Primate cytomegalovirus assembly: Evidence that DNA packaging occurs subsequent to B capsid assembly

Lee, Joanna Y.; Irmiere, A; Gibson, Wade

doi:10.1016/0042-6822(88)90057-8

Cited by 51 publications

(54 citation statements)

References 47 publications

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“…The unusual viral particles we observed may thus reflect nonspecific effects of impaired viral DNA synthesis (70,71) or, instead, may reflect roles of UL97 during virion morphogenesis, possibly in regulating the solubility or trafficking of the pp65 tegument protein (22,72). Meanwhile, the lack of enveloped C-capsids in the cytoplasm in UL97-deficient TB40/E infections is consistent with the reported nuclear egress defects of ⌬97 AD169 (7).…”

Section: Discussionsupporting

confidence: 78%

“…Our observation of increased levels of noninfectious enveloped particles (NIEPs) and other morphologically unusual particles in the cytoplasm of UL97-deficient TB40/E infections is consistent with reports that inhibition of DNA synthesis in laboratory strain AD169 and in simian cytomegalovirus strain Colburn is associated with increased levels of NIEPs and/or other unusual particles (Table 2) (70,71). The unusual viral particles we observed may thus reflect nonspecific effects of impaired viral DNA synthesis (70,71) or, instead, may reflect roles of UL97 during virion morphogenesis, possibly in regulating the solubility or trafficking of the pp65 tegument protein (22,72).…”

Section: Discussionsupporting

confidence: 75%

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The ULb′ Region of the Human Cytomegalovirus Genome Confers an Increased Requirement for the Viral Protein Kinase UL97

Wang

Schauflinger³

et al. 2013

J Virol

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bWe report a requirement for the viral protein kinase UL97 in human cytomegalovirus (HCMV) replication that maps to the ULb= region of the viral genome. A UL97-null (⌬97) mutant of strain TB40/E, which encodes a full-length ULb= region, exhibited replication defects, particularly in production of cell-free virus, that were more severe than those seen with a ⌬97 mutant of laboratory strain AD169, which harbors extensive deletions in its ULb= region. These differences were recapitulated with additional HCMV strains by treatment with a UL97 kinase inhibitor, 1-(␤-L-ribofuranosyl)-2-isopropylamino-5,6-dichlorobenzimidazole (maribavir). We observed lower levels of viral DNA synthesis and an increased requirement for UL97 in viral late gene expression in strains with full-length ULb= regions. Analysis of UL97-deficient TB40/E infections by electron microscopy revealed fewer C-capsids in nuclei, unusual viral particles in the cytoplasmic assembly compartment, and defective viral nuclear egress. Partial inhibition of viral DNA synthesis caused defects in production of cell-free virus that were up to ϳ100-fold greater than those seen with cell-associated virus in strains TB40/E and TR, suggesting that UL97-dependent defects in cell-free virus production in strains with full-length ULb= regions were secondary to DNA synthesis defects. Accordingly, a chimeric virus in which the ULb= region of TB40/E was replaced with that of AD169 showed reduced effects of UL97 inhibition on viral DNA synthesis, late gene expression, and production of cell-free virus compared to parental TB40/E. Together, these results argue that the ULb= region encodes a factor(s) which invokes an increased requirement for UL97 during viral DNA synthesis.

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Section: Discussionsupporting

confidence: 78%

Section: Discussionsupporting

confidence: 75%

The ULb′ Region of the Human Cytomegalovirus Genome Confers an Increased Requirement for the Viral Protein Kinase UL97

Wang

Schauflinger³

et al. 2013

J Virol

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“…This species is referred to as the scaffolding protein in bacteriophage (9) and as the assembly protein in herpesviruses (10). Unlike the other B-capsid proteins, the assembly protein is phosphorylated (3,11,12) and undergoes proteolytic processing (1,5,12) that eliminates its C-terminal end (13,14), but the enzymes responsible have not been identified.…”

mentioning

confidence: 99%

“…These particles lack DNA and accumulate in the nucleus of infected cells that have been blocked chemically (ref. 3, and references therein) or genetically (4)(5)(6) at the level of viral DNA synthesis. Like bacteriophage proheads, herpesvirus B-capsids or procapsids contain an abundant protein constituent that is not found in mature virions (1,7,8).…”

mentioning

confidence: 99%

A herpesvirus maturational proteinase, assemblin: identification of its gene, putative active site domain, and cleavage site.

Welch

Woods

McNally

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

174

210

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A herpesvirus proteinase activity has been identified and partially characterized by using the cloned enzyme and substrate genes in transient transfection assays. Evidence is presented that the proteinase gene of cytomegalovirus strain Colburn encodes a 590-amino acid protein whose N-terminal 249 residues contain the proteolytic activity and two domains that are highly conserved in the homologous protein ofother herpesviruses. Insertion ofa short amino acid sequence between these domains abolished proteinase activity, suggesting that this region constitutes part or all of the enzyme active site. Plasma desorption mass spectrometry was used to identify the C terminus of the mature assembly protein as alanine, enabling the recognition of a consensus proteinase cleavage sequence of V/L-X-A 4 S/V, near the C-terminal end of all herpesvirus assembly protein homologs. Interestingly, the proteinase and its substrate, the assembly protein precursor, are encoded by opposite halves of the same open reading frame.

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“…Radiolabeling was for 3 days in complete medium containing 400 Ci of 32 P i (ICN, Costa Mesa, Calif.) per milliliter. B-capsids were recovered from the cells by rate-velocity centrifugation, as described previously (16,31), but with phosphatase inhibitors (1 mM sodium vanadate, 10 mM sodium pyrophosphate, 50 mM NaF, 1 M okadaic acid, and 5 M cyclosporine A) (9,30) included in the NP-40 cell disruption solution.…”

Section: Cells and Virusesmentioning

confidence: 99%

Assembly Protein Precursor (pUL80.5 Homolog) of Simian Cytomegalovirus Is Phosphorylated at a Glycogen Synthase Kinase 3 Site and Its Downstream “Priming” Site: Phosphorylation Affects Interactions of Protein with Itself and with Major Capsid Protein

Casaday

Bailey

Kalb

et al. 2004

J Virol

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Capsid assembly among the herpes-group viruses is coordinated by two related scaffolding proteins. In cytomegalovirus (CMV), the main scaffolding constituent is called the assembly protein precursor (pAP). Like its homologs in other herpesviruses, pAP is modified by proteolytic cleavage and phosphorylation. Cleavage is essential for capsid maturation and production of infectious virus, but the role of phosphorylation is undetermined. As a first step in evaluating the significance of this modification, we have identified the specific sites of phosphorylation in the simian CMV pAP. Two were established previously to be adjacent serines (Ser156 and Ser157) in a casein kinase II consensus sequence. The remaining two, identified here as Thr231 and Ser235, are within consensus sequences for glycogen synthase kinase 3 (GSK-3) and mitogen-activated protein kinase, respectively. Consistent with Thr231 being a GSK-3 substrate, its phosphorylation required a downstream "priming" phosphate (i.e., Ser235) and was reduced by a GSK-3-specific inhibitor. Phosphorylation of Ser235 converts pAP to an electrophoretically slower-mobility isoform, pAP*; subsequent phosphorylation of pAP* at Thr231 converts pAP* to a still-slower isoform, pAP**. The mobility shift to pAP* was mimicked by substituting an acidic amino acid for either Thr231 or Ser235, but the shift to pAP** required that both positions be phosphorylated. Glu did not substitute for pSer235 in promoting phosphorylation of Thr231. We suggest that phosphorylation of Thr231 and Ser235 causes charge-driven conformational changes in pAP, and we demonstrate that preventing these modifications alters interactions of pAP with itself and with major capsid protein, suggesting a functional significance.Formation of herpes virions begins with the assembly of procapsids in the nucleus of infected cells (17,47,54). Organization and maturation of these particles requires the involvement of an abundant protein, called the assembly protein precursor (pAP) in cytomegalovirus (CMV) (6,17,56,57). pAP and its genetically related maturational protease have key roles in both early and late steps of the assembly process. One of the first is translocating the major capsid protein (MCP) into the nucleus, which is initiated by self-interaction of pAP through its amino-conserved domain (5, 40, 61). This self-interaction potentiates or stabilizes the interaction of pAP, through its carboxyl-conserved domain, with MCP (2, 24, 40, 61). By forming a complex with MCP, pAP provides the nuclear localization sequences that MCP lacks, enabling the complex to enter the nucleus (42). Within the nucleus, pAP further associates with itself and with the protease precursor to form an internal scaffolding structure that helps organize MCP into the procapsid shell. Ultimately, in preparation for packaging viral DNA into the nascent capsid, pAP is cleaved by the protease (33,45,60) and eliminated from the capsid cavity. In addition to this cleavage, pAP and its homologs in other herpesviruses are phosphorylated (14,...

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Primate cytomegalovirus assembly: Evidence that DNA packaging occurs subsequent to B capsid assembly

Cited by 51 publications

References 47 publications

The ULb′ Region of the Human Cytomegalovirus Genome Confers an Increased Requirement for the Viral Protein Kinase UL97

The ULb′ Region of the Human Cytomegalovirus Genome Confers an Increased Requirement for the Viral Protein Kinase UL97

A herpesvirus maturational proteinase, assemblin: identification of its gene, putative active site domain, and cleavage site.

Assembly Protein Precursor (pUL80.5 Homolog) of Simian Cytomegalovirus Is Phosphorylated at a Glycogen Synthase Kinase 3 Site and Its Downstream “Priming” Site: Phosphorylation Affects Interactions of Protein with Itself and with Major Capsid Protein

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