Human cytomegalovirus UL28 and UL29 open reading frames encode a spliced mRNA and stimulate accumulation of immediate-early RNAs.

Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA.
Journal of Virology (Impact Factor: 4.65). 08/2009; 83(19):10187-97. DOI: 10.1128/JVI.00396-09
Source: PubMed

ABSTRACT We have identified a spliced transcript that contains sequences from the HCMV UL29 and UL28 open reading frames. It contains amino-terminal UL29 sequences followed by UL28 sequences, and it includes a poly(A) signal derived from the 3'-untranslated region following the UL26 open reading frame. UL29/28 RNA is expressed with early kinetics, and a virus containing a FLAG epitope inserted at the amino terminus of UL29 expressed a tagged approximately 79-kDa protein, pUL29/28, that was detected at 6 h postinfection. The virus also expressed a less-abundant tagged 41-kDa protein, which corresponds in size to a protein that could be produced by translation of an unspliced UL29/28 transcript. Consistent with this prediction, both unspliced and spliced UL29/28 transcript was present in RNA isolated from polysomes. FLAG-tagged protein from the UL29/28 locus accumulated within nuclear viral replication centers during the early phase of infection. Late after infection it was present in the cytoplasm as well, and the protein was present and resistant to proteinase treatment in partially purified preparations of viral particles. Disruption of the UL29/28 locus by mutation resulted in a 10-fold decrease in the levels of DNA replication along with a similar reduction in virus yield. Quantitative reverse transcription-PCR analysis revealed an approximately 2-fold decrease in immediate-early gene expression at 4 to 10 h postinfection compared to the wild-type virus, and transient expression of pUL29/28 activated the major immediate-early promoter. Our results argue that the UL29/28 locus contributes to activation of immediate-early gene expression.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The human cytomegalovirus (HCMV) UL26 gene encodes a virion protein that is important for high titer viral replication. To identify specific domains within the UL26 protein that contribute to viral infection, we created a panel of site-directed UL26 mutant viruses and assessed their impact on phenotypes attributed to UL26. We find that the C-terminal 38 amino acids of the UL26 protein are absolutely necessary for UL26 function. A stop-insertion mutant that produced a truncated UL26 protein lacking this region behaved identically to UL26-null viruses. This included reduced accumulation of IE1 protein at early time points, smaller plaque size, reduced virion stability, and growth with similarly attenuated kinetics. This C-terminal truncation decreased the amount of UL26 packaged into the virion resulting in reduced delivery of UL26 to newly infected cells. Further, this C-terminal truncated UL26 exhibited substantially reduced nuclear localization compared to wildtype UL26. Translation of UL26 mRNA is initiated from two separate in frame methionines that give rise to a long and a short isoform of UL26. We find that the N-terminal 34 amino acids, which are unique to the long isoform of UL26, are also important for the function of the UL26 protein. A viral mutant that produces only the short isoform of UL26 and lacks these N-terminal 34 amino acids exhibits delayed IE1 accumulation, and demonstrates intermediate defects in viral plaque size, virion stability and viral growth kinetics. Ablation of the short UL26 isoform in the presence of the long UL26 isoform did not impact any of the in vitro phenotypes tested. These experiments highlight important domains within the UL26 protein that contribute to HCMV infection.
    PLoS ONE 02/2014; 9(2):e88101. · 3.53 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Human cytomegalovirus is an opportunistic double-stranded DNA virus with one of the largest viral genomes known. The 235 kB genome is divided in a unique long (UL) and a unique short (US) region which are flanked by terminal and internal repeats. The expression of HCMV genes is highly complex and involves the production of protein coding transcripts, polyadenylated long non-coding RNAs, polyadenylated anti-sense transcripts and a variety of non-polyadenylated RNAs such as microRNAs. Although the function of many of these transcripts is unknown, they are suggested to play a direct or regulatory role in the delicately orchestrated processes that ensure HCMV replication and life-long persistence. This review focuses on annotating the complete viral genome based on three sources of information. First, previous reviews were used as a template for the functional keywords to ensure continuity; second, the Uniprot database was used to further enrich the functional database; and finally, the literature was manually curated for novel functions of HCMV gene products. Novel discoveries were discussed in light of the viral life cycle. This functional annotation highlights still poorly understood regions of the genome but more importantly it can give insight in functional clusters and/or may be helpful in the analysis of future transcriptomics and proteomics studies.
    Frontiers in Microbiology 05/2014; 5:218. · 3.94 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Human cytomegalovirus (HCMV) infection, a worldwide contagion, causes a serious disorder in infected individuals. Analysis of codon usage can reveal much molecular information about this virus. The effective number of codon (ENC) values, relative synonymous codon usage (RSCU) values, codon adaptation index (CAI), and nucleotide contents was investigated in approximately 160 coding sequences (CDS) among 17 human cytomegalovirus genomes using the software CodonW. Linear regression analysis and logistic regression were performed to explore the preliminary data. The results showed that, overall, HCMV genomes had low codon usage bias (mean ENC=47.619). However, the ENC of individual CDS varied widely and was distributed unevenly between host-related genes and viral-self-function genes (P=0.002, odds ratio (OR)=3.194), as did the GC content (P=0.016, OR=2.178). The ENC values correlated with CAI, GC content, and the nucleotide composing at the 3rd codon position (GC3s) (P<0.001). There was a significant variation in the codon preference that depended on the RSCU data. The predicted ENC curve suggested that mutational pressure, rather than natural selection, was one of the main factors that determined the codon usage bias in HCMV. Among 123 genes with known function, the genes related to viral self-replication and viral-host interaction showed different ENC and CAI values, and GC and GC3s contents. In conclusion, the detailed codon usage bias theoretically revealed information concerning HCMV evolution and could be a valuable additional parameter for HCMV gene function research.
    Gene 05/2014; · 2.20 Impact Factor

Full-text (2 Sources)

Available from
May 16, 2014