Recovery of an HMWP/hmwBP (pUL48/pUL47) complex from virions of human cytomegalovirus: subunit interactions, oligomer composition, and deubiquitylase activity.

Unlabelled: We report that the human cytomegalovirus (HCMV) high-molecular-weight tegument protein (HMWP, pUL48; 253 kDa) and the HMWP-binding protein (hmwBP, pUL47; 110 kDa) can be recovered as a complex from virions disrupted by treatment with 50 mM Tris (pH 7.5), 0.5 M NaCl, 0.

Nelfinavir inhibits maturation and export of herpes simplex virus 1.

Unlabelled: Nelfinavir (NFV) is an HIV-1 protease inhibitor with demonstrated antiviral activity against herpes simplex virus 1 (HSV-1) and several other herpesviruses. However, the stages of HSV-1 replication inhibited by NFV have not been explored. In this study, we investigated the effects of NFV on capsid assembly and envelopment.

Cytomegalovirus capsid protease: biological substrates are cleaved more efficiently by full-length enzyme (pUL80a) than by the catalytic domain (assemblin).

We compared the full-length capsid maturational protease (pPR, pUL80a) of human cytomegalovirus with its proteolytic domain (assemblin) for the ability to cleave two biological substrates, and we found that pPR is more efficient with both. Affinity-purified, refolded enzymes and substrates were combined under defined reaction conditions, and cleavage was monitored and quantified following staining of the resulting electrophoretically separated fragments. The enzymes were stabilized against self-cleavage by a single point mutation in each cleavage site (ICRMT-pPR and IC-assemblin).

Cleavage specificity of the UL48 deubiquitinating protease activity of human cytomegalovirus and the growth of an active-site mutant virus in cultured cells.

The human cytomegalovirus (HCMV) open reading frame UL48 encodes a 253-kDa tegument protein that is closely associated with the capsid and was recently shown to have ubiquitin-specific protease activity (J. Wang, A. N.

Nuclear localization sequences in cytomegalovirus capsid assembly proteins (UL80 proteins) are required for virus production: inactivating NLS1, NLS2, or both affects replication to strikingly different extents.

Scaffolding proteins of spherical prokaryotic and eukaryotic viruses have critical roles in capsid assembly. The primary scaffolding components of cytomegalovirus, called the assembly protein precursor (pAP, pUL80.5) and the maturational protease precursor (pPR, pUL80a), contain two nuclear localization sequences (NLS1 and NLS2), at least one of which is required in coexpression experiments to translocate the major capsid protein (MCP, pUL85) into the nucleus.

Enzymatic activities of human cytomegalovirus maturational protease assemblin and its precursor (pPR, pUL80a) are comparable: [corrected] maximal activity of pPR requires self-interaction through its scaffolding domain.

Herpesviruses encode an essential, maturational serine protease whose catalytic domain, assemblin (28 kDa), is released by self-cleavage from a 74-kDa precursor (pPR, pUL80a). Although there is considerable information about the structure and enzymatic characteristics of assemblin, a potential pharmacologic target, comparatively little is known about these features of the precursor. To begin studying pPR, we introduced five point mutations that stabilize it against self-cleavage at its internal (I), cryptic (C), release (R), and maturational (M) sites and at a newly discovered "tail" (T) site.

The amino-conserved domain of human cytomegalovirus UL80a proteins is required for key interactions during early stages of capsid formation and virus production.

Assembly of many spherical virus capsids is guided by an internal scaffolding protein or group of proteins that are often cleaved and eliminated in connection with maturation and incorporation of the genome. In cytomegalovirus there are at least two proteins that contribute to this scaffolding function; one is the maturational protease precursor (pUL80a), and the other is the assembly protein precursor (pUL80.5) encoded by a shorter genetic element within UL80a.

The chemokine receptor homologue encoded by US27 of human cytomegalovirus is heavily glycosylated and is present in infected human foreskin fibroblasts and enveloped virus particles.

Human cytomegalovirus (HCMV), a member of the beta-herpesvirus family, encodes four homologues of cellular G protein-coupled receptors (GPCRs). One of these, the protein product of HCMV open reading frame (ORF) UL33, has been identified in HCMV-infected cells and virus particles and shown to be heat-aggregatable and N-glycosylated. Another, the product of ORF US28, has been functionally characterized as a beta-chemokine receptor.

Cleavage of human cytomegalovirus protease pUL80a at internal and cryptic sites is not essential but enhances infectivity.

The cytomegalovirus (CMV) maturational protease, assemblin, contains an "internal" (I) cleavage site absent from its homologs in other herpesviruses. Blocking this site for cleavage did not prevent replication of the resulting I(-) mutant virus. However, cells infected with the I(-) virus showed increased amounts of a fragment produced by cleavage at the nearby "cryptic" (C) site, suggesting that its replication may bypass the I-site block by using the C site as an alternate cleavage pathway.

Chemical rescue of I-site cleavage in living cells and in vitro discriminates between the cytomegalovirus protease, assemblin, and its precursor, pUL80a.

Chemical rescue is an established approach that offers a directed strategy for designing mutant enzymes in which activity can be restored by supplying an appropriate exogenous compound. This method has been used successfully to study a broad range of enzymes in vitro, but its application to living systems has received less attention. We have investigated the feasibility of using chemical rescue to make a conditional-lethal mutant of the cytomegalovirus (CMV) maturational protease.

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.

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.

Cytomegalovirus assemblin (pUL80a): cleavage at internal site not essential for virus growth; proteinase absent from virions.

The human cytomegalovirus (HCMV) maturational proteinase is synthesized as an enzymatically active 74-kDa precursor that cleaves itself at four sites. Two of these, called the maturational (M) and release (R) sites, are conserved in the homologs of all herpesviruses. The other two, called the internal (I) and cryptic (C) sites, have recognized consensus sequences only among cytomegalovirus (CMV) homologs and are located in the 28-kDa proteolytic portion of the precursor, called assemblin.