Dominant Negative Mutants of the Murine Cytomegalovirus M53 Gene Block Nuclear Egress and Inhibit Capsid Maturation

The alphaherpesvirus proteins UL31 and UL34 and their homologues in other herpesvirus subfamilies cooperate at the nuclear membrane in the export of nascent herpesvirus capsids. We studied the respective betaherpesvirus proteins M53 and M50 in mouse cytomegalovirus (MCMV). Recently, we established a random approach to identify dominant negative (DN) mutants of essential viral genes and isolated DN mutants of M50 (B. Rupp, Z. Ruzsics, C. Buser, B. Adler, P. Walther and U. H. Koszinowski, J. Virol 81:5508-5517). Here, we report the identification and phenotypic characterization of DN alleles of its partner, M53. While mutations in the middle of the M53 open reading frame (ORF) resulted in DN mutants inhibiting MCMV replication by ∼100-fold, mutations at the C terminus resulted in up to 1,000,000-fold inhibition of virus production. C-terminal DN mutants affected nuclear distribution and steady-state levels of the nuclear egress complex and completely blocked export of viral capsids. In addition, they induced a marked maturation defect of viral capsids, resulting in the accumulation of nuclear capsids with aberrant morphology. This was associated with a two-thirds reduction in the total amount of unit length genomes, indicating an accessory role for M53 in DNA packaging.Our understanding of herpesvirus morphogenesis is mainly derived from studies of Alphaherpesvirinae, such as herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PrV). A faster replication cycle and a more productive infection in tissue culture aided genetic analysis of alphaherpesvirus morphogenesis. In addition, deletion mutants of key morphogenesis genes in alphaherpesviruses often maintain basic replication capacity, whereas the mutations of their homologues in Betaherpesvirinae or Gammaherpesvirinae mostly result in a lethal phenotype (for the UL31 and the UL34 family, see references 3, 6, 9-11, 16, 20, 21, and 42). These genes became amenable to comprehensive genetic analysis in betaherpesviruses only after their genomes were cloned as infectious bacterial artificial chromosomes (BACs), which obviated the need to generate replication-competent intermediates or complementing cell lines (3, 21, 23). BAC-based mutagenesis allowed viability screens mapping essential genes (8, 43) or even functional sites of essential genes in cytomegaloviruses (3, 21). However, these approaches cannot easily be applied to reveal the null phenotypes in the context of virus replication, as mutant viruses are not easily reconstituted. In addition, deletion of an essential viral gene can reveal the null phenotype of only the first of perhaps several essential functions during virus morphogenesis. This problem can be addressed to some extent by using dominant negative (DN) mutations (36). DN mutants are loss-of-function mutants that induce a null phenotype in the presence of the wild-type (wt) allele (14). Analysis of phenotypes induced by DN mutants proved to be extremely useful in genetics and cell biology, signaling, and biochemistry. Such inhibitory mutants of cellular proteins are often designed based on knowledge on the structural or functional role of a well-characterized protein domain. Unfortunately, we lack the structural information that would allow knowledge-based design of viral DN mutants for the majority of herpesvirus gene products. Thus, we established a random screen consisting of three steps to identify mutants of viral genes with DN potential (36): (i) a library of mutants is generated by random insertion of 5 amino acids (aa) or a stop codon into the open reading frame (ORF) of interest using transposon mutagenesis, (ii) nonfunctional mutants are identified by cis complementation of the respective deletion mutant mouse cytomegalovirus (MCMV) BAC, and (iii) nonfunctional mutants are tested for their inhibitory potential upon reconstitution of the wt BAC cloned genomes. In the last screen, mutants that have a specific inhibitory effect on the activity of the wt allele are selected. The specific phenotype obtained upon induction of the inhibitory mutants in the context of virus replication is then verified and further characterized using a tetracycline (Tet) regulon-based viral conditional expression system (36, 37).One intriguing aspect of herpesvirus morphogenesis is the transition of capsids from the nuclear to the cytoplasmic phase of virus morphogenesis. Two conserved nonstructural proteins, the homologues of the membrane protein pUL34 and its nuclear partner protein pUL31, form a nuclear egress complex (NEC) (18, 27, 42), which is required for primary envelopment and export of nuclear capsids to the cytoplasm (reviewed in references 24 and 25). Recent studies have revealed that the homologues of alphaherpesvirus pUL34 and pUL31, the M50 and the M53 gene products of the betaherpesvirus MCMV (pM50 and pM53, respectively) and the BFRF1 and the BFLF2 gene products of the gammaherpesvirus Epstein-Barr virus (EBV), apparently share the major functions of these two proteins. The lack of one or both proteins of the NEC generally results in the retention of viral capsids in the nucleus. This is lethal for beta- and gammaherpesvirus production (3, 9-11, 16, 18, 21, 27, 35, 42).The details of the mechanisms by which the NEC proteins mediate capsid export through the nuclear envelope are poorly understood. We (3, 21, 36, 38) and others (1, 19, 34) have started to dissect details of the NEC function using a genetic approach based on subtle mutagenesis of the respective genes. Analysis of the MCMV M50 gene by comprehensive mutagenesis localized two different functional sites. They were the M53 binding site within the N-terminal domain of M50, as well as the transmembrane region at its C terminus (3). Liang and Baines located the respective binding site in HSV-1 UL34 at aa 137 to 181 (19). Our approach, based on screens for DN mutants, identified a proline-rich sequence (aa 179 to 207) in the M50 gene product as an additional essential region (36). A recombinant virus expressing an M50 mutant lacking this site was defective in capsid egress from the nucleus despite the presence of the wt M50 protein. Consequently, the production of infectious particles after infection was reduced by more than 2 orders of magnitude. The UL34 homologues of alpha- and gammaherpesviruses lack a similar polyproline motif, but the result was confirmed by mutating the human cytomegalovirus (HCMV) homologue UL50 at the corresponding region, which is conserved within betaherpesviruses (36). The M50 mutants lacking the proline-rich motif still bind and colocalize to their respective NEC partner, pM53. Interestingly, Bjerke and coworkers also provided genetic evidence for the existence of at least one additional, yet-unknown, but essential functional entity in pUL34 of HSV-1, besides its known pUL31 binding activity, using a screen based on charged-cluster mutations (1). Further analysis of one of the noncomplementing charged-cluster mutants carrying the defect in the N-terminal domain of pUL34 also revealed a DN activity and suggested a new functional site involved in membrane curvature formation, together with the C-terminal domain of UL31 (34).The genetic analysis of M53 by Tn7-based linker scanning mutagenesis, followed by a cis complementation assay, localized the M50-binding site between aa 112 and 137 within the first of the four conserved regions (CRs) shared among the herpesvirus UL31 homologues (21). This analysis, together with a study we performed for further characterization of pM50/pM53 interaction, revealed that the large C-terminal part of pM53, comprising CR2 to -4, must carry at least one additional, yet-unknown, but essential functional site (21, 38).Here, we screened loss-of-function mutants of the MCMV M53 gene to retrieve M53 alleles with DN activity to localize this new functional domain. Mutants with a very strong inhibitory potential accumulated within CR4 of pM53 close to its C terminus. These CR4 mutants induced a block of capsid export from the nucleus. In addition, we could associate these mutations with the induction of a defect in capsid maturation and/or DNA packaging. These data suggested that pM53 is not only crucial for nuclear egress, but also involved in earlier steps of MCMV morphogenesis.     

Murine Cytomegalovirus US22 Protein pM140 Protects Its Binding Partner,pM141, from Proteasome-Dependent but Ubiquitin-Independent Degradation

Stable assembly of murine cytomegalovirus (MCMV) virions in differentiated macrophages is dependent upon the expression of US22 family gene M140. The M140 protein (pM140) exists in complex with products of neighboring US22 genes. Here we report that pM140 protects its binding partner, pM141, from ubiquitin-independent proteasomal degradation. Protection is conferred by a stabilization domain mapping to amino acids 306 to 380 within pM140, and this domain is functionally independent from the region that confers binding of pM140 to pM141. The M140 protein thus contains multiple domains that collectively confer a structure necessary to function in virion assembly in macrophages.Murine cytomegalovirus (MCMV) US22 family genes M36, M139, M140, and M141 promote efficient replication of the virus in macrophages (1, 8, 12, 17). The M139, M140, and M141 genes are clustered within the MCMV genome and appear to function cooperatively (10, 12). During infection, the protein M140 (pM140) forms a stable complex with pM141, and one or more larger complexes are formed by the addition of M139 gene products (15). Although these complexes are evident in infected fibroblasts as well as macrophages, they are required for optimal MCMV replication selectively in macrophages (1, 17). In the absence of M140, virion assembly in macrophages is defective, likely due to the reduced levels of the major capsid protein and tegument protein M25 (11). pM140 also confers stability to its binding partner, pM141; in the absence of the M140 gene, the half-life of pM141 is reduced from 2 h to 1 h (12). Deletion of M141 compromises virus replication in macrophages (12), and pM141 directs pM140 to a perinuclear region of infected macrophages adjacent to an enlarged microtubule organizing center with characteristics of an aggresome (11, 15). Aggresomes are sites where proteins are targeted for degradation by either the proteasome or autophagy (3, 6, 19). We therefore hypothesized that complexing of pM141 to pM140 rescues pM141 from degradation by the proteasome and/or autophagy.     

Crystal Structure of the Human Cytomegalovirus pUL50-pUL53 Core Nuclear Egress Complex Provides Insight into a Unique Assembly Scaffold for Virus-Host Protein Interactions

Nuclear replication of cytomegalovirus relies on elaborate mechanisms of nucleocytoplasmic egress of viral particles. Thus, the role of two essential and conserved viral nuclear egress proteins, pUL50 and pUL53, is pivotal. pUL50 and pUL53 heterodimerize and form a core nuclear egress complex (NEC), which is anchored to the inner nuclear membrane and provides a scaffold for the assembly of a multimeric viral-cellular NEC. Here, we report the crystal structure of the pUL50-pUL53 heterodimer (amino acids 1–175 and 50–292, respectively) at 2.44 Å resolution. Both proteins adopt a globular fold with mixed α and β secondary structure elements. pUL53-specific features include a zinc-binding site and a hook-like N-terminal extension, the latter representing a hallmark element of the pUL50-pUL53 interaction. The hook-like extension (amino acids 59–87) embraces pUL50 and contributes 1510 Ų to the total interface area (1880 Ų). The pUL50 structure overall resembles the recently published NMR structure of the murine cytomegalovirus homolog pM50 but reveals a considerable repositioning of the very C-terminal α-helix of pUL50 upon pUL53 binding. pUL53 shows structural resemblance with the GHKL domain of bacterial sensory histidine kinases. A close examination of the crystal structure indicates partial assembly of pUL50-pUL53 heterodimers to hexameric ring-like structures possibly providing additional scaffolding opportunities for NEC. In combination, the structural information on pUL50-pUL53 considerably improves our understanding of the mechanism of HCMV nuclear egress. It may also accelerate the validation of the NEC as a unique target for developing a novel type of antiviral drug and improved options of broad-spectrum antiherpesviral therapy.     

Skeletal Characterization of Smurf2-Deficient Mice and In Vitro Analysis of Smurf2-Deficient Chondrocytes

Overexpression of Smad ubiquitin regulatory factor 2 (Smurf2) in chondrocytes was reported to cause spontaneous osteoarthritis (OA) in mice. However, it is unclear whether Smurf2 is involved in bone and cartilage homeostasis and if it is required for OA pathogenesis. Here we characterized age-related changes in the bone and articular cartilage of Smurf2-deficient (MT) mice by microCT and histology, and examined whether reduced Smurf2 expression affected the severity of OA upon surgical destabilization of the medial meniscus (DMM). Using immature articular chondrocytes (iMAC) from MT and wild-type (WT) mice, we also examined how Smurf2 deficiency affects chondrogenic and catabolic gene expressions and Smurf2 and Smurf1 proteins upon TGF-β3 or IL-1β treatment in culture. We found no differences in cortical, subchondral and trabecular bone between WT and MT in young (4 months) and old mice (16–24 months). The articular cartilage and age-related alterations between WT and MT were also similar. However, 2 months following DMM, young MT showed milder OA compared to WT (~70% vs ~30% normal or exhibiting only mild OA cartilage phenotype). The majority of the older WT and MT mice developed moderate/severe OA 2 months after DMM, but a higher subset of aged MT cartilage (27% vs. 9% WT) remained largely normal. Chondrogenic gene expression (Sox9, Col2, Acan) trended higher in MT iMACs than WT with/without TGF-β3 treatment. IL-1β treatment suppressed chondrgenic gene expression, but Sox9 expression in MT remained significantly higher than WT. Smurf2 protein in WT iMACs increased upon TGF-β3 treatment and decreased upon IL-1β treatment in a dose-dependent manner. Smurf1 protein elevated more in MT than WT upon TGF-β3 treatment, suggesting a potential, but very mild compensatory effect. Overall, our data support a role of Smurf2 in regulating OA development but suggest that inhibiting Smurf2 alone may not be sufficient to prevent or consistently mitigate post-traumatic OA across a broad age range.     

Characterization of a Human Cytomegalovirus with Phosphorylation Site Mutations in the Immediate-Early 2 Protein

A human cytomegalovirus mutant (TNsubIE2P) was constructed with alanine substitutions of four residues (T27, S144, T233, and S234) previously shown to be phosphorylated in the immediate-early 2 (IE2) protein. This mutant grew as well as the wild type at both low and high multiplicities of infection. The mutant activated the major immediate-early, UL4, and UL44 promoters to similar levels, and with similar kinetics, as wild-type virus. However, the TNsubIE2P mutant virus transactivated an endogenous simian virus 40 early promoter 4 h earlier and to higher levels than the wild-type virus in infected human fibroblasts. The modification of the IE2 protein by SUMO-1 (i.e., its sumoylated state) was also examined.     

Proteomic Analysis of the Multimeric Nuclear Egress Complex of Human Cytomegalovirus

Herpesviral capsids are assembled in the host cell nucleus before being translocated into the cytoplasm for further maturation. The crossing of the nuclear envelope represents a major event that requires the formation of the nuclear egress complex (NEC). Previous studies demonstrated that human cytomegalovirus (HCMV) proteins pUL50 and pUL53, as well as their homologs in all members of Herpesviridae, interact with each other at the nuclear envelope and form the heterodimeric core of the NEC. In order to characterize further the viral and cellular protein content of the multimeric NEC, the native complex was isolated from HCMV-infected human primary fibroblasts at various time points and analyzed using quantitative proteomics. Previously postulated components of the HCMV-specific NEC, as well as novel potential NEC-associated proteins such as emerin, were identified. In this regard, interaction and colocalization between emerin and pUL50 were confirmed by coimmunoprecipitation and confocal microscopy analyses, respectively. A functional validation of viral and cellular NEC constituents was achieved through siRNA-mediated knockdown experiments. The important role of emerin in NEC functionality was demonstrated by a reduction of viral replication when emerin expression was down-regulated. Moreover, under such conditions, reduced production of viral proteins and deregulation of viral late cytoplasmic maturation were observed. Combined, these data prove the functional importance of emerin as an NEC component, associated with pUL50, pUL53, pUL97, p32/gC1qR, and further regulatory proteins. Summarized, our findings provide the first proteomics-based characterization and functional validation of the HCMV-specific multimeric NEC.Viruses are tightly linked to the regulatory processes governing the metabolic state of their host cells. This regulatory linkage is reflected by viral activation or silencing of gene expression and productive replication in response to cellular changes in signaling, cell cycle, apoptosis, differentiation, and other parameters. Viruses also tend to exert a strong influence on regulatory cellular pathways and the developmental fate of virus-infected tissues (1, 2). These examples of virus-cell interregulation have been studied in detail, but in many cases the essential molecular mechanisms are still poorly understood. In the field of herpesviruses, profound efforts in molecular research have been undertaken to characterize those direct protein–protein interactions that regulate cross-talk between the virus and its host. Multi-protein complexes composed of both viral and cellular constituents were identified in several stages of herpesviral lytic replication. In particular, detailed studies on the replication of human cytomegalovirus (HCMV)¹ in primary fibroblasts and other permissive cell types have provided very interesting insights into the nature of chimeric multi-protein complexes. These examples were described for viral entry, viral response to intrinsic immunity, intracellular transport of viral products, nucleocytoplasmic egress of viral capsids, and other processes (3–6). In classical approaches, protein–protein interaction was studied by means of approved methods including yeast two-hybrid, coimmunoprecipitation (CoIP), and pulldown analyses with purified proteins. More recently, very sensitive methods have been introduced into this field, such as proteomic analysis using tandem mass spectrometry (MS/MS), confocal imaging techniques, surface plasmon resonance analysis, and others.During HCMV replication, the translocation of genome-containing viral capsids from the nucleus to the cytoplasm (nuclear egress) is one of the most crucial steps. In this process, the nuclear envelope represents a barrier consisting of three distinct elements: nuclear membranes, nuclear pores, and the proteinaceous network of the nuclear lamina. The viral capsids traverse the nuclear envelope by budding through nuclear membranes. Importantly, HCMV capsids access the inner nuclear membrane by overcoming the proteinaceous network of the nuclear lamina. To regulate the serial steps in this procedure, a multimeric protein complex is formed, termed the nuclear egress complex (NEC) (4, 7). One of the main tasks of the NEC is the distortion of the nuclear lamina. Our recent studies identified the formation of lamina-depleted areas that result from the recruitment of sophisticated enzymatic activities to these specific sites at the lamina (8). Viral and cellular effectors, such as protein kinases, a proline cis/trans isomerase, and possibly further regulatory proteins, are involved in this process (4). It is commonly accepted that the core NEC is composed of two viral proteins, namely, pUL50 and pUL53 (9–13). Moreover, the association of pUL50–pUL53 with a number of viral and cellular proteins supports the concept of a multimeric NEC that may include the viral protein kinase pUL97, multi-ligand binding protein p32/gC1qR, lamin B receptor, and protein kinase C (PKC) (14).In this work, we first confirmed the major role played by pUL50 and pUL53 in NEC formation. The pUL50–pUL53 core NEC was then used as bait for the identification of other NEC components at different time points post-infection. Quantitative MS-based proteomics confirmed known members of the multimeric NEC and also identified the cellular inner nuclear membrane protein emerin as a novel NEC constituent. Importantly, colocalization of emerin with the HCMV-specific NEC and its interaction with pUL50 were demonstrated for the first time. Knockdown experiments provided functional validation of the importance of emerin and other NEC proteins for HCMV replication. Together, these data provide an extended mechanistic model for the composition and function of the HCMV-specific NEC.     

Human Cytomegalovirus UL50 and UL53 Recruit Viral Protein Kinase UL97, Not Protein Kinase C,for Disruption of Nuclear Lamina and Nuclear Egress in Infected Cells

Herpesvirus nucleocapsids traverse the nuclear envelope into the cytoplasm in a process called nuclear egress that includes disruption of the nuclear lamina. In several herpesviruses, a key player in nuclear egress is a complex of two proteins, whose homologs in human cytomegalovirus (HCMV) are UL50 and UL53. However, their roles in nuclear egress during HCMV infection have not been shown. Based largely on transfection studies, UL50 and UL53 have been proposed to facilitate disruption of the nuclear lamina by recruiting cellular protein kinase C (PKC), as occurs with certain other herpesviruses, and/or the viral protein kinase UL97 to phosphorylate lamins. To investigate these issues during HCMV infection, we generated viral mutants null for UL50 or UL53. Correlative light electron microscopic analysis of null mutant-infected cells showed the presence of intranuclear nucleocapsids and the absence of cytoplasmic nucleocapsids. Confocal immunofluorescence microscopy revealed that UL50 and UL53 are required for disruption of the nuclear lamina. A subpopulation of UL97 colocalized with the nuclear rim, and this was dependent on UL50 and, to a lesser extent, UL53. However, PKC was not recruited to the nuclear rim, and its localization was not affected by the absence of UL50 or UL53. Immunoprecipitation from cells infected with HCMV expressing tagged UL53 detected UL97 but not PKC. In summary, HCMV UL50 and UL53 are required for nuclear egress and disruption of nuclear lamina during HCMV infection, and they recruit UL97, not PKC, for these processes. Thus, despite the strong conservation of herpesvirus nuclear egress complexes, a key function can differ among them.     

Amino Acids of Conserved Kinase Motifs of Cytomegalovirus Protein UL97 Are Essential for Autophosphorylation

Thirteen point mutations targeting predicted domains conserved in homologous protein kinases were introduced into the UL97 coding region of the human cytomegalovirus. All mutagenized proteins were expressed in cells infected with recombinant vaccinia viruses (rVV). Several mutations drastically reduced ganciclovir (GCV) phosphorylation. Mutations at amino acids G340, A442, L446, and F523 resulted in a complete loss of pUL97 phosphorylation, which was strictly associated with a loss of GCV phosphorylation. Our results confirm that in rVV-infected cells pUL97 phosphorylation is due to autophosphorylation and show that several amino acids conserved within domains of protein kinases are essential for this pUL97 phosphorylation. GCV phosphorylation is dependent on pUL97 phosphorylation.     

Comparison of Effects of Inhibitors of Viral and Cellular Protein Kinases on Human Cytomegalovirus Disruption of Nuclear Lamina and Nuclear Egress

Human cytomegalovirus (HCMV) kinase UL97 is required for efficient nuclear lamina disruption during nuclear egress. However, cellular protein kinase C (PKC) has been implicated in this process in other systems. Comparing the effects of UL97 and cellular kinase inhibitors on HCMV nuclear egress confirms a role for UL97 in lamina disruption and nuclear egress. A pan-PKC inhibitor did not affect lamina disruption but did reduce the number of cytoplasmic capsids more than the number of nuclear capsids.     

PAI—2及其突变体的生物化学性质

为研究和比较纤深酶原激活剂抑制物２型（ｐｌａｓｍｉｎｏｇｅｎ ａｃｔｉｖａｏｒ ｉｎｈｉｂｉｔｏｒ ｔｙｐｅ ２,ＰＡＩ－２）及其突变体的生物化学性质,用ＰＣＲ、体外定点突变技术构建ＰＡＩ－２突变体ＰＡＩ－２ＣＤ和ＰＡＩ－２ＱｃＤＮＡ,将突变体ｃＤＮＡ与原核表达载体重组并转化怕杆菌。经诱导表达,ＳＤＳ－ＰＡＧＥ证实表达出相应蛋白质,均占全菌总蛋白的１４％。Ｗｅｔｅｒｎ印迹、溶圈抑制及纤维蛋白翻转板     

Experimental Preemptive Immunotherapy of Murine Cytomegalovirus Disease with CD8 T-Cell Lines Specific for ppM83 and pM84, the Two Homologs of Human Cytomegalovirus Tegument Protein ppUL83 (pp65)

CD8 T cells are the principal antiviral effectors controlling cytomegalovirus (CMV) infection. For human CMV, the virion tegument protein ppUL83 (pp65) has been identified as a source of immunodominant peptides and is regarded as a candidate for cytoimmunotherapy and vaccination. Two sequence homologs of ppUL83 are known for murine CMV, namely the virion protein ppM83 (pp105) expressed late in the viral replication cycle and the nonstructural protein pM84 (p65) expressed in the early phase. Here we show that ppM83, unlike ppUL83, is not delivered into the antigen presentation pathway after virus penetration before or in absence of viral gene expression, while other virion proteins of murine CMV are processed along this route. In cytokine secretion-based assays, ppM83 and pM84 appeared to barely contribute to the acute immune response and to immunological memory. Specifically, the frequencies of M83 and M84 peptide-specific CD8 T cells were low and undetectable, respectively. Nonetheless, in a murine model of cytoimmunotherapy of lethal CMV disease, M83 and M84 peptide-specific cytolytic T-cell lines proved to be highly efficient in resolving productive infection in multiple organs of cell transfer recipients. These findings demonstrate that proteins which fail to prime a quantitatively dominant immune response can nevertheless represent relevant antigens in the effector phase. We conclude that quantitative and qualitative immunodominance are not necessarily correlated. As a consequence of these findings, there is no longer a rationale for considering T-cell abundance as the key criterion for choosing specificities to be included in immunotherapy and immunoprophylaxis of CMV disease and of viral infections in general.