Article

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.

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    • "Two additional viral products, i.e., pp71 (UL82), a tegument protein; and pp65 (UL83), mostly known for its properties as an immune modulator, were found to transactivate or regulate the MIEP (Homer et al., 1999; Hofmann et al., 2002; Cristea et al., 2010; Arcangeletti et al., 2011; Tomtishen, 2012). Other proteins such as UL97, products of the UL28-UL29 locus and US28 mediate the MIEP via cellular transcription factors (i.e., US28), via HDACs (UL97 and UL28-UL29) or via modulation of the host (UL32/pp150) (Mitchell et al., 2009; Wen et al., 2009; Terhune et al., 2010; Bigley et al., 2013; Bogdanow et al., 2013). Also US3 gene expression is tightly regulated. "
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    • "These viruses grow to reduced titers, have decreased virion stability and exhibit decreased plaque size [23], [24]. The UL26 protein is expressed from a spliced mRNA transcript that is also responsible for the expression of the UL29, UL28, UL27, and UL29/28 open reading frames [25], [30] (Fig. 1A). Previously characterized UL26-null viruses contain large deletions within the UL26 open reading frame which could impact the expression of the other open reading frames that are expressed from this mRNA transcript. "
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    • "Primers for p21 were 5 0 -GGCGTTTGGAGTGGTAGAAAT-3 0 and 5 0 -TGGA GACTCTCAGGGTCGAAA-3 0 . RACE analysis for HCMV transcripts has been previously described (Mitchell et al., 2009). "
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