Decoding human cytomegalovirus

Abstract

The human cytomegalovirus (HCMV) genome was sequenced 20 years ago. However, like those of other complex viruses, our understanding of its protein coding potential is far from complete. We used ribosome profiling and transcript analysis to experimentally define the HCMV translation products and follow their temporal expression. We identified hundreds of previously unidentified open reading frames and confirmed a fraction by means of mass spectrometry. We found that regulated use of alternative transcript start sites plays a broad role in enabling tight temporal control of HCMV protein expression and allowing multiple distinct polypeptides to be generated from a single genomic locus. Our results reveal an unanticipated complexity to the HCMV coding capacity and illustrate the role of regulated changes in transcript start sites in generating this complexity.

Fig. 1

Ribosome profiling of HCMV infected cells. (A) Ribosome occupancies following various treatments (illustrated on the left); cycloheximide (CHX), No-drug, harringtonine (Harr) and LTM together with mRNA profiles of the UL25 gene at 72 hpi. An arrow marks the mRNA start. (B,C) Ribosome occupancy profiles for UL38 (B) and UL10(C) genes that contain internal initiations. The grey area symbolizes a low complexity region.

Fig. 2

Many ribosome footprints do not correspond to previously annotated ORFs. (A) Ribosome occupancy profiles for the leader region of UL139 gene. (B) Ribosome occupancy profiles of plus and minus strands (red and blue respectively) for the UL91 gene. (C) mRNA and ribosome occupancy profiles for a novel short ORF. (D) Ribosome occupancies around a short ORF that initiates at a CUG codon. (E) Ribosome occupancy profiles for RNA β2.7. The upper panels show the annotated MS/MS spectra of two unique peptides originating from ORFL6C and ORFL7C.

Fig. 3

Annotating the HCMV translated ORFs. (A) Fold enrichment of AUG and near-cognate codons at predicted sites of translation initiation compared to their genomic distribution. (B) The ribosome footprints occupancy after LTM treatment at each start codon (relative to the median density across the gene) is depicted for the previously annotated ORFs (blue) and newly identified ORFs (red and empty red for ORFs that were removed). The occupancy at a codon five positions downstream of the start codon is depicted as a control (green). (C) Venn diagram summarizing the HCMV translated ORFs. Note 53 ORFs were initially identified by manual inspection (see text). (D) The lengths distribution of newly identified ORFs (red) and previously annotated ORFs (blue). (E) Position of 30-nt ribosome footprints relative to the reading frame in the newly identified ORFs (red) and previously annotated ORFs (blue). (F) MRC-5 cells were mock-treated or infected with TB40-US33A-HA and protein lysates were analyzed by western blotting with indicated antibodies. (G) HeLa cells were transfected with GFP fusion proteins together with an ER marker (KDEL-mCherry) or stained with MitoTracker Red and imaged by confocal microscopy.

Fig. 4

A major source of ORFs diversity during infection originates from alternative transcripts starts. (A) The mRNA and ribosome occupancy profiles around US18-US20 loci at different infection times (marked on the left). Small arrows denote the different mRNA starts and the corresponding mRNAs are illustrated (upper part). The lower panel shows an expanded view of the US18 locus at 72 hpi and includes the harringtonine and LTM profiles (the internal initiation is marked with a star). (B) Total RNA extracted at different time points during infection was subjected to Northern blotting for ORFS346C.1