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

Abstract

The host eIF4F translation initiation complex plays a critical role the translation of capped mRNAs. Although human cytomegalovirus (HCMV) infection increases the abundance and activity of the host eIF4F complex, the requirement for eIF4F components in HCMV replication and mRNA translation has not been directly tested. In this study, we found that decreasing the abundance or activity of eIF4F from the start of infection inhibits HCMV replication. However, as infection progresses, viral mRNA translation and replication becomes increasingly resistant to eIF4F inhibition. During the late stage of infection the association of representative immediate-early, early, and late mRNAs with polysomes was not affected by eIF4F disruption. In contrast, eIF4F inhibition decreased the translation of representative host eIF4F-dependent mRNAs during the late stage of infection. A global analysis of the translation efficiency of HCMV mRNAs during the late stage of infection found that eIF4F disruption had a minimal impact on the association of HCMV mRNAs with polysomes but significantly diminished the translation efficiency of eIF4F-dependent host transcripts. Together, our data show that the translation of host eIF4F-dependent mRNAs remains dependent on eIF4F activity during HCMV infection. However, during the late stage of infection the translation efficiency of viral mRNAs does not correlate with the abundance or activity of the host eIF4F complex.

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“…The locations of ribosomal subunits, monosomes, and polysomes in the gradient were determined by continuous monitoring of the absorbance at an optical density of 254 nm (OD 254 ) during gradient fractionation using a Brandel gradient fractionator system coupled to a spectrophotometer (ISCO). We routinely monitored the efficiency of fractionation by performing Western blotting for the nuclear protein lamin A/C and the cytoplasmic protein tubulin (38). Total RNA was extracted from an equal volume of each gradient fraction using TRIzol, and contaminating DNA was removed with DNase (Ambion, Austin, TX).…”

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“…Unless otherwise noted, all infections were performed at a multiplicity of infection of 1 in 300 l of medium. The amount of cell-free virus in the supernatant was quantified by the TCID 50 method on MRC-5 fibroblasts as previously described (50).…”

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“…Total RNA was extracted using TRIzol according to the manufacturer's directions as described previously (47,48). Briefly, cell pellets were resuspended in TRIzol and extracted with chloroform, and RNA was precipitated with isopropanol.…”

“…5= rapid amplification of cDNA ends (5= RACE) was performed using the RLM-RACE (Ambion) kit according to the manufacturer's protocol as previously described (47). Briefly, total or polysome-associated RNA was treated with calf intestinal phosphatase (CIP) to dephosphorylate uncapped or degraded RNAs.…”

“…Polysomes and polysome-associated RNAs were isolated as described previously (47,48). Briefly, at the time of harvest, cells were incubated in normal growth medium containing 0.1 mg/ml cycloheximide (CHX) for 10 min.…”