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Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins
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A mutation in the DE loop of the VP1 protein that prevents polyomavirus transcription and replication

M I Garcia1, M Perez, M Caruso

  • 1Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Biotecnologie Cellulari, Sezione di Genetica Molecolare, Università di Roma La Sapienza, Viale Regina Elena 324, Rome, 00161, Italy.

Virology
|June 30, 2000
PubMed

Insights

A Polyomavirus (Py) mutant with a deleted DE loop in its VP1 protein cannot replicate or initiate transcription. This mutation affects viral gene expression, highlighting the DE loop's crucial role in Py infectivity.

Area of Science:

  • Virology
  • Molecular Biology
  • Structural Biology

Background:

  • The DE loop of Polyomavirus (Py) major coat protein VP1 is known to influence host specificity.
  • Understanding the VP1 DE loop's function is key to deciphering Py infectivity mechanisms.

Purpose of the Study:

  • To investigate the role of the VP1 DE loop in Py infectivity by creating and analyzing a specific mutant.
  • To elucidate the function of the VP1 DE loop in viral replication and early gene expression.

Main Methods:

  • Construction and characterization of a Py mutant (Py M17) with a 7 amino acid deletion in the VP1 DE loop.
  • Transfection of fibroblast cells with mutant and wild-type (wt) Py DNA.
  • Complementation experiments to assess cis-dominance of the mutation.
  • In situ cell fractionation to track viral DNA and VP1 protein localization.

Main Results:

  • Py M17 mutant virions failed to replicate and initiate early transcription in fibroblast cells.
  • Complementation experiments demonstrated the cis-dominant nature of the M17 mutation.
  • Both mutant and wt Py entered cells, reached the nucleus, and associated viral DNA and VP1 protein with the nuclear matrix.

Conclusions:

  • The VP1 protein, when bound to viral DNA, influences early viral gene expression.
  • The DE loop of the VP1 protein is essential for this process and thus critical for Polyomavirus infectivity.

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