Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Mar 28;90(8):3839-3848.
doi: 10.1128/JVI.02714-15. Print 2016 Apr.

Human Cytomegalovirus pTRS1 and pIRS1 Antagonize Protein Kinase R To Facilitate Virus Replication

Benjamin Ziehr  1   2 , Heather A Vincent  1   2 , Nathaniel J Moorman  3   2
Affiliations

Human Cytomegalovirus pTRS1 and pIRS1 Antagonize Protein Kinase R To Facilitate Virus Replication

Benjamin Ziehr et al. J Virol. .

Abstract

Human cytomegalovirus (HCMV) counteracts host defenses that otherwise act to limit viral protein synthesis. One such defense is the antiviral kinase protein kinase R (PKR), which inactivates the eukaryotic initiation factor 2 (eIF2) translation initiation factor upon binding to viral double-stranded RNAs. Previously, the viral TRS1 and IRS1 proteins were found to antagonize the antiviral kinase PKR outside the context of HCMV infection, and the expression of either pTRS1 or pIRS1 was shown to be necessary for HCMV replication. In this study, we found that expression of either pTRS1 or pIRS1 is necessary to prevent PKR activation during HCMV infection and that antagonism of PKR is critical for efficient viral replication. Consistent with a previous study, we observed decreased overall levels of protein synthesis, reduced viral protein expression, and diminished virus replication in the absence of both pTRS1 and pIRS1. In addition, both PKR and eIF2α were phosphorylated during infection when pTRS1 and pIRS1 were absent. We also found that expression of pTRS1 was both necessary and sufficient to prevent stress granule formation in response to eIF2α phosphorylation. Depletion of PKR prevented eIF2α phosphorylation, rescued HCMV replication and protein synthesis, and reversed the accumulation of stress granules in infected cells. Infection with an HCMV mutant lacking the pTRS1 PKR binding domain resulted in PKR activation, suggesting that pTRS1 inhibits PKR through a direct interaction. Together our results show that antagonism of PKR by HCMV pTRS1 and pIRS1 is critical for viral protein expression and efficient HCMV replication.

Importance: To successfully replicate, viruses must counteract host defenses that limit viral protein synthesis. We have identified inhibition of the antiviral kinase PKR by the viral proteins TRS1 and IRS1 and shown that this is a critical step in HCMV replication. Our results suggest that inhibiting pTRS1 and pIRS1 function or restoring PKR activity during infection may be a successful strategy to limit HCMV disease.

PubMed Disclaimer

Figures

FIG 1
FIG 1
Expression of either pTRS1or pIRS1 is necessary for efficient HCMV replication. (A) Control cells (Scr) or shIRS1-HFs were infected with wild-type HCMV at an MOI of 3. Cells were harvested at the indicated times and analyzed by Western blotting (n = 3). (B) Control cells and shIRS1-HFs were infected with HCMV lacking TRS1 (HCMVΔTRS1) at an MOI of 3. Cells were harvested at the indicated times and analyzed by Western blotting (n = 3). (C) Cells were infected as described for panel A, and the amount of virus in cell free supernatants was measured by the TCID50 method (n = 3).
FIG 2
FIG 2
HCMV pTRS1 or pIRS1 is necessary to antagonize PKR, maintain infected-cell protein synthesis, and inhibit stress granule formation. (A) Control cells (Scr) or shIRS1-HFs (shIRS1) were infected with either wild-type virus or HCMVΔTRS1 at an MOI of 3. Cells were harvested at 24 h after infection and analyzed by Western blotting (n = 3). Asterisks indicate nonspecific background bands. (B) Cells were infected as described for panel A, and the amount of radiolabeled amino acids incorporated into acid-insoluble protein in 30 min was quantified at 24 h after infection (n = 3; P < 0.05). Filled bars indicate wild-type infection, and open bars indicate HCMVΔTRS1 infection. (C) Control cells or shIRS1-HFs were infected with HCMVΔTRS1, and the formation of G3BP1 puncta was measured by indirect immunofluorescence at 24 h after infection. A representative image from one of three independent experiments is shown.
FIG 3
FIG 3
pTRS1 is sufficient to inhibit stress granule formation. (A) HeLa cells were transfected with a GFP or pTRS1 expression vector. Twenty-four hours after transfection, cells were treated with sodium arsenite (0.5 mM) for 1 h, and G3BP1 punctum formation was measured by indirect immunofluorescence. (B) PKR-deficient HeLa cells were transfected with a GFP or pTRS1 expression vector. Cells were treated with arsenite as described for panel A and analyzed by indirect immunofluorescence as above. (C) Western blotting was used to measure PKR expression in PKR-deficient HeLa cells. PKR-deficient cells were transfected and treated as described for panel A. The presence of G3BP1-positive puncta in transfected cells was determined by indirect immunofluorescence. The range of transgene expression was divided into quartiles (1 to 25% of maximum, 26 to 50% of maximum, etc.), and the results are shown as the percentage of GFP-expressing (top panel) or pTRS1-expressing (bottom panel) cells containing two or more G3BP1 puncta.
FIG 4
FIG 4
PKR inhibits viral protein synthesis and induces stress granule formation in the absence of pTRS1 and pIRS1. (A) shIRS1-HFs were transfected with scrambled or PKR-specific siRNAs prior to infection with HCMVΔTRS1. The formation of G3BP1 (+) puncta was monitored by indirect immunofluorescence at 24 h after infection. A representative image from one of three independent experiments is shown. (B) shIRS1-HFs were transfected with scrambled (scr) or PKR-specific (PKR) siRNAs prior to infection with HCMVΔTRS1. Expression of the indicated proteins was measured by Western blotting at 96 h after infection, except for eIF2α-P and total eIF2α, which were measured at 24 h after infection (n = 3). (C) Cells stably expressing scrambled (Scr) or pIRS1-specific (IRS1) shRNAs were transfected with scrambled (scr) or PKR-specific (PKR) siRNAs and then infected with HCMVΔTRS1. The amount of radiolabeled amino acids incorporated into acid-insoluble protein in 30 min was quantified at 24 h after infection (n = 3). (D) Cells were treated and infected as described for panel C. The amount of virus in the culture supernatants at 96 h after infection was determined by the TCID50 method (n = 3).
FIG 5
FIG 5
The pTRS1 PKR binding domain is required for efficient virus replication. (A) Cartoon showing the region of pTRS1 deleted in the HCMVΔPBD virus. (B) Western blot showing expression of pTRS1 after infection with wild-type HCMV or HCMVΔPBD virus at 72 h after infection. (C) Control HFs (scr; filled bars) or shIRS1-HFs (open bars) were infected with HCMVΔTRS1 or HCMVΔPBD virus at an MOI of 1, and the amount of cell-free virus in the culture supernatants was quantified by the TCID50 method (n = 3).
FIG 6
FIG 6
The pTRS1 PKR binding domain is necessary to antagonize PKR in the absence of pIRS1. (A) Control HFs (Scr) or shIRS1-HFs (shIRS1) were infected with the HCMVΔPBD virus at an MOI of 1. Cells were harvested at the indicated times after infection, and the expression of the indicated proteins was measured by Western blotting (n = 3). (B) Control HFs or shIRS1-HFs were infected with the indicated virus as described for panel A. Cells were harvested at 24 h after infection and analyzed by Western blotting (n = 3). (C) Control HFs (filled bars) or shIRS1-HFs (open bars) were infected with HCMVΔTRS1 or HCMVΔPBD virus, and the amount of radiolabeled amino acids incorporated into acid-insoluble protein in 30 min was quantified 24 h after infection (n = 3). (D) Control HFs or shIRS1-HFs were infected with HCMVΔPBD, and the presence of G3BP1 puncta was determined by indirect immunofluorescence 24 h after infection. Representative images from one of three independent experiments are shown. (E) Cells were transfected with a pTRS1ΔPBD expression vector and treated and analyzed as described for Fig. 3. Representative results from one of three independent experiments are shown.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Black TL, Safer B, Hovanessian A, Katze MG. 1989. The cellular 68,000-Mr protein kinase is highly autophosphorylated and activated yet significantly degraded during poliovirus infection: implications for translational regulation. J Virol 63:2244–2251. - PMC - PubMed
    1. Katze MG, DeCorato D, Safer B, Galabru J, Hovanessian AG. 1987. Adenovirus VAI RNA complexes with the 68 000 Mr protein kinase to regulate its autophosphorylation and activity. EMBO J 6:689–697. - PMC - PubMed
    1. Romano PR, Garcia-Barrio MT, Zhang X, Wang Q, Taylor DR, Zhang F, Herring C, Mathews MB, Qin J, Hinnebusch AG. 1998. Autophosphorylation in the activation loop is required for full kinase activity in vivo of human and yeast eukaryotic initiation factor 2alpha kinases PKR and GCN2. Mol Cell Biol 18:2282–2297. doi:10.1128/MCB.18.4.2282. - DOI - PMC - PubMed
    1. Taylor DR, Lee SB, Romano PR, Marshak DR, Hinnebusch AG, Esteban M, Mathews MB. 1996. Autophosphorylation sites participate in the activation of the double-stranded-RNA-activated protein kinase PKR. Mol Cell Biol 16:6295–6302. doi:10.1128/MCB.16.11.6295. - DOI - PMC - PubMed
    1. Samuel CE. 1979. Mechanism of interferon action: phosphorylation of protein synthesis initiation factor eIF-2 in interferon-treated human cells by a ribosome-associated kinase processing site specificity similar to hemin-regulated rabbit reticulocyte kinase. Proc Natl Acad Sci U S A 76:600–604. doi:10.1073/pnas.76.2.600. - DOI - PMC - PubMed

Publication types