Wade Gibson

Isolation and characterization of a noninfectious virion-like particle released from cells infected with human strains of cytomegalovirus

Three types of virus particles have been recovered from the culture medium of human foreskin fibroblasts infected with human strains of cytomegalovirus (HCMV). Two of these, virions and dense bodies, are routinely observed and have been described by others. The third, produced in lesser amounts, has not been previously characterized. This particle, separable from virions by rate-velocity sedimentation, is morphologically distinguished from them only by core structure. Radiolabeling and biological assays have established that these particles, like dense bodies, lack DNA and are not infectious. Based on these properties, we have designated this virion-like structure as a noninfectious enveloped particle (NIEP). Comparisons of the protein constituents of these three particles has shown that dense bodies have the simplest composition. Approximately 95% of their protein mass is represented by a 69,000 Da (69K) matrix-like protein. While dense bodies appear to have a normal complement of virion glycoproteins, they completely lack other predominant virion species. The protein compositions of virions and NIEPs are more complex than that of dense bodies, and are distinguished from one another by the presence in NIEPs of a 35,000 Da (35K) protein absent from the two other particles. Biosynthetic radiolabeling and cell fractionation experiments have demonstrated that this 35K protein is produced only in infected cells, is phosphorylated and partitions with the nuclear fraction. These and other results suggest that this protein is the HCMV counterpart of the previously described B-capsid proteins VP22a of herpes simplex and 37K of CMV (strain Colburn). NIEPs are produced by all HCMV strains examined and have not been observed in preparations of herpes simplex virus- or Old World monkey CMV-infected cells. Although this particle is generally present in much lower amounts than virions, strain AD169 overproduces NIEPs by approximately 10-fold. We have also found that the additional NIEP protein of AD169 has an apparently larger size (i.e., 36K) than the corresponding protein of other strains. The correlation between AD169 NIEP overproduction and its altered protein suggests that the two may be causally related.

Structure and Assembly of the Virion

This article briefly catalogues most of the recognized virion proteins of cytomegalovirus (CMV), summarizes general features of the virion assembly pathway, and outlines some early protein-protein interactions that appear to play key roles in initiating and guiding the capsid assembly process. As more is learned about the structure and assembly of herpes group viruses in general, and CMV in particular, new targets and strategies for antiviral intervention can be anticipated, and insight into potentially similar mechanisms used in the formation of macromolecular structures in eukaryotic cells may be obtained.

Protein counterparts of human and simian cytomegaloviruses

Cytomegalovirus (CMV) proteins from isolates of both human (HCMV) and simian (SCMV) origin have been compared. Three classes were analyzed: the immediate-early (IE) proteins, other infected-cell-specific proteins not present in virus particles, and the proteins that constitute the mature extracellular virion. Comparisons were based on one- and two-dimensional (charge-size) separations in denaturing polyacrylamide gels, and on the selectivity of biosynthetic radiolabeling with [32P]orthophosphate and [3H]glucosamine. Results indicate that most, if not all, of the HCMV and SCMV proteins recognized, have counterparts in strain Colburn. As a group, the simian strains exhibit protein similarities that distinguish them from the human strains. Among the most diagnostic of these are the 205K and 145K virion proteins, each of which is about 7K smaller than its HCMV counterpart, and the predominant IE proteins, which are 10K to 20K (depending upon the strain) larger than their HCMV counterparts. The proteins of strain Colburn are shown to be more like those of the simian isolates than the human, and more like those of a vervet strain than rhesus. Leads provided by experiments using strain Colburn have aided in the identification of a previously unrecognized, abundant virion protein that is a principal phosphate acceptor, both in vivo and in vitro. Three additional phosphorylated proteins are identified in HCMV virions, as well as three glycoproteins. Only two HCMV strain-specific protein differences were detected by comparisons based on separation in SDS-containing polyacrylamide gels--one in the IE protein of strains Towne and Davis; the other in a virus capsid protein of strain AD169.

High-Molecular-Weight Protein (pUL48) of Human Cytomegalovirus Is a Competent Deubiquitinating Protease: Mutant Viruses Altered in Its Active-Site Cysteine or Histidine Are Viable

ABSTRACT We show here that the high-molecular-weight protein (HMWP or pUL48; 253 kDa) of human cytomegalovirus (HCMV) is a functionally competent deubiquitinating protease (DUB). By using a suicide substrate probe specific for ubiquitin-binding cysteine proteases (DUB probe) to screen lysates of HCMV-infected cells, we found just one infected-cell-specific DUB. Characteristics of this protein, including its large size, expression at late times of infection, presence in extracellular virus particles, and reactivity with an antiserum to the HMWP, identified it as the HMWP. This was confirmed by constructing mutant viruses with substitutions in two of the putative active-site residues, Cys24Ile and His162Ala. HMWP with these mutations either failed to bind the DUB probe (C24I) or had significantly reduced reactivity with it (H162A). More compellingly, the deubiquitinating activity detected in wild-type virus particles was completely abolished in both the C24I and H162A mutants, thereby directly linking HMWP with deubiquitinating enzyme activity. Mutations in these active-site residues were not lethal to virus replication but slowed production of infectious virus relative to wild type and mutations of other conserved residues. Initial studies, by electron microscopy, of cells infected with the mutants revealed no obvious differences at late times of replication in the general appearance of the cells or in the distribution, relative numbers, or appearance of virus particles in the cytoplasm or nucleus.

Structural and nonstructural proteins of strain Colburn cytomegalovi

Abstract Studies to investigate the involvement of cytomegalovirus (CMV, strain Colburn) proteins in the infection process have led to the following observations. First, four types of virus particles can be recovered from infected cells. These particles are distinguished on the basis of their intracellular compartmentalization, sedimentation properties in rate-velocity sucrose gradients, protein composition, and infectivity. By analogy with their apparent counterparts in herpes simplex virus (HSV)-infected cells, these particles have been designated as A-, B-, C-capsids and virions. A-capsids, composed of three protein species (i.e., 145K, 34K, and 28K daltons), have the simplest structure; virions, containing at least 20 protein species ranging in molecular weight from approximately 200K to 20K, are structurally the most complex. Among the protein constituents of these virus particles, the 145K was found to be the major structural element of the capsid; a 45K and a 37K B-capsid-specific protein were shown to be closely related and, like their HSV counterparts, absent from the mature virion; and a 66K protein is suggested to serve as a matrix, interfacing the nucleocapsid with the outer envelope. Second, evidence based on peptide analyses, pulse—chase radiolabeling experiments, and comparisons of intracellular viral proteins with those present in virus particles, indicates that several strain Colburn proteins undergo post-translational modifications. And third, these studies have established that, in addition to the proteins identified as structural elements of virus particles, strain Colburn-infected cells contain at least five nonstructural proteins. Most of the structural as well as these nonstructural proteins partitioned strongly with the NP-40 nuclear fraction, but were found to enter that fraction at markedly different rates.

Protease-facilitated transfer of high-molecular-weight proteins during electrotransfer to nitrocellulose

Abstract A procedure is described to enhance the electrophoretic transfer of high-molecular-weight proteins from polyacrylamide gels onto nitrocellulose. The technique takes advantage of in situ proteolysis to reduce the size of large proteins. No apparent loss of low-molecular-weight protein bands was observed, and transferred proteins remained immunoreactive.

Cleavage Specificity of the UL48 Deubiquitinating Protease Activity of Human Cytomegalovirus and the Growth of an Active-Site Mutant Virus in Cultured Cells

ABSTRACT 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. Loveland, L. M. Kattenhorn, H. L. Ploegh, and W. Gibson, J. Virol. 80:6003-6012, 2006). Here, we examined the cleavage specificity of this deubiquitinase (DUB) and replication characteristics of an active-site mutant virus. The purified catalytic domain of the UL48 DUB (1 to 359 amino acids), corresponding to the herpes simplex virus UL36USP DUB (L. M. Kattenhorn, G. A. Korbel, B. M. Kessler, E. Spooner, and H. L. Ploegh, Mol. Cell 19:547-557, 2005), efficiently released ubiquitin but not ubiquitin-like modifications from a hemagglutinin peptide substrate. Mutating the active-site residues Cys24 or His162 (C24S and H162A, respectively) abolished this activity. The HCMV UL48 and HSV UL36USP DUBs cleaved both Lys48- and Lys63-linked ubiquitin dimers and oligomers, showing more activity toward Lys63 linkages. The DUB activity of the full-length UL48 protein immunoprecipitated from virus-infected cells also showed a better cleavage of Lys63-linked ubiquitinated substrates. An HCMV (Towne) mutant virus in which the UL48 DUB activity was destroyed [UL48(C24S)] produced 10-fold less progeny virus and reduced amounts of viral proteins compared to wild-type virus at a low multiplicity of infection. The mutant virus also produced perceptibly less overall deubiquitination than the wild-type virus. Our findings demonstrate that the HCMV UL48 DUB contains both a ubiquitin-specific carboxy-terminal hydrolase activity and an isopeptidase activity that favors ubiquitin Lys63 linkages and that these activities can influence virus replication in cultured cells.

Structure and Formation of the Cytomegalovirus Virion

Transport and protection of the nuclear-replicating double-stranded DNA genome of herpesviruses is accomplished by the virion and its substructures. Studies of the composition, organization, and formation of these particles have provided insight into the molecular mechanisms of virus assembly, leads for antiviral strategies, and information about cellular processes that are required for, resemble, or antagonize virus replication. This chapter updates earlier reviews on the structure and formation human cytomegalovirus (HCMV) virions (Gibson 1996, 2006; Eickmann et al. 2006), and complements several other reviews on herpesvirus structure and replication presented in this volume (see the chapters by E. Murphy and T. Shenk, Z. Ruzsics and U. Koszinowski, R. Kalejta, and G.S. Pari) and elsewhere (Rixon 1993; Steven and Spear 1997; Brown et al. 2002; Varnum et al. 2004; Liu and Zhou 2007).

Cytomegalovirus Basic Phosphoprotein (pUL32) Binds to Capsids In Vitro through Its Amino One-Third

ABSTRACT The cytomegalovirus (CMV) basic phosphoprotein (BPP) is a component of the tegument. It remains with the nucleocapsid fraction under conditions that remove most other tegument proteins from the virion, suggesting a direct and perhaps tight interaction with the capsid. As a step toward localizing this protein within the molecular structure of the virion and understanding its function during infection, we have investigated the BPP-capsid interaction. In this report we present evidence that the BPP interacts selectively, through its amino one-third, with CMV capsids. Radiolabeled simian CMV (SCMV) BPP, synthesized in vitro, bound to SCMV B-capsids, and C-capsids to a lesser extent, following incubation with either isolated capsids or lysates of infected cells. Human CMV (HCMV) BPP (pUL32) also bound to SCMV capsids, and SCMV BPP likewise bound to HCMV capsids, indicating that the sequence(s) involved is conserved between the two proteins. Analysis of SCMV BPP truncation mutants localized the capsid-binding region to the amino one-third of the molecule—the portion of BPP showing the greatest sequence conservation between the SCMV and HCMV homologs. This general approach may have utility in studying the interactions of other proteins with conformation-dependent binding sites.

Assemblin, a herpes virus serine maturational proteinase and new molecular target for antivirals

Herpes group viruses encode a serine proteinase that is essential for the production of infectious virus. This chapter summarizes our current understanding of its biochemical and enzymatic properties, and considers its potential as a target for antivirals.