Major Tegument Protein pp65 of Human Cytomegalovirus Is Required for the Incorporation of pUL69 and pUL97 into the Virus Particle and for Viral Growth in Macrophages

The tegument protein pp65 of human cytomegalovirus (HCMV) represents the major component of mature virus particles. Nevertheless, deletion of pp65 has been shown to have no effects on virus replication and morphogenesis in fibroblasts in vitro. We have studied the HCMV virion composition in the absence of pp65 and viral growth of a pp65 stop mutant in different cell types, including monocyte-derived macrophages. Two stop codons at amino acids 11 and 12 of pp65 were introduced by bacterial artificial chromosome mutagenesis into the endotheliotropic strain TB40/E. Clear changes of the tegument composition could be observed in purified mutant virus particles, where the amount of tegument protein pUL25 was drastically reduced. In addition, pUL69 and the virally encoded protein kinase UL97 were undetectable in the pp65 stop mutant. Expression of pUL69 in infected cells was unaltered while pUL25 accumulated in the absence of pp65, thus demonstrating that only incorporation into virus particles is dependent on pp65. Coimmunoprecipitation experiments using lysates of infected cells revealed an interaction between pUL69 and pp65. This interaction was verified in pull-down experiments using transfected cells, which showed that pp65 and pUL69 do not require the presence of other viral proteins for their interaction. We conclude that pp65 is required for the incorporation of other viral proteins into the virus particle and thus is involved in the protein-protein interaction network leading to normal tegument formation. When studying growth kinetics of the pp65 stop mutant in different cell types, we found a severe impairment of viral growth in monocyte-derived macrophages, showing for the first time a strong cell-specific role of pp65 in viral growth.Human cytomegalovirus (HCMV), a member of the Betaherpesvirinae subfamily, is a threatening pathogen for immunocompromised patients, such as transplant recipients, AIDS patients, and conatally infected infants (15). HCMV infection of individuals with a compromised immune system causes considerable morbidity and mortality after primary infection or reactivation from latency.Mature HCMV virions comprise four distinct structures: core, capsid, tegument, and envelope. The nucleocapsid consists of the core containing the approximately 240-kb linear double-stranded DNA genome, which is embedded in an icosahedral capsid. Between the envelope, a cellularly derived lipid membrane containing viral glycoproteins, and the nucleocapsid, a protein layer called tegument (26), is located. The tegument of HCMV is composed of at least 25 viral proteins. Tegument proteins have been proposed to act in several processes, such as immune evasion (reviewed in reference 30), release of viral DNA into the nucleus (6), and initiation and regulation of the viral replication cycle (3, 7, 16, 31, 41). However, for many of the tegument proteins, the morphogenetic or regulatory functions are unknown. An increasing number of host cell proteins, e.g., cytoskeletal proteins such as α- and β-actin, have also been detected in HCMV particles (4, 39). In addition to infectious virions, HCMV-infected cells generate two types of aberrant particles: noninfectious enveloped particles (NIEPs) and dense bodies (DBs) (18). The protein composition and morphology of NIEPs are nearly identical to those of mature virions; however, their lack of an electron-dense DNA-containing core allows discrimination of NIEPs from infectious virions by electron microscopy (18). DBs are fusion-competent enveloped particles lacking a nucleocapsid. They are composed primarily of the tegument protein pp65 (ppUL83) (4, 18, 39).For a long time, the herpesvirus tegument has been considered to be unstructured. Data mainly from alphaherpesviruses indicate that morphogenesis depends on an intricate network of tegument protein-protein interactions (reviewed in reference 23). Interestingly, for most tegument proteins of alphaherpesviruses relevant for primary tegumentation and envelopment, homologues have been found in HCMV, whereas there is much less homology between the proteins involved in secondary tegumentation and envelopment. Cryoelectron microscopic analyses of herpesvirus particles, including HCMV, provide evidence for an icosahedral symmetry and protein-protein complexes forming substructures, at least for the innermost part of the tegument (11).Remarkably, the most abundant tegument protein and major constituent of extracellular virions, pp65, is not essential for virus replication in fibroblasts in vitro. Deletion of pp65 in HCMV strain AD169 causes a complete loss of DB formation, while production of infectious virus in fibroblasts appears to be unaffected (34). Wild-type virus particle-associated pp65 is rapidly translocated to the nuclei of infected cells after penetration of the incoming virus (4, 33). Newly synthesized pp65 accumulates in both nucleus and cytoplasm at later stages of infection. In all, the precise function of pp65 during infection is not clear.During HCMV infection, pp65 is a major antigen for cellular immune responses. Besides its function as a structural component of the virus, pp65 seems to be involved in manipulation of the host''s immune system. Recent reports provide evidence that pp65 is involved in subverting the host immune response by mediating a decreased expression of major histocompatibility complex class II molecules (27). Microarray studies demonstrating an increase in the cellular antiviral cytokine response during infection with a pp65 deletion mutant suggested that pp65 is involved in downmodulation of beta interferon and of a number of chemokines (1, 8). However, most recent work demonstrates that not pp65 but the immediate-early 2 (IE2) gene product IE86 is responsible for the block of beta interferon induction during HCMV infection and that IE86 expression is delayed in the pp65 deletion mutant due to a decreased expression of pp71 (36). It has also been shown that pp65 can directly interact with NKp30, the natural killer (NK) cell-activating receptor, and that this interaction leads to a general inhibition of the killing ability of NK cells (2). Because of the requirement of cell-free pp65, the relevance of this interaction during HCMV infection in vivo is not entirely clear and needs to be investigated in more detail.Another feature of pp65 is the ability to interact with cellular as well as viral proteins. The interaction of pp65 with the cellular Polo-like kinase 1 (Plk1) results in an incorporation of Plk1 into virus particles. Plk1 is able to phosphorylate pp65 in vitro (14). Recently, it has been shown that pp65 interacts directly with the viral protein kinase pUL97 (20). pUL97 seems to be required for normal intranuclear distribution of pp65. Inhibition of the pUL97 kinase activity with maribavir or mutation of an essential amino acid in the kinase domain results in accumulation of pp65 in characteristic inclusions in the nuclei of infected as well as transfected cells (28).To extend our knowledge about pp65 and its function, we investigated the composition of endotheliotropic HCMV particles in the absence of the most abundant tegument protein, pp65. We hypothesized that other viral or cellular proteins might compensate for the lack of pp65 in virus particles, as described for tegument mutants of pseudorabies virus (25). The results presented here, using a pp65 stop codon mutant of the endotheliotropic HCMV strain TB40/E, demonstrate that in contrast to our hypothesis, incorporation of at least three other HCMV tegument proteins, pUL25, pUL69, and pUL97, is severely impaired when pp65 is lacking. For pUL69, a direct interaction with pp65 could be shown in infected as well as transfected cells. These results show that pp65 interacts with other viral tegument proteins during infection, which in turn is important for the incorporation of these proteins into mature virus particles. Finally, for the first time, we could show a cell-specific biological relevance of pp65 for growth of HCMV in monocyte-derived macrophages (MDM).     

Phosphorylation-Mediated Control of Histone Chaperone ASF1 Levels by Tousled-Like Kinases

Histone chaperones are at the hub of a diverse interaction networks integrating a plethora of chromatin modifying activities. Histone H3/H4 chaperone ASF1 is a target for cell-cycle regulated Tousled-like kinases (TLKs) and both proteins cooperate during chromatin replication. However, the precise role of post-translational modification of ASF1 remained unclear. Here, we identify the TLK phosphorylation sites for both Drosophila and human ASF1 proteins. Loss of TLK-mediated phosphorylation triggers hASF1a and dASF1 degradation by proteasome-dependent and independent mechanisms respectively. Consistent with this notion, introduction of phosphorylation-mimicking mutants inhibits hASF1a and dASF1 degradation. Human hASF1b is also targeted for proteasome-dependent degradation, but its stability is not affected by phosphorylation indicating that other mechanisms are likely to be involved in control of hASF1b levels. Together, these results suggest that ASF1 cellular levels are tightly controlled by distinct pathways and provide a molecular mechanism for post-translational regulation of dASF1 and hASF1a by TLK kinases.     

The Novel Structural Protein of Human Cytomegalovirus, pUL25, Is Localized in the Viral Tegument

Human cytomegalovirus UL25 codes for a structural phosphoprotein of 85 kDa (C. J. Baldick and T. Shenk, J. Virol. 70:6097-6105, 1996; M. C. Battista et al., J. Virol. 73:3800-3809, 1999). In this study we analyzed the intracellular and intraviral localization of pUL25 by confocal and immunoelectron microscopy and found that pUL25 is a component of the viral tegument and the dense body matrix, acquired during the late cytoplasmic phase of virus maturation.     

Histone Deacetylase Inhibitor SAHA Induces Expression of Fatty Acid-Binding Protein 4 and Inhibits Replication of Human Cytomegalovirus

Virologica Sinica - Suberoylanilide hydroxamic acid (SAHA) is a histone deacetylase inhibitor that shows marked efficacy against many types of cancers and is approved to treat severe metastatic...     

Viral Mimicry of Cdc2/Cyclin-Dependent Kinase 1 Mediates Disruption of Nuclear Lamina during Human Cytomegalovirus Nuclear Egress

The nuclear lamina is a major obstacle encountered by herpesvirus nucleocapsids in their passage from the nucleus to the cytoplasm (nuclear egress). We found that the human cytomegalovirus (HCMV)-encoded protein kinase UL97, which is required for efficient nuclear egress, phosphorylates the nuclear lamina component lamin A/C in vitro on sites targeted by Cdc2/cyclin-dependent kinase 1, the enzyme that is responsible for breaking down the nuclear lamina during mitosis. Quantitative mass spectrometry analyses, comparing lamin A/C isolated from cells infected with viruses either expressing or lacking UL97 activity, revealed UL97-dependent phosphorylation of lamin A/C on the serine at residue 22 (Ser²²). Transient treatment of HCMV-infected cells with maribavir, an inhibitor of UL97 kinase activity, reduced lamin A/C phosphorylation by approximately 50%, consistent with UL97 directly phosphorylating lamin A/C during HCMV replication. Phosphorylation of lamin A/C during viral replication was accompanied by changes in the shape of the nucleus, as well as thinning, invaginations, and discrete breaks in the nuclear lamina, all of which required UL97 activity. As Ser²² is a phosphorylation site of particularly strong relevance for lamin A/C disassembly, our data support a model wherein viral mimicry of a mitotic host cell kinase activity promotes nuclear egress while accommodating viral arrest of the cell cycle.