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Binding and nuclear relocalization of protein kinase R by human cytomegalovirus TRS1

Morgan Hakki1, Emily E Marshall, Katherine L De Niro

  • 1Division of Human Biology, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., Seattle, WA 98109, USA.

Journal of Virology
|September 22, 2006
PubMed

Insights

Human cytomegalovirus (HCMV) TRS1 and IRS1 proteins prevent host protein synthesis shutoff by binding to and sequestering protein kinase R (PKR) in the nucleus, inhibiting its activation.

Area of Science:

  • Virology
  • Molecular Biology
  • Immunology

Background:

  • Human cytomegalovirus (HCMV) infection can lead to shutoff of cellular protein synthesis.
  • This shutoff is mediated by the phosphorylation of eukaryotic initiation factor 2 alpha (eIF2alpha).
  • HCMV TRS1 and IRS1 genes counteract this shutoff.

Purpose of the Study:

  • To elucidate the mechanism by which HCMV TRS1 and IRS1 block eIF2alpha phosphorylation and protein synthesis shutoff.
  • To investigate the interaction between HCMV proteins and protein kinase R (PKR).

Main Methods:

  • Recombinant vaccinia virus (VV) systems were used, including VV deleted of E3L (VVDeltaE3L).
  • Immunoblot analysis and immunofluorescence were employed to detect protein localization and levels.
  • PKR-EGFP fusion protein was used to visualize PKR relocalization.
  • Binding assays (mammalian cell and in vitro) were performed to confirm protein interactions.

Main Results:

  • HCMV pTRS1 expression caused nuclear accumulation of PKR and decreased cytoplasmic PKR levels in VVDeltaE3L-infected cells.
  • PKR in both nuclear and cytoplasmic fractions was unactivated (nonphosphorylated) in the presence of pTRS1.
  • pTRS1 directly interacted with PKR, requiring its carboxy-terminal region.
  • pIRS1 also interacted with PKR and mediated rescue of VVDeltaE3L replication.
  • HCMV infection itself led to PKR nuclear accumulation.

Conclusions:

  • HCMV TRS1 and IRS1 proteins directly interact with PKR.
  • This interaction sequesters PKR in the nucleus, away from cytoplasmic dsRNA (activator) and eIF2alpha (substrate).
  • Nuclear sequestration inhibits PKR activation, thereby preventing eIF2alpha phosphorylation and host protein synthesis shutoff during HCMV infection.

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