High-resolution human cytomegalovirus transcriptome

Abstract

Deep sequencing was used to bring high resolution to the human cytomegalovirus (HCMV) transcriptome at the stage when infectious virion production is under way, and major findings were confirmed by extensive experimentation using conventional techniques. The majority (65.1%) of polyadenylated viral RNA transcription is committed to producing four noncoding transcripts (RNA2.7, RNA1.2, RNA4.9, and RNA5.0) that do not substantially overlap designated protein-coding regions. Additional noncoding RNAs that are transcribed antisense to protein-coding regions map throughout the genome and account for 8.7% of transcription from these regions. RNA splicing is more common than recognized previously, which was evidenced by the identification of 229 potential donor and 132 acceptor sites, and it affects 58 protein-coding genes. The great majority (94) of 96 splice junctions most abundantly represented in the deep-sequencing data was confirmed by RT-PCR or RACE or supported by involvement in alternative splicing. Alternative splicing is frequent and particularly evident in four genes (RL8A, UL74A, UL124, and UL150A) that are transcribed by splicing from any one of many upstream exons. The analysis also resulted in the annotation of four previously unrecognized protein-coding regions (RL8A, RL9A, UL150A, and US33A), and expression of the UL150A protein was shown in the context of HCMV infection. The overall conclusion, that HCMV transcription is complex and multifaceted, has implications for the potential sophistication of virus functionality during infection. The study also illustrates the key contribution that deep sequencing can make to the genomics of nuclear DNA viruses.

Fig. 1.

Transcription and splicing of the HCMV genome. The genome is shown in five sections, with the inverted repeats (ab/b′a′ and a′c′/ca) shaded gray. CRs are shown by color-shaded arrows and NNTs as white-shaded arrows, with gene nomenclature below. Introns connecting CRs or the RNA5.0 exons are shown as narrow white bars. Colors applied to CRs indicate conservation among α-, β-, and γ-herpesviruses (core genes) or between β- and γ-herpesviruses (subcore genes), with subsets of the noncore genes grouped into families of related genes. UL72 is both a member of the deoxyuridine triphosphatase-related protein (DURP) gene family and a core gene. The light yellow windows depict transcription profiles (Methods) as explained in the key in the bottom right corner. The plots in the lower windows show rightward and leftward transcripts combined: the NDS profile on linear and logarithmic scales (right and left, respectively) and the DDS profile on a logarithmic scale. The plots in the upper windows show the DDS profiles for rightward and leftward transcripts separately, on a logarithmic scale (right and left, respectively). The horizontal bar above the gene nomenclature shows regions where rightward (magenta) or leftward (cyan) transcription predominates. The vertical lines in the lower windows show the locations of the subset of splice sites supported by >10 reads, oriented rightward or leftward as explained in the key. The height of the line depicts the number of reads representing the site, plotted on a logarithmic scale (left). The complete list of splice sites is provided in Dataset S1.

Fig. 2.

Immunoblot analysis of V5-tagged UL150A proteins expressed in the context of HCMV. Proteins harvested at 1, 3, and 6 d after infection from mock-infected cells (MI) or cells infected with the HCMV recombinant (UL150A) were probed using a V5-Tag antibody. Estimated masses (kDa) are shown.