Translational regulation of the human cytomegalovirus pp28 (UL99) late gene

Human cytomegalovirus-encoded UL92 plays an essential role in viral replication that has not been resolved. We show here that this gene controls the accumulation of true late (γ2) viral transcripts, a property shared with several other recently evaluated genes (UL79, UL87, UL91, and UL95) conserved among beta- and gammaherpesviruses. When the UL92 mutant virus was evaluated, function was fully complemented by either the natural protein or the homologous Rh127 protein from rhesus cytomegalovirus. N-terminal epitope-tagged UL92 protein is functional, follows complex early-late expression kinetics, and localizes in the nucleus within viral replication compartments. UL92 severely impacts the late (72-h postinfection) expression of nine genes encoding virion proteins (UL32, UL55, UL73, UL75, UL80, UL86, UL99, and UL115), as well as UL91 and itself, but does not influence the levels of UL44, UL82, or UL83 accumulation. Although viral DNA is made at normal levels, viral capsid accumulation in the nucleus is severely compromised in UL92 mutant virus-infected cells, and mature virions are not observed in the cytoplasm. Taken together, UL92 is a key regulator of late viral gene expression, apparently functioning with four other beta- or gammaherpesvirus gene products in a pattern that appears reminiscent of gene regulation in T4 DNA bacteriophage.

Section: Discussionmentioning

confidence: 99%

“…Many other viral transcripts are detected at late times (37). True late gene regulation in HCMV (35,38,39), like HSV-1 (40) is controlled by a small, TATA box-proximal region. An unusual, TATT sequence predominates in HCMV ␥ 2 genes (27,29,33,37,39,41).…”

mentioning

confidence: 99%

Transcription of True Late (γ2) Cytomegalovirus Genes Requires UL92 Function That Is Conserved among Beta- and Gammaherpesviruses

Omoto

Mocarski

2014

“…Lastly, UL99-encoded pp28 is expressed with true late kinetics and has been studied as the prototypical gene for this class in promoter studies (26,28). It was not known whether such restricted expression was required or, alternatively, whether misregulation is deleterious.…”

Section: Discussionmentioning

confidence: 99%

“…Unlike the other major phosphoproteins products of UL82 and UL83, which have early-late kinetics, the UL99 phosphoprotein is expressed with strict late kinetics (34). Sequences within 40 bases upstream of the UL99 coding region were identified to be sufficient for its expression with late kinetics (26,28). The UL99 ORF is the 3Ј most ORF in the UL93-UL99 transcription unit (55).…”

mentioning

confidence: 99%

An Acidic Cluster of Human Cytomegalovirus UL99 Tegument Protein Is Required for Trafficking and Function

Jones¹,

Lee²

2004

The human cytomegalovirus (HCMV) virion is comprised of a linear double-stranded DNA genome, proteinaceous capsid and tegument, and a lipid envelope containing virus-encoded glycoproteins. Of these components, the tegument is the least well defined in terms of both protein content and function. Several of the major tegument proteins are phosphoproteins (pp), including pp150, pp71, pp65, and pp28. pp28, encoded by the UL99 open reading frame (ORF), traffics to vacuole-like cytoplasmic structures and was shown recently to be essential for envelopment. To elucidate the UL99 amino acid sequences necessary for its trafficking and function in the HCMV replication cycle, two types of viral mutants were analyzed. Using a series of recombinant viruses expressing various UL99-green fluorescent protein fusions, we demonstrate that myristoylation at glycine 2 and an acidic cluster (AC; amino acids 44 to 57) are required for the punctate perinuclear and cytoplasmic (vacuole-like) localization observed for wild-type pp28. A second approach involving the generation of several UL99 deletion mutants indicated that at least the C-terminal two-thirds of this ORF is nonessential for viral growth. Furthermore, the data suggest that an N-terminal region of UL99 containing the AC is required for viral growth. Regarding virion incorporation or UL99-encoded proteins, we provide evidence that suggests that a hypophosphorylated form of pp28 is incorporated, myristoylation is required, and sequences within the first 57 amino acids are sufficient.

“…The major tegument constituents are pUL83 (the lower matrix protein) (for nomenclature of HCMV proteins, see reference 32), of 65 kDa (24), which constitutes 90% of the dense-body protein mass (17); pUL32 (basic phosphoprotein), of 150 kDa (18), which appears to be most tightly associated with the capsid (14); and pUL82 (the upper matrix protein), of 71 kDa (16,24), which is a transcriptional activator that is able to upregulate the HCMV immediate-early promoter (21). A less abundant tegument protein, which is absent from dense bodies (20,28), is pUL99 (28 kDa) (19,23,29). More recently identified tegument proteins are p130 (pUL56) (3,5,14), p212 (highmolecular-weight protein), pUL48 together with pUL47 (highmolecular-weight-protein-binding protein, 110 kDa) (1,5,14), and finally a subform of the transactivator protein pUL69, the homologue of herpes simplex virus ICP27 (35,36).…”

mentioning

confidence: 99%

Expression and Characterization of a Novel Structural Protein of Human Cytomegalovirus, pUL25

Battista

Bergamini

Boccuni

et al. 1999

Human cytomegalovirus (HCMV) UL25 has recently been found to encode a new structural protein that is present in both virion and defective viral particles (C. J. Baldick and T. Shenk, J. Virol. 70:6097–6105, 1996). In the present work a polyclonal antibody was raised against a prokaryotic pUL25 fusion protein in order to investigate the biosynthesis and localization of the UL25 product (pUL25) during HCMV replication in human fibroblasts. Furthermore, pUL25 was transiently expressed in its native form and fused to the FLAG epitope, in COS7 and U373MG cells, in order to compare the properties of the isolated protein and that produced during infection. Immunoblotting analysis revealed a group of polypeptides, ranging from 80 to 100 kDa, in both transfected and infected cells; in vivo labeling experiments with infected cells demonstrated they are posttranslationally modified by phosphorylation. The transcriptional analysis of the UL25 open reading frame combined with the study of pUL25 biosynthesis showed true late kinetics for this protein in infected human fibroblasts. By indirect immunofluorescence both recombinant and viral pUL25 were detected exclusively in the cytoplasm of transfected or infected cells. Interestingly, pUL25 was shown to localize in typical condensed structures in the perinuclear region as already observed for other HCMV tegument proteins. Colocalization of ppUL99 in the same vacuoles suggests that these structure are endosomal cisternae, which are proposed to be a preferential site of viral particle envelopment. Our data suggest that pUL25 is most likely a novel tegument protein and possibly plays a key role in the process of envelopment.