Alphavirus positive and negative strand RNA synthesis and the role of polyproteins in formation of viral replication complexes

  • D. L. Sawicki
  • S. G. Sawicki
Conference paper
Part of the Archives of Virology Supplementum book series (ARCHIVES SUPPL, volume 9)


The genome of alphaviruses is translated into polyproteins that are processed into a viral replicase that produces both negative and positive strands. In infected cells, negative strand synthesis is short-lived and occurs only early, whereas positive strand synthesis is stable and occurs both early and late. Analysis of temperature sensitive mutants indicated: nsP1 functioned in the initiation of transcription; nsP3 acted to form initial transcription complexes; and nsP2 and nsP4 first recognized positive strands as templates and then made negative strands the preferred templates. While nsP4 and nsP1 individually rescued early defects in transcription, nsP2 and nsP3 acted initially in cis. We interpret our results to suggest nsP1234 was cleaved to nsP4, nsP1 and nsP23, bound a positive strand and synthesized a negative strand. Cleavage of P23 or other modifications to nsP2 and nsP4 convert the initial transcription complex to a stable complex that synthesizes positive strands. Negative strand synthesis is unstable because of the failure to form initial transcription complexes after host factors that are part of the replicase are depleted or the half-life of polyprotein precursors like P23 is shortened.


Strand Synthesis Replication Complex Sindbis Virus Negative Strand Positive Strand 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Goldbach R (1990) Genome similarities between positive-strand RNA viruses from plants and animals. In: Brinton MA, Heinz FX (eds) New aspects of positive- strand RNA viruses. ASM Press, Washington, pp 3–11Google Scholar
  2. 2.
    Koonin EV, Gorbalenya AE, Purdy MA, Rozanov MN, Reyes GR, Bradley DW (1992) Computer-assisted assignment of functional domains in the nonstructural polyprotein of hepatitis E virus: delineation of an additional group of positive- strand RNA plant and animal viruses. Proc Natl Acad Sci USA 89: 8259–8263Google Scholar
  3. 3.
    Strauss JH, Strauss EG (1991) Alphavirus proteinases. Semin Virol 1: 347–356Google Scholar
  4. 4.
    Mi S, Stollar V (1991) Expression of Sindbis virus nsPl and methyltransferase activity in E. coli. Virology 184: 423–427PubMedCrossRefGoogle Scholar
  5. 5.
    Kääriäinen L, Peränen J (1992) Pers. comm.Google Scholar
  6. 6.
    Wang YF, Sawicki SG, Sawicki DL (1991) Sindbis nsPl functions in negative strand synthesis. J Virol 65: 985–988PubMedGoogle Scholar
  7. 7.
    Gorbalenya AE, Blinov VM, Dochenko AP, Koonin EV (1989) An NTP-binding motif is the most conserved sequence in a highly diverged monophyletic group of proteins involved in positive strand RNA viral replication. J Mol Evol 28: 256–268PubMedCrossRefGoogle Scholar
  8. 8.
    Kroner PA, Young BM, Ahlquist P (1990) Analysis of the role of brome mosaic virus la protein domains in RNA replication, using linker insertion mutagenesis. J Virol 64: 6110–6120PubMedGoogle Scholar
  9. 9.
    Gorbalenya AE, Koonin EV, Lai MMC (1991) Putative papain-related thiol pro¬teases of positive strand RNA viruses. FEBS Lett 288: 210–205CrossRefGoogle Scholar
  10. 10.
    Sawicki DL, Sawicki SG (1985) Functional analysis of the A complementation group mutants of Sindbis HR virus. Virology 144: 20–34PubMedCrossRefGoogle Scholar
  11. 11.
    Hahn YS, Strauss EG, Strauss JH (1989) Mapping of RNA-negative temperature- sensitive mutants of Sindbis virus: assignment of complementation group A, B and G to nonstructural proteins. J Virol 63: 3142–3150Google Scholar
  12. 12.
    Sawicki DL, Sawicki SG (1992) A second nonstructural protein functions in the regulation of alphavirus negative strand RNA synthesis. J Virol 67: 3605–3610Google Scholar
  13. 13.
    Peränen J, Rikkonen M, Liljeström P, Kääriäinen L (1990) Nuclear localization of Semliki Forest virus-specific nonstructural protein nsP2. J Virol 64: 1888–1896PubMedGoogle Scholar
  14. 14.
    Li G, LaStarza M, Hardy WR, Strauss JH, Rice CM (1990) Phosphorylation of Sindbis virus nsP3 in vivo and in vitro. Virology 179: 416–427PubMedCrossRefGoogle Scholar
  15. 15.
    Barton DJ, Sawicki SG, Sawicki DL (1991) Solubilization and immunoprecipitation of alphavirus replication complexes. J Virol 65: 1496–1506PubMedGoogle Scholar
  16. 16.
    Wang YF, Sawicki SG, Sawicki DL (1993) Initiation of alphavirus negative strand synthesis requires two different nonstructural proteins, nsP1 and nsP3. J Virol (submitted)Google Scholar
  17. 17.
    Hodgman TC (1988) A new superfamily of replicative proteins. Nature 332: 22–23CrossRefGoogle Scholar
  18. 18.
    Barton DJ, Sawicki SG, Sawicki DL (1988) Demonstration in vitro of temperature-sensitive elongation of RNA in Sindbis virus mutant ts6. J Virol 62: 3597–3602PubMedGoogle Scholar
  19. 19.
    Sawicki DL, Barkhimer DB, Sawicki SG, Rice CM, Schlesinger S (1990) Temperature sensitive shutoff of alphavirus minus strand RNA synthesis maps to a nonstructural protein, nsP4. Virology 174: 43–52PubMedCrossRefGoogle Scholar
  20. 20.
    Lemm JA, Durbin RK, Stollar V, Rice CM (1990) Mutations which alter the level or structure of nsP4 can affect the efficiency of Sindbis virus replication in a host- dependent manner. J Virol 64: 3001–3011PubMedGoogle Scholar
  21. 21.
    Sawicki DL, Sawicki SG (1987) Alphavirus plus and minus strand RNA synthesis. In: Brinton M, Ruckert R (eds) Positive-strand RNA viruses. Alan R. Liss, New York, pp 251–259Google Scholar
  22. 22.
    Sawicki DL, Kääriäinen L, Lambek C, Gomatos PG (1978) Mechanism for control of synthesis of Semliki Forest virus 26S and 42S RNA. J Virol 25: 19–27PubMedGoogle Scholar
  23. 23.
    Sawicki SG, Sawicki DL (1986) The effect of overproduction of nonstructural proteins on alphavirus plus-strand and minus-strand RNA synthesis. Virology 152: 507–512PubMedCrossRefGoogle Scholar
  24. 24.
    Rice CM, Levis R, Strauss JH, Huang HV (1987) Production of infectious RNA transcripts from Sindbis virus cDNA clones: mapping of lethal mutations, rescue of a temperature-sensitive marker, and in vitro mutagenesis to generate defined mutants. J Virol 61: 3809–3819PubMedGoogle Scholar
  25. 25.
    Ishikawa M, Mesi T, Ohno T, Okada Y (1991) Specific cessation of minus strand RNA accumulation at an early stage of Tobacco mosaic virus infection. J Virol 65: 861–868PubMedGoogle Scholar
  26. 26.
    Sawicki SG, Sawicki DL (1986) The effect of loss of regulation of minus strand RNA synthesis on Sindbis virus replication. Virology 151: 339–349PubMedCrossRefGoogle Scholar
  27. 27.
    deGroot RJ, Hardy WR, Shirako Y, Strauss JH (1990) Cleavage-site preferences of Sindbis virus polyproteins containing the nonstructural proteinase. Evidence for temporal regulation of polyprotein processing in vivo. EMBO J 9: 2631–2638Google Scholar
  28. 28.
    Lemm J, Rice CM (1993) Roles of nonstructural polyproteins and cleavage products in regulating Sindbis virus RNA replication and transcription. J Virol 67: 1916–1926PubMedGoogle Scholar

Copyright information

© Springer-Verlag 1994

Authors and Affiliations

  • D. L. Sawicki
    • 1
  • S. G. Sawicki
    • 1
  1. 1.Department of MicrobiologyMedical College of OhioToledoUSA

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