Dataset Information

Regulation of the translation initiation factor eIF4F by multiple mechanisms in human cytomegalovirus-infected cells.

ABSTRACT: As a viral opportunistic pathogen associated with serious disease among the immunocompromised and congenital defects in newborns, human cytomegalovirus (HCMV) must engage the translational machinery within its host cell to synthesize the viral proteins required for its productive growth. However, unlike many viruses, HCMV does not suppress the translation of host polypeptides. Here, we examine how HCMV regulates the cellular cap recognition complex eIF4F, a critical component of the cellular translation initiation apparatus that recruits the 40S ribosome to the 5' end of the mRNA. This study establishes that the cap binding protein eIF4E, together with the translational repressor 4E-BP1, are both phosphorylated early in the productive viral growth cycle and that the activity of the cellular eIF4E kinase, mnk, is critical for efficient viral replication. Furthermore, HCMV replication also induces an increase in the overall abundance of eIF4F components and promotes assembly of eIF4F complexes. Notably, increasing the abundance of select eIF4F core components and associated factors alters the ratio of active eIF4F complexes in relation to the 4E-BP1 translational repressor, illustrating a new strategy through which members of the herpesvirus family enhance eIF4F activity during their replicative cycle.

SUBMITTER: Walsh D

PROVIDER: S-EPMC1143722 | biostudies-literature | 2005 Jul

REPOSITORIES: biostudies-literature

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Regulation of the translation initiation factor eIF4F by multiple mechanisms in human cytomegalovirus-infected cells.

Walsh Derek D Perez Cesar C Notary Joanna J Mohr Ian I

Journal of virology 20050701 13

As a viral opportunistic pathogen associated with serious disease among the immunocompromised and congenital defects in newborns, human cytomegalovirus (HCMV) must engage the translational machinery within its host cell to synthesize the viral proteins required for its productive growth. However, unlike many viruses, HCMV does not suppress the translation of host polypeptides. Here, we examine how HCMV regulates the cellular cap recognition complex eIF4F, a critical component of the cellular tra ...[more]

PMID: 15956551

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Project description:Eukaryotic translation initiation factor 4GI (eIF4GI) is an essential protein that is the target for translational regulation in many cellular processes and viral systems. It has been shown to function in both cap-dependent and cap-independent translation initiation by recruiting the 40S ribosomal subunit to the mRNA cap structure or internal ribosome entry site (IRES) element, respectively. Interestingly eIF4GI mRNA itself has been reported to contain an IRES element in its 5' end that facilitates eIF4GI protein synthesis via a cap-independent mechanism. In HeLa cells, eIF4GI exists as several isoforms that differ in their migration in sodium dodecyl sulfate (SDS) gels; however, the nature of these isoforms was unclear. Here, we report a new cDNA clone for eIF4GI that extends the 5' sequence 340 nucleotides beyond the previously published sequence. The new extended sequence of eIF4GI is located on chromosome 3, within two additional exons immediately upstream of the previously published eIF4GI sequence. When mRNA transcribed from this cDNA clone was translated in vitro, five eIF4GI polypeptides were generated that comigrated in SDS-polyacrylamide gels with the five isoforms of native eIF4GI. Furthermore, translation of eIF4GI-enhanced green fluorescent protein fusion constructs in vitro or in vivo generated five isoforms of fusion polypeptides, suggesting that multiple isoforms of eIF4GI are generated by alternative translation initiation in vitro and in vivo. Mutation of two of the five in-frame AUG residues in the eIF4GI cDNA sequence resulted in loss of corresponding polypeptides after translation in vitro, confirming alternate use of AUGs as the source of the multiple polypeptides. The 5' untranslated region of eIF4GI mRNA also contains an out-of-frame open reading frame (ORF) that may down-regulate expression of eIF4GI. Further, data are presented to suggest that a proposed IRES embedded in the eIF4GI ORF is able to catalyze synthesis of multiple eIF4GI isoforms as well. Our data suggest that expression of the eIF4GI isoforms is partly controlled by a complex translation strategy involving both cap-dependent and cap-independent mechanisms.

Project description:Human cytomegalovirus (HCMV) utilizes peripheral blood monocytes as a means to systemically disseminate throughout the host. Following viral entry, HCMV stimulates non-canonical Akt signaling leading to the activation of mTORC1 and the subsequent translation of select antiapoptotic proteins within infected monocytes. However, the full extent to which the HCMV-initiated Akt/mTORC1 signaling axis reshapes the monocyte translatome is unclear. We found HCMV entry alone was able to stimulate widescale changes to mRNA translation levels and that inhibition of mTOR, a component of mTORC1, dramatically attenuated HCMV-induced protein synthesis. Although monocytes treated with normal myeloid growth factors also exhibited increased levels of translation, mTOR inhibition had no effect, suggesting HCMV activation of mTOR stimulates the acquisition of a unique translatome within infected monocytes. Indeed, polyribosomal profiling of HCMV-infected monocytes identified distinct prosurvival transcripts that were preferentially loaded with ribosomes when compared to growth factor-treated cells. Sirtuin 1 (SIRT1), a deacetylase that exerts prosurvival effects through regulation of the PI3K/Akt pathway, was found to be highly enriched following HCMV infection in an mTOR-dependent manner. Importantly, SIRT1 inhibition led to the death of HCMV-infected monocytes while having minimal effect on uninfected cells. SIRT1 also supported a positive feedback loop to sustain Akt/mTORC1 signaling following viral entry. Taken together, HCMV profoundly reshapes mRNA translation in an mTOR-dependent manner to enhance the synthesis of select factors necessary for the survival of infected monocytes.IMPORTANCEHuman cytomegalovirus (HCMV) infection is a significant cause of morbidity and mortality among the immunonaïve and immunocompromised. Peripheral blood monocytes are a major cell type responsible for disseminating the virus from the initial site of infection. In order for monocytes to mediate viral spread within the host, HCMV must subvert the naturally short lifespan of these cells. In this study, we performed polysomal profiling analysis, which demonstrated HCMV to globally redirect mRNA translation toward the synthesis of cellular prosurvival factors within infected monocytes. Specifically, HCMV entry into monocytes induced the translation of cellular SIRT1 to generate an antiapoptotic state. Defining the precise mechanisms through which HCMV stimulates survival will provide insight into novel anti-HCMV drugs able to target infected monocytes.

Dataset Information

Regulation of the translation initiation factor eIF4F by multiple mechanisms in human cytomegalovirus-infected cells.

Publications

Regulation of the translation initiation factor eIF4F by multiple mechanisms in human cytomegalovirus-infected cells.

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