No virus encodes a ribosome, thus all viruses require the host translation machinery for the synthesis of viral proteins. This fundamental host:pathogen interface creates competition for the host translation machinery, which is a critical barrier to human cytomegalovirus (HCMV) mRNA translation. Many viruses prevent competition for the translation machinery by inhibiting host translation, however host translation is maintained during HCMV infection. How HCMV mRNAs successfully compete for access to host translation machinery to ensure efficient viral protein synthesis is poorly understood, highlighting a critical gap in our knowledge of the mechanisms controlling HCMV mRNA translation. How might HCMV mRNAs compete with host mRNAs for access to ribosomes? Based on our data we hypothesize that HCMV avoids competition by using a unique complement of host and viral translation initiation factors to facilitate viral mRNA translation. Formation of the eIF4F translation initiation complex is the limiting step in host mRNA translation, thus competition between host and viral mRNAs would be most intense for eIF4F components. We showed that the eIF4F complex is necessary for the continued translation of host mRNAs during infection; however HCMV mRNAs translate efficiently when the eIF4F complex is disrupted. We found that translation of the HCMV IE1 and IE2 mRNAs, which encode critical regulators of virus replication, is resistant to eIF4F disruption, depletion or inhibition. How HCMV mRNAs translate in the absence of eIF4F is unknown. As translation initiation factors assemble on the mRNA 5' untranslated region (5'UTR), in Aim1 we determine how RNA sequence and structure in the shared 5'UTR of the IE1 and IE2 mRNAs recruit cellular factors to allow for their eIF4F-independent translation. We also determine how these factors contribute to the widespread eIF4F-independent translation of HCMV mRNAs as a whole.
Aim 2 builds on our published results suggesting that the HCMV TRS1 protein (pTRS1) acts as a viral translation initiation factor to facilitate viral protein synthesis. While pTRS1 antagonizes the antiviral kinase PKR, which inhibits protein synthesis, we showed that pTRS1 stimulates translation in PKR deficient cells. Further, we found that pTRS1 binds the mRNA cap independent of the eIF4F complex and co- purifies with 40S ribosomal subunits, suggesting a role in translation initiation. We now show that pTRS1 interacts with the DHX29 RNA helicase, a translation initiation factor that can recruit ribosomes to mRNAs in the absence of the eIF4F complex, and that DHX29 is necessary for efficient IE1/2 mRNA translation and virus replication.
In Aim 2 we role of pTRS1 and DHX29 in translation initiation complex on HCMV mRNAs, and the role of their interaction in the ability of pTRS1 to enhance translation independent of PKR antagonism. We further determine how pTRS1 binding to DHX29 impacts virus replication and the translation of HCMV mRNAs in the absence of the eIF4F complex. Together these aims will identify regulatory events unique to HCMV mRNA translation that could be targeted to limit HCMV disease.

Public Health Relevance

HCMV is a significant human pathogen that causes severe disease in children and immunocompromised adults. By defining the mechanism(s) by which host and viral factors regulate HCMV mRNA translation, we will uncover new targets for novel antiviral therapeutics that specifically target viral protein expression, and thus inhibit virus replication and limit HCMV disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI103311-07
Application #
9743685
Study Section
Virology - B Study Section (VIRB)
Program Officer
Beisel, Christopher E
Project Start
2012-12-01
Project End
2023-06-30
Budget Start
2019-07-01
Budget End
2020-06-30
Support Year
7
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Arend, Kyle C; Lenarcic, Erik M; Moorman, Nathaniel J (2018) The 5' Untranslated Region of the Major Immediate Early mRNA Is Necessary for Efficient Human Cytomegalovirus Replication. J Virol 92:
Kutchko, Katrina M; Madden, Emily A; Morrison, Clayton et al. (2018) Structural divergence creates new functional features in alphavirus genomes. Nucleic Acids Res 46:3657-3670
Hawman, David W; Carpentier, Kathryn S; Fox, Julie M et al. (2017) MUTATIONS IN THE E2 GLYCOPROTEIN AND THE 3' UNTRANSLATED REGION ENHANCE CHIKUNGUNYA VIRUS VIRULENCE IN MICE. J Virol :
Feng, Hui; Lenarcic, Erik M; Yamane, Daisuke et al. (2017) NLRX1 promotes immediate IRF1-directed antiviral responses by limiting dsRNA-activated translational inhibition mediated by PKR. Nat Immunol 18:1299-1309
Borchardt, Erin K; Meganck, Rita M; Vincent, Heather A et al. (2017) Inducing circular RNA formation using the CRISPR endoribonuclease Csy4. RNA 23:619-627
Westdorp, Kristen N; Sand, Andrea; Moorman, Nathaniel J et al. (2017) Cytomegalovirus Late Protein pUL31 Alters Pre-rRNA Expression and Nuclear Organization during Infection. J Virol 91:
Vincent, Heather A; Ziehr, Benjamin; Moorman, Nathaniel J (2017) Mechanism of Protein Kinase R Inhibition by Human Cytomegalovirus pTRS1. J Virol 91:
Arend, Kyle C; Lenarcic, Erik M; Vincent, Heather A et al. (2017) Kinome Profiling Identifies Druggable Targets for Novel Human Cytomegalovirus (HCMV) Antivirals. Mol Cell Proteomics 16:S263-S276
Corley, Meredith; Solem, Amanda; Phillips, Gabriela et al. (2017) An RNA structure-mediated, posttranscriptional model of human ?-1-antitrypsin expression. Proc Natl Acad Sci U S A 114:E10244-E10253
Peppenelli, Megan A; Arend, Kyle C; Cojohari, Olesea et al. (2016) Human Cytomegalovirus Stimulates the Synthesis of Select Akt-Dependent Antiapoptotic Proteins during Viral Entry To Promote Survival of Infected Monocytes. J Virol 90:3138-47

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