1887

Abstract

The replicase polyproteins of equine arteritis virus (EAV; family , order ) are processed by three viral proteases to yield 12 non-structural proteins (nsps). The nsp2 and nsp3 cleavage products have previously been found to interact, a property that allows nsp2 to act as a co-factor in the processing of the downstream part of the polyprotein by the nsp4 protease. Remarkably, upon infection of Vero cells, but not of BHK-21 or RK-13 cells, EAV nsp2 is now shown to be subject to an additional, internal, cleavage. In Vero cells, approximately 50% of nsp2 (61 kDa) was cleaved into an 18 kDa N-terminal part and a 44 kDa C-terminal part, most likely by a host cell protease that is absent in BHK-21 and RK-13 cells. Although the functional consequences of this additional processing step are unknown, the experiments in Vero cells revealed that the C-terminal part of nsp2 interacts with nsp3. Most EAV nsps localize to virus-induced double-membrane structures in the perinuclear region of the infected cell, where virus RNA synthesis takes place. It is now shown that, in an expression system, the co-expression of nsp2 and nsp3 is both necessary and sufficient to induce the formation of double-membrane structures that strikingly resemble those found in infected cells. Thus, the nsp2 and nsp3 cleavage products play a crucial role in two processes that are common to positive-strand RNA viruses that replicate in mammalian cells: controlled proteolysis of replicase precursors and membrane association of the virus replication complex.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-82-5-985
2001-05-01
2019-09-21
Loading full text...

Full text loading...

/deliver/fulltext/jgv/82/5/0820985a.html?itemId=/content/journal/jgv/10.1099/0022-1317-82-5-985&mimeType=html&fmt=ahah

References

  1. Bredenbeek, P. J., Frolov, I., Rice, C. M. & Schlesinger, S. ( 1993; ). Sindbis virus expression vectors: packaging of RNA replicons by using defective helper RNAs. Journal of Virology 67, 6439-6446.
    [Google Scholar]
  2. Carette, J. E., Stuiver, M., Van Lent, J., Wellink, J. & Van Kammen, A. ( 2000; ). Cowpea mosaic virus infection induces a massive proliferation of endoplasmic reticulum but not Golgi membranes and is dependent on de novo membrane synthesis. Journal of Virology 74, 6556-6563.[CrossRef]
    [Google Scholar]
  3. Cavanagh, D. ( 1997; ). Nidovirales: a new order comprising Coronaviridae and Arteriviridae. Archives of Virology 142, 629-633.
    [Google Scholar]
  4. Chen, J. & Ahlquist, P. ( 2000; ). Brome mosaic virus polymerase-like protein 2a is directed to the endoplasmic reticulum by helicase-like viral protein 1a. Journal of Virology 74, 4310-4318.[CrossRef]
    [Google Scholar]
  5. den Boon, J. A., Snijder, E. J., Chirnside, E. D., de Vries, A. A. F., Horzinek, M. C. & Spaan, W. J. M. ( 1991; ). Equine arteritis virus is not a togavirus but belongs to the coronaviruslike superfamily. Journal of Virology 65, 2910-2920.
    [Google Scholar]
  6. de Vries, A. A. F., Chirnside, E. D., Horzinek, M. C. & Rottier, P. J. M. ( 1992; ). Structural proteins of equine arteritis virus. Journal of Virology 66, 6294-6303.
    [Google Scholar]
  7. Doll, E. R., Bryans, J. T., McCollum, W. H. & Crowe, M. E. W. ( 1957; ). Isolation of a filterable agent causing arteritis of horses and abortion by mares. Its differentiation from the equine abortion (influenza) virus. Cornell Veterinarian 47, 3-41.
    [Google Scholar]
  8. Egger, D., Teterina, N., Ehrenfeld, E. & Bienz, K. ( 2000; ). Formation of the poliovirus replication complex requires coupled viral translation, vesicle production, and viral RNA synthesis. Journal of Virology 74, 6570-6580.[CrossRef]
    [Google Scholar]
  9. Lai, M. M. C. & Cavanagh, D. ( 1997; ). The molecular biology of coronaviruses. Advances in Virus Research 48, 1-100.[CrossRef]
    [Google Scholar]
  10. Mackenzie, J. M., Jones, M. K. & Westaway, E. G. ( 1999; ). Markers for trans-Golgi membranes and the intermediate compartment localize to induced membranes with distinct replication functions in flavivirus-infected cells. Journal of Virology 73, 9555-9567.
    [Google Scholar]
  11. Pedersen, K. W., van der Meer, Y., Roos, N. & Snijder, E. J. ( 1999; ). Open reading frame 1a-encoded subunits of the arterivirus replicase induce endoplasmic reticulum-derived double-membrane vesicles which carry the viral replication complex. Journal of Virology 73, 2016-2026.
    [Google Scholar]
  12. Ryan, M. D., Monaghan, S. & Flint, M. ( 1998; ). Virus-encoded proteinases of the Flaviviridae. Journal of General Virology 79, 947-959.
    [Google Scholar]
  13. Schaad, M. C., Jensen, P. E. & Carrington, J. C. ( 1997; ). Formation of plant RNA virus replication complexes on membranes: role of an endoplasmic reticulum-targeted viral protein. EMBO Journal 16, 4049-4059.[CrossRef]
    [Google Scholar]
  14. Schlegel, A., Giddings, T. H.Jr, Ladinsky, M. S. & Kirkegaard, K. ( 1996; ). Cellular origin and ultrastructure of membranes induced during poliovirus infection. Journal of Virology 70, 6576-6588.
    [Google Scholar]
  15. Seybert, A., van Dinten, L. C., Snijder, E. J. & Ziebuhr, J. ( 2000; ). Biochemical characterization of the equine arteritis virus helicase suggests a close functional relationship between arterivirus and coronavirus helicases. Journal of Virology 74, 9586-9593.[CrossRef]
    [Google Scholar]
  16. Snijder, E. J. & Meulenberg, J. J. M. ( 1998; ). The molecular biology of arteriviruses. Journal of General Virology 79, 961-979.
    [Google Scholar]
  17. Snijder, E. J., Wassenaar, A. L. M. & Spaan, W. J. M. ( 1992; ). The 5’ end of the equine arteritis virus replicase gene encodes a papainlike cysteine protease. Journal of Virology 66, 7040-7048.
    [Google Scholar]
  18. Snijder, E. J., Wassenaar, A. L. M. & Spaan, W. J. M. ( 1994; ). Proteolytic processing of the replicase ORF1a protein of equine arteritis virus. Journal of Virology 68, 5755-5764.
    [Google Scholar]
  19. Snijder, E. J., Wassenaar, A. L. M., Spaan, W. J. M. & Gorbalenya, A. E. ( 1995; ). The arterivirus Nsp2 protease. An unusual cysteine protease with primary structure similarities to both papain-like and chymotrypsin-like proteases. Journal of Biological Chemistry 270, 16671-16676.[CrossRef]
    [Google Scholar]
  20. Snijder, E. J., Wassenaar, A. L. M., van Dinten, L. C., Spaan, W. J. M. & Gorbalenya, A. E. ( 1996; ). The arterivirus nsp4 protease is the prototype of a novel group of chymotrypsin-like enzymes, the 3C-like serine proteases. Journal of Biological Chemistry 271, 4864-4871.[CrossRef]
    [Google Scholar]
  21. van der Meer, Y., van Tol, H., Krijnse Locker, J. & Snijder, E. J. ( 1998; ). ORF1a-encoded replicase subunits are involved in the membrane association of the arterivirus replication complex. Journal of Virology 72, 6689-6698.
    [Google Scholar]
  22. van der Meer, Y., Snijder, E. J., Dobbe, J. C., Schleich, S., Denison, M. R., Spaan, W. J. M. & Krijnse Locker, J. ( 1999; ). Localization of mouse hepatitis virus nonstructural proteins and RNA synthesis indicates a role for late endosomes in viral replication. Journal of Virology 73, 7641-7657.
    [Google Scholar]
  23. van Dinten, L. C., Wassenaar, A. L. M., Gorbalenya, A. E., Spaan, W. J. M. & Snijder, E. J. ( 1996; ). Processing of the equine arteritis virus replicase ORF1b protein: identification of cleavage products containing the putative viral polymerase and helicase domains. Journal of Virology 70, 6625-6633.
    [Google Scholar]
  24. van Dinten, L. C., den Boon, J. A., Wassenaar, A. L. M., Spaan, W. J. M. & Snijder, E. J. ( 1997; ). An infectious arterivirus cDNA clone: identification of a replicase point mutation that abolishes discontinuous mRNA transcription. Proceedings of the National Academy of Sciences, USA 94, 991-996.[CrossRef]
    [Google Scholar]
  25. van Dinten, L. C., Rensen, S., Gorbalenya, A. E. & Snijder, E. J. ( 1999; ). Proteolytic processing of the open reading frame 1b-encoded part of arterivirus replicase is mediated by nsp4 serine protease and is essential for virus replication. Journal of Virology 73, 2027-2037.
    [Google Scholar]
  26. van Dinten, L. C., van Tol, H., Gorbalenya, A. E. & Snijder, E. J. ( 2000; ). The predicted metal-binding region of the arterivirus helicase protein is involved in subgenomic mRNA synthesis, genome replication, and virion biogenesis. Journal of Virology 74, 5213-5223.[CrossRef]
    [Google Scholar]
  27. Vaux, D., Tooze, J. & Fuller, S. ( 1990; ). Identification by anti-idiotype antibodies of an intracellular membrane protein that recognizes a mammalian endoplasmic reticulum retention signal. Nature 345, 495-502.[CrossRef]
    [Google Scholar]
  28. Wassenaar, A. L. M., Spaan, W. J. M., Gorbalenya, A. E. & Snijder, E. J. ( 1997; ). Alternative proteolytic processing of the arterivirus replicase ORF1a polyprotein: evidence that NSP2 acts as a cofactor for the NSP4 serine protease. Journal of Virology 71, 9313-9322.
    [Google Scholar]
  29. Zentgraf, H., Frey, M., Schwinn, S., Tessmer, C., Willemann, B., Samstag, Y. & Velhagen, I. ( 1995; ). Detection of histidine-tagged fusion proteins by using a high-specific mouse monoclonal anti-histidine tag antibody. Nucleic Acids Research 23, 3347-3348.[CrossRef]
    [Google Scholar]
  30. Ziebuhr, J., Snijder, E. J. & Gorbalenya, A. E. ( 2000; ). Virus-encoded proteinases and proteolytic processing in the Nidovirales. Journal of General Virology 81, 853-879.
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-82-5-985
Loading
/content/journal/jgv/10.1099/0022-1317-82-5-985
Loading

Data & Media loading...

Most Cited This Month

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error