the synthesis of cellular macromolecules by herpes simplex virus. J Gen Virol. 1978 Oct;41(1):37–51. doi: 10.1099/0022-1317-41-1-37. [DOI] [PubMed] [Google Scholar]
  20. Fenwick M., Morse L. S., Roizman B. Anatomy of herpes simplex virus DNA. XI. Apparent clustering of functions effecting rapid inhibition of host DNA and protein synthesis. J Virol. 1979 Feb;29(2):825–827. doi: 10.1128/jvi.29.2.825-827.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gay D. A., Yen T. J., Lau J. T., Cleveland D. W. Sequences that confer beta-tubulin autoregulation through modulated mRNA stability reside within exon 1 of a beta-tubulin mRNA. Cell. 1987 Aug 28;50(5):671–679. doi: 10.1016/0092-8674(87)90325-4. [DOI] [PubMed] [Google Scholar]
  22. Graves R. A., Pandey N. B., Chodchoy N., Marzluff W. F. Translation is required for regulation of histone mRNA degradation. Cell. 1987 Feb 27;48(4):615–626. doi: 10.1016/0092-8674(87)90240-6. [DOI] [PubMed] [Google Scholar]
  23. Guyette W. A., Matusik R. J., Rosen J. M. Prolactin-mediated transcriptional and post-transcriptional control of casein gene expression. Cell. 1979 Aug;17(4):1013–1023. doi: 10.1016/0092-8674(79)90340-4. [DOI] [PubMed] [Google Scholar]
  24. Harris-Hamilton E., Bachenheimer S. L. Accumulation of herpes simplex virus type 1 RNAs of different kinetic classes in the cytoplasm of infected cells. J Virol. 1985 Jan;53(1):144–151. doi: 10.1128/jvi.53.1.144-151.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hill T. M., Sinden R. R., Sadler J. R. Herpes simplex virus types 1 and 2 induce shutoff of host protein synthesis by different mechanisms in Friend erythroleukemia cells. J Virol. 1983 Jan;45(1):241–250. doi: 10.1128/jvi.45.1.241-250.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Jones T. R., Cole M. D. Rapid cytoplasmic turnover of c-myc mRNA: requirement of the 3' untranslated sequences. Mol Cell Biol. 1987 Dec;7(12):4513–4521. doi: 10.1128/mcb.7.12.4513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kass S., Tyc K., Steitz J. A., Sollner-Webb B. The U3 small nucleolar ribonucleoprotein functions in the first step of preribosomal RNA processing. Cell. 1990 Mar 23;60(6):897–908. doi: 10.1016/0092-8674(90)90338-f. [DOI] [PubMed] [Google Scholar]
  28. Krikorian C. R., Read G. S. Proteins associated with mRNA in cells infected with herpes simplex virus. Biochem Biophys Res Commun. 1989 Oct 16;164(1):355–361. doi: 10.1016/0006-291x(89)91726-9. [DOI] [PubMed] [Google Scholar]
  29. Kwong A. D., Frenkel N. Herpes simplex virus-infected cells contain a function(s) that destabilizes both host and viral mRNAs. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1926–1930. doi: 10.1073/pnas.84.7.1926. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kwong A. D., Frenkel N. The herpes simplex virus virion host shutoff function. J Virol. 1989 Nov;63(11):4834–4839. doi: 10.1128/jvi.63.11.4834-4839.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kwong A. D., Kruper J. A., Frenkel N. Herpes simplex virus virion host shutoff function. J Virol. 1988 Mar;62(3):912–921. doi: 10.1128/jvi.62.3.912-921.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lasater L. S., Eichler D. C. Isolation and properties of a single-strand 5'----3' exoribonuclease from Ehrlich ascites tumor cell nucleoli. Biochemistry. 1984 Sep 11;23(19):4367–4373. doi: 10.1021/bi00314a019. [DOI] [PubMed] [Google Scholar]
  33. Leder A., Pattengale P. K., Kuo A., Stewart T. A., Leder P. Consequences of widespread deregulation of the c-myc gene in transgenic mice: multiple neoplasms and normal development. Cell. 1986 May 23;45(4):485–495. doi: 10.1016/0092-8674(86)90280-1. [DOI] [PubMed] [Google Scholar]
  34. Lee W. M., Schwab M., Westaway D., Varmus H. E. Augmented expression of normal c-myc is sufficient for cotransformation of rat embryo cells with a mutant ras gene. Mol Cell Biol. 1985 Dec;5(12):3345–3356. doi: 10.1128/mcb.5.12.3345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Lüscher B., Stauber C., Schindler R., Schümperli D. Faithful cell-cycle regulation of a recombinant mouse histone H4 gene is controlled by sequences in the 3'-terminal part of the gene. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4389–4393. doi: 10.1073/pnas.82.13.4389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. McGeoch D. J., Dalrymple M. A., Davison A. J., Dolan A., Frame M. C., McNab D., Perry L. J., Scott J. E., Taylor P. The complete DNA sequence of the long unique region in the genome of herpes simplex virus type 1. J Gen Virol. 1988 Jul;69(Pt 7):1531–1574. doi: 10.1099/0022-1317-69-7-1531. [DOI] [PubMed] [Google Scholar]
  37. Müllner E. W., Kühn L. C. A stem-loop in the 3' untranslated region mediates iron-dependent regulation of transferrin receptor mRNA stability in the cytoplasm. Cell. 1988 Jun 3;53(5):815–825. doi: 10.1016/0092-8674(88)90098-0. [DOI] [PubMed] [Google Scholar]
  38. Müllner E. W., Neupert B., Kühn L. C. A specific mRNA binding factor regulates the iron-dependent stability of cytoplasmic transferrin receptor mRNA. Cell. 1989 Jul 28;58(2):373–382. doi: 10.1016/0092-8674(89)90851-9. [DOI] [PubMed] [Google Scholar]
  39. Nishioka Y., Silverstein S. Degradation of cellular mRNA during infection by herpes simplex virus. Proc Natl Acad Sci U S A. 1977 Jun;74(6):2370–2374. doi: 10.1073/pnas.74.6.2370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Nishioka Y., Silverstein S. Requirement of protein synthesis for the degradation of host mRNA in Friend erythroleukemia cells infected wtih herpes simplex virus type 1. J Virol. 1978 Sep;27(3):619–627. doi: 10.1128/jvi.27.3.619-627.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Oroskar A. A., Read G. S. A mutant of herpes simplex virus type 1 exhibits increased stability of immediate-early (alpha) mRNAs. J Virol. 1987 Feb;61(2):604–606. doi: 10.1128/jvi.61.2.604-606.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Oroskar A. A., Read G. S. Control of mRNA stability by the virion host shutoff function of herpes simplex virus. J Virol. 1989 May;63(5):1897–1906. doi: 10.1128/jvi.63.5.1897-1906.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Oshimura M., Gilmer T. M., Barrett J. C. Nonrandom loss of chromosome 15 in Syrian hamster tumours induced by v-Ha-ras plus v-myc oncogenes. Nature. 1985 Aug 15;316(6029):636–639. doi: 10.1038/316636a0. [DOI] [PubMed] [Google Scholar]
  44. Paek I., Axel R. Glucocorticoids enhance stability of human growth hormone mRNA. Mol Cell Biol. 1987 Apr;7(4):1496–1507. doi: 10.1128/mcb.7.4.1496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Pandey N. B., Marzluff W. F. The stem-loop structure at the 3' end of histone mRNA is necessary and sufficient for regulation of histone mRNA stability. Mol Cell Biol. 1987 Dec;7(12):4557–4559. doi: 10.1128/mcb.7.12.4557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Pei R., Calame K. Differential stability of c-myc mRNAS in a cell-free system. Mol Cell Biol. 1988 Jul;8(7):2860–2868. doi: 10.1128/mcb.8.7.2860. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Pelham H. R., Jackson R. J. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem. 1976 Aug 1;67(1):247–256. doi: 10.1111/j.1432-1033.1976.tb10656.x. [DOI] [PubMed] [Google Scholar]
  48. Pellegrini S., Basilico C. Rat fibroblasts expressing high levels of human c-myc transcripts are anchorage-independent and tumorigenic. J Cell Physiol. 1986 Jan;126(1):107–114. doi: 10.1002/jcp.1041260115. [DOI] [PubMed] [Google Scholar]
  49. Peltz S. W., Brewer G., Kobs G., Ross J. Substrate specificity of the exonuclease activity that degrades H4 histone mRNA. J Biol Chem. 1987 Jul 5;262(19):9382–9388. [PubMed] [Google Scholar]
  50. Peltz S. W., Ross J. Autogenous regulation of histone mRNA decay by histone proteins in a cell-free system. Mol Cell Biol. 1987 Dec;7(12):4345–4356. doi: 10.1128/mcb.7.12.4345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Piechaczyk M., Yang J. Q., Blanchard J. M., Jeanteur P., Marcu K. B. Posttranscriptional mechanisms are responsible for accumulation of truncated c-myc RNAs in murine plasma cell tumors. Cell. 1985 Sep;42(2):589–597. doi: 10.1016/0092-8674(85)90116-3. [DOI] [PubMed] [Google Scholar]
  52. Rabbitts P. H., Forster A., Stinson M. A., Rabbitts T. H. Truncation of exon 1 from the c-myc gene results in prolonged c-myc mRNa stability. EMBO J. 1985 Dec 30;4(13B):3727–3733. doi: 10.1002/j.1460-2075.1985.tb04141.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Read G. S., Frenkel N. Herpes simplex virus mutants defective in the virion-associated shutoff of host polypeptide synthesis and exhibiting abnormal synthesis of alpha (immediate early) viral polypeptides. J Virol. 1983 May;46(2):498–512. doi: 10.1128/jvi.46.2.498-512.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Read G. S., Sharp J. A., Summers W. C. In vitro and in vivo transcription initiation sites on the TK-encoding BamHI Q fragment of HSV-1 DNA. Virology. 1984 Oct 30;138(2):368–372. doi: 10.1016/0042-6822(84)90363-5. [DOI] [PubMed] [Google Scholar]
  55. Read G. S., Summers W. C. In vitro transcription of the thymidine kinase gene of herpes simplex virus. Proc Natl Acad Sci U S A. 1982 Sep;79(17):5215–5219. doi: 10.1073/pnas.79.17.5215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Roizman B., Borman G. S., Rousta M. K. Macromolecular synthesis in cells infected with herpes simplex virus. Nature. 1965 Jun 26;206(991):1374–1375. doi: 10.1038/2061374a0. [DOI] [PubMed] [Google Scholar]
  57. Ross J., Kobs G., Brewer G., Peltz S. W. Properties of the exonuclease activity that degrades H4 histone mRNA. J Biol Chem. 1987 Jul 5;262(19):9374–9381. [PubMed] [Google Scholar]
  58. Ross J., Kobs G. H4 histone messenger RNA decay in cell-free extracts initiates at or near the 3' terminus and proceeds 3' to 5'. J Mol Biol. 1986 Apr 20;188(4):579–593. doi: 10.1016/s0022-2836(86)80008-0. [DOI] [PubMed] [Google Scholar]
  59. Ross J. Messenger RNA turnover in eukaryotic cells. Mol Biol Med. 1988 Feb;5(1):1–14. [PubMed] [Google Scholar]
  60. Ross J., Peltz S. W., Kobs G., Brewer G. Histone mRNA degradation in vivo: the first detectable step occurs at or near the 3' terminus. Mol Cell Biol. 1986 Dec;6(12):4362–4371. doi: 10.1128/mcb.6.12.4362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Rouault T. A., Hentze M. W., Haile D. J., Harford J. B., Klausner R. D. The iron-responsive element binding protein: a method for the affinity purification of a regulatory RNA-binding protein. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5768–5772. doi: 10.1073/pnas.86.15.5768. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Saha B. K., Graham M. Y., Schlessinger D. Acid ribonuclease from HeLa cell lysosomes. J Biol Chem. 1979 Jul 10;254(13):5951–5957. [PubMed] [Google Scholar]
  63. Schek N., Bachenheimer S. L. Degradation of cellular mRNAs induced by a virion-associated factor during herpes simplex virus infection of Vero cells. J Virol. 1985 Sep;55(3):601–610. doi: 10.1128/jvi.55.3.601-610.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Schuler G. D., Cole M. D. GM-CSF and oncogene mRNA stabilities are independently regulated in trans in a mouse monocytic tumor. Cell. 1988 Dec 23;55(6):1115–1122. doi: 10.1016/0092-8674(88)90256-5. [DOI] [PubMed] [Google Scholar]
  65. Shaw G., Kamen R. A conserved AU sequence from the 3' untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell. 1986 Aug 29;46(5):659–667. doi: 10.1016/0092-8674(86)90341-7. [DOI] [PubMed] [Google Scholar]
  66. Strom T., Frenkel N. Effects of herpes simplex virus on mRNA stability. J Virol. 1987 Jul;61(7):2198–2207. doi: 10.1128/jvi.61.7.2198-2207.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Sunitha I., Slobin L. I. An in vitro system derived from Friend erythroleukemia cells to study messenger RNA stability. Biochem Biophys Res Commun. 1987 Apr 29;144(2):560–568. doi: 10.1016/s0006-291x(87)80003-7. [DOI] [PubMed] [Google Scholar]
  68. Sydiskis R. J., Roizman B. Polysomes and protein synthesis in cells infected with a DNA virus. Science. 1966 Jul 1;153(3731):76–78. doi: 10.1126/science.153.3731.76. [DOI] [PubMed] [Google Scholar]
  69. Weinheimer S. P., McKnight S. L. Transcriptional and post-transcriptional controls establish the cascade of herpes simplex virus protein synthesis. J Mol Biol. 1987 Jun 20;195(4):819–833. doi: 10.1016/0022-2836(87)90487-6. [DOI] [PubMed] [Google Scholar]
  70. Wilson T., Treisman R. Removal of poly(A) and consequent degradation of c-fos mRNA facilitated by 3' AU-rich sequences. Nature. 1988 Nov 24;336(6197):396–399. doi: 10.1038/336396a0. [DOI] [PubMed] [Google Scholar]
  71. Yager D. R., Coen D. M. Analysis of the transcript of the herpes simplex virus DNA polymerase gene provides evidence that polymerase expression is inefficient at the level of translation. J Virol. 1988 Jun;62(6):2007–2015. doi: 10.1128/jvi.62.6.2007-2015.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  72. Yager D. R., Marcy A. I., Coen D. M. Translational regulation of herpes simplex virus DNA polymerase. J Virol. 1990 May;64(5):2217–2225. doi: 10.1128/jvi.64.5.2217-2225.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  73. Yen T. J., Machlin P. S., Cleveland D. W. Autoregulated instability of beta-tubulin mRNAs by recognition of the nascent amino terminus of beta-tubulin. Nature. 1988 Aug 18;334(6183):580–585. doi: 10.1038/334580a0. [DOI] [PubMed] [Google Scholar]

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