Translational Control of the Abundance of Cytoplasmic Poly(A) Binding Protein in Human Cytomegalovirus-Infected Cells

Abstract

Irrespective of their effects on ongoing host protein synthesis, productive replication of the representative alphaherpesvirus herpes simplex virus type 1, the representative gammaherpesvirus Kaposi's sarcoma herpesvirus, and the representative betaherpesvirus human cytomegalovirus [HCMV] stimulates the assembly of the multisubunit, cap-binding translation factor eIF4F. However, only HCMV replication is associated with an increased abundance of eIF4F core components (eIF4E, eIF4G, eIF4A) and the eIF4F-associated factor poly(A) binding protein (PABP). Here, we demonstrate that the increase in translation factor concentration was readily detected in an asynchronous population of HCMV-infected primary human fibroblasts, abolished by prior UV inactivation of virus, and genetically dependent upon viral immediate-early genes. Strikingly, while increased mRNA steady-state levels accompanied the rise in eIF4E and eIF4G protein levels, the overall abundance of PABP mRNA, together with the half-life of the polypeptide it encodes, remained relatively unchanged by HCMV infection. Instead, HCMV-induced PABP accumulation resulted from new protein synthesis and was sensitive to the mTORC1-selective inhibitor rapamycin, which interferes with phosphorylation of the mTORC1 substrate p70 S6K and the translational repressor 4E-BP1. While virus-induced PABP accumulation did not require p70 S6K, it was inhibited by the expression of a dominant-acting 4E-BP1 variant unable to be inactivated by mTORC1. Finally, unlike the situation in alpha-or gammaherpesvirus-infected cells, where PABP is redistributed to nuclei, PABP accumulated in the cytoplasm of HCMV-infected cells. Thus, cytoplasmic PABP accumulation is translationally controlled in HCMV-infected cells via a mechanism requiring mTORC1-mediated inhibition of the cellular 4E-BP1 translational repressor.Herpesvirus mRNAs contain methyl-7-GTP caps and 3Ј polyadenylate tails like their host cell counterparts and are primarily translated by a cap-dependent mechanism. Assembly of the cap-binding protein eIF4E, eIF4G, and the RNA helicase eIF4A into the active, cap-binding, multisubunit translation initiation factor eIF4F represents a key step regulating translation (reviewed in reference 33). In addition to controlling small ribosome subunit recruitment to the mRNA 5Ј end, whereupon a scanning mechanism commences to locate the initiator AUG codon, eIF4F assembly is responsive to a diverse assortment of cell stress and signaling inputs, including viral infection (24). Typically, eIF4E is bound to the translational repressor 4E-BP1. Hyperphosphorylation of 4E-BP1 by activated mTORC1 relieves this repression, releasing eIF4E and exposing the binding site for eIF4G, a large assembly platform bound to eIF4A. eIF4G also binds eIF3, which directly associates with the 40S ribosome subunit. The cellular poly(A) binding protein (PABP) and the eIF4E kinase Mnk are eIF4F-associated proteins that physically associate with eIF4G and act to stimulate translation. Bound to both the 3Ј poly(A) tail and...

A new role for the cellular PABP repressor Paip2 as an innate restriction factor capable of limiting productive cytomegalovirus replication

“…The HCMV-induced increase in EDD1 protein abundance was not accompanied by a detectable corresponding increase in steady-state mRNA levels, whereas the increase in Paip2 protein was only accompanied by a modest increase in mRNA (Fig. 1C), suggesting an underlying post-transcriptional control strategy similar to what we previously defined for PABP1 (Perez et al 2011). Importantly, while reducing PABP1 abundance has been shown to trigger a corresponding decrease in Paip2 and EDD1 levels (Yoshida et al 2006), this is the first demonstration that these factors are coordinately up-regulated.…”

“…To ensure that viral mRNAs (which contain 7-methyl guanosine [m 7 G]-capped 59 and 39 polyadenylated termini) effectively compete for limiting translation factors, HCMV infection triggers an increase in the intracellular concentration of key initiation factors, including the cellular polyadenylate-binding protein PABPC1 (PABP1) and the multisubunit cap-binding complex eIF4F (Kudchodkar et al 2004;Isler et al 2005;Walsh et al 2005;Perez et al 2011). Comprised of the cap-binding protein eIF4E and the RNA helicase eIF4A bound to the large molecular scaffold eIF4G, eIF4F complex assembly is regulated on the m 7 GTP cap structure, facilitating 40S ribosome subunit loading onto the mRNA 59 end (Jackson et al 2010).…”

The Changing Role of mTOR Kinase in the Maintenance of Protein Synthesis during Human Cytomegalovirus Infection

“…We also examined the expression of eIF4E and eIF4G in the input samples. Here, we reiterate previous findings that both of these proteins are increased in HCMV-infected cells under normal conditions (27). In addition, the data suggest that the levels of both eIF4E and eIF4G are diminished in mock-infected cells as a result of 4E-BP1 depletion and Torin1 treatment.…”

Differential Role for Host Translation Factors in Host and Viral Protein Synthesis during Human Cytomegalovirus Infection

“…8A), which are translated in an eIF4F-dependent manner. We found that several host mRNAs previously shown to require eIF4F for their translation during HCMV infection were translated less efficiently in the presence of Torin1 (31). In contrast, the relative translation efficiency of HCMV mRNAs was for the most part similar in untreated and Torin1-treated cells (Fig.…”