1887

Abstract

Most chloroviruses encode small K channels, which are functional in electrophysiological assays. The experimental finding that initial steps in viral infection exhibit the same sensitivity to channel inhibitors as the viral K channels has led to the hypothesis that the channels are structural proteins located in the internal membrane of the virus particles. This hypothesis was questioned recently because proteomic studies failed to detect the channel protein in virions of the prototype chlorovirus chlorella virus 1 (PBCV-1). Here, we used a mAb raised against the functional K channel from chlorovirus MA-1D to search for the viral K channel in the virus particle. The results showed that the antibody was specific and bound to the tetrameric channel on the extracellular side. The antibody reacted in a virus-specific manner with protein extracts from chloroviruses that encoded channels similar to that from MA-1D. There was no cross-reactivity with chloroviruses that encoded more diverse channels or with a chlorovirus that lacked a K channel gene. Together with electron microscopic imaging, which revealed labelling of individual virus particles with the channel antibody, these results establish that the viral particles contain an active K channel, presumably located in the lipid membrane that surrounds the DNA in the mature virions.

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2013-11-01
2024-11-14
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References

  1. Agarkova I., Dunigan D. D., Gurnon J., Greiner T., Barres J., Thiel G., Van Etten J. L. 2008; Chlorovirus-mediated membrane depolarization of Chlorella alters secondary active transport of solutes. J Virol 82:12181–12190 [View Article][PubMed]
    [Google Scholar]
  2. Balss J., Papatheodorou P., Mehmel M., Baumeister D., Hertel B., Delaroque N., Chatelain F. C., Minor D. L. Jr, Van Etten J. L.other authors 2008; Transmembrane domain length of viral K+ channels is a signal for mitochondria targeting. Proc Natl Acad Sci U S A 105:12313–12318 [View Article][PubMed]
    [Google Scholar]
  3. Dunigan D. D., Cerny R. L., Bauman A. T., Roach J. C., Lane L. C., Agarkova I. V., Wulser K., Yanai-Balser G. M., Gurnon J. R.other authors 2012; Paramecium bursaria chlorella virus 1 proteome reveals novel architectural and regulatory features of a giant virus. J Virol 86:8821–8834 [View Article][PubMed]
    [Google Scholar]
  4. Fischer F., Poetsch A. 2006; Protein cleavage strategies for an improved analysis of the membrane proteome. Proteome Sci 4:2 [View Article][PubMed]
    [Google Scholar]
  5. Fitzgerald L. A., Graves M. V., Li X., Feldblyum T., Hartigan J., Van Etten J. L. 2007a; Sequence and annotation of the 314-kb MT325 and the 321-kb FR483 viruses that infect Chlorella Pbi. Virology 358:459–471 [View Article][PubMed]
    [Google Scholar]
  6. Fitzgerald L. A., Graves M. V., Li X., Feldblyum T., Nierman W. C., Van Etten J. L. 2007b; Sequence and annotation of the 369-kb NY-2A and the 345-kb AR158 viruses that infect Chlorella NC64A. Virology 358:472–484 [View Article][PubMed]
    [Google Scholar]
  7. Frohns F., Käsmann A., Kramer D., Schäfer B., Mehmel M., Kang M., Van Etten J. L., Gazzarrini S., Moroni A., Thiel G. 2006; Potassium ion channels of Chlorella viruses cause rapid depolarization of host cells during infection. J Virol 80:2437–2444 [View Article][PubMed]
    [Google Scholar]
  8. Gazzarrini S., Severino M., Lombardi M., Morandi M., DiFrancesco D., Van Etten J. L., Thiel G., Moroni A. 2003; The viral potassium channel Kcv: structural and functional features. FEBS Lett 552:12–16 [View Article][PubMed]
    [Google Scholar]
  9. Gazzarrini S., Kang M., Epimashko S., Van Etten J. L., Dainty J., Thiel G., Moroni A. 2006; Chlorella virus MT325 encodes water and potassium channels that interact synergistically. Proc Natl Acad Sci U S A 103:5355–5360 [View Article][PubMed]
    [Google Scholar]
  10. Gazzarrini S., Kang M., Abenavoli A., Romani G., Olivari C., Gaslini D., Ferrara G., van Etten J. L., Kreim M.other authors 2009; Chlorella virus ATCV-1 encodes a functional potassium channel of 82 amino acids. Biochem J 420:295–303 [View Article][PubMed]
    [Google Scholar]
  11. Greiner T., Frohns F., Kang M., Van Etten J. L., Käsmann A., Moroni A., Hertel B., Thiel G. 2009; Chlorella viruses prevent multiple infections by depolarizing the host membrane. J Gen Virol 90:2033–2039 [View Article][PubMed]
    [Google Scholar]
  12. Greiner T., Ramos J., Alvarez M. C., Gurnon J. R., Kang M., Van Etten J. L., Moroni A., Thiel G. 2011; Functional HAK/KUP/KT-like potassium transporter encoded by chlorella viruses. Plant J 68:977–986 [View Article][PubMed]
    [Google Scholar]
  13. Harlow E., Lane D. 1989 Antibodies: a Laboratory Manual Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  14. Hillmer S., Viotti C., Robinson D. G. 2012; An improved procedure for low-temperature embedding of high-pressure frozen and freeze-substituted plant tissues resulting in excellent structural preservation and contrast. J Microsc 247:43–47 [View Article][PubMed]
    [Google Scholar]
  15. Jeanniard A., Dunigan D. D., Gurnon J. R., Agarkova I. V., Kang M., Vitek J., Duncan G., McClung O. W., Larsen M.other authors 2013; Towards defining the chloroviruses: a genomic journey through a genus of large DNA viruses. BMC Genomics 14:158 [View Article][PubMed]
    [Google Scholar]
  16. Kang M., Moroni A., Gazzarrini S., DiFrancesco D., Thiel G., Severino M., Van Etten J. L. 2004; Small potassium ion channel proteins encoded by chlorella viruses. Proc Natl Acad Sci U S A 101:5318–5324 [View Article][PubMed]
    [Google Scholar]
  17. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 [View Article][PubMed]
    [Google Scholar]
  18. Mehmel M., Rothermel M., Meckel T., Van Etten J. L., Moroni A., Thiel G. 2003; Possible function for virus encoded K+ channel Kcv in the replication of chlorella virus PBCV-1. FEBS Lett 552:7–11 [View Article][PubMed]
    [Google Scholar]
  19. Moroni A., Viscomi C., Sangiorgio V., Pagliuca C., Meckel T., Horvath F., Gazzarrini S., Valbuzzi P., Van Etten J. L.other authors 2002; The short N-terminus is required for functional expression of the virus-encoded miniature K+ channel Kcv. FEBS Lett 530:65–69 [View Article][PubMed]
    [Google Scholar]
  20. Neupärtl M., Meyer C., Woll I., Frohns F., Kang M., Van Etten J. L., Kramer D., Hertel B., Moroni A., Thiel G. 2008; Chlorella viruses evoke a rapid release of K+ from host cells during the early phase of infection. Virology 372:340–348 [View Article][PubMed]
    [Google Scholar]
  21. Pagliuca C., Goetze T. A., Wagner R., Thiel G., Moroni A., Parcej D. 2007; Molecular properties of Kcv, a virus encoded K+ channel. Biochemistry 46:1079–1090 [View Article][PubMed]
    [Google Scholar]
  22. Plugge B., Gazzarrini S., Nelson M., Cerana R., Van Etten J. L., Derst C., DiFrancesco D., Moroni A., Thiel G. 2000; A potassium channel protein encoded by chlorella virus PBCV-1. Science 287:1641–1644 [View Article][PubMed]
    [Google Scholar]
  23. Shim J. W., Yang M., Gu L. Q. 2007; In vitro synthesis, tetramerization and single channel characterization of virus-encoded potassium channel Kcv. FEBS Lett 581:1027–1034 [View Article][PubMed]
    [Google Scholar]
  24. Skrdla M. P., Burbank D. E., Xia Y., Meints R. H., Van Etten J. L. 1984; Structural proteins and lipids in a virus, PBCV-1, which replicates in a Chlorella-like alga. Virology 135:308–315 [View Article][PubMed]
    [Google Scholar]
  25. Tayefeh S., Kloss T., Thiel G., Hertel B., Moroni A., Kast S. M. 2007; Molecular dynamics simulation of the cytosolic mouth in Kcv-type potassium channels. Biochemistry 46:4826–4839 [View Article][PubMed]
    [Google Scholar]
  26. Thiel G., Moroni A., Dunigan D., Van Etten J. L. 2010; Initial events associated with virus PBCV-1 infection of Chlorella NC64A. Prog Bot 71:169–183 [View Article][PubMed]
    [Google Scholar]
  27. Thiel G., Baumeister D., Schroeder I., Kast S. M., Van Etten J. L., Moroni A. 2011; Minimal art: or why small viral K+ channels are good tools for understanding basic structure and function relations. Biochim Biophys Acta 1808:580–588 [View Article][PubMed]
    [Google Scholar]
  28. Van Etten J. L. 2003; Unusual life style of giant chlorella viruses. Annu Rev Genet 37:153–195 [View Article][PubMed]
    [Google Scholar]
  29. Van Etten J. L., Dunigan D. D. 2012; Chloroviruses: not your everyday plant virus. Trends Plant Sci 17:1–8 [View Article][PubMed]
    [Google Scholar]
  30. Van Etten J. L., Burbank D. E., Xia Y., Meints R. H. 1983; Growth cycle of a virus, PBCV-1, that infects Chlorella-like algae. Virology 126:117–125 [View Article][PubMed]
    [Google Scholar]
  31. Wang K., Xie S., Sun B. 2011; Viral proteins function as ion channels. Biochim Biophys Acta 1808:510–515 [View Article][PubMed]
    [Google Scholar]
  32. Yamada T., Onimatsu H., Van Etten J. L. 2006; Chlorella viruses. Adv Virus Res 66:293–336 [View Article][PubMed]
    [Google Scholar]
  33. Yan X., Olson N. H., Van Etten J. L., Bergoin M., Rossmann M. G., Baker T. S. 2000; Structure and assembly of large lipid-containing dsDNA viruses. Nat Struct Biol 7:101–103 [View Article][PubMed]
    [Google Scholar]
  34. Zhang X., Xiang Y., Dunigan D. D., Klose T., Chipman P. R., Van Etten J. L., Rossmann M. G. 2011; Three-dimensional structure and function of the Paramecium bursaria chlorella virus capsid. Proc Natl Acad Sci U S A 108:14837–14842 [View Article][PubMed]
    [Google Scholar]
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