Jens Milbradt

Novel mode of phosphorylation-triggered reorganization of the nuclear lamina during nuclear egress of human cytomegalovirus

The nucleocytoplasmic egress of viral capsids is a rate-limiting step in the replication of the human cytomegalovirus (HCMV). As reported recently, an HCMV-specific nuclear egress complex is composed of viral and cellular proteins, in particular protein kinases with the capacity to induce destabilization of the nuclear lamina. Viral protein kinase pUL97 and cellular protein kinase C (PKC) play important roles by phosphorylating several types of nuclear lamins. Using pUL97 mutants, we show that the lamin-phosphorylating activity of pUL97 is associated with a reorganization of nuclear lamin A/C. Either pUL97 or PKC has the potential to induce distinct punctate lamina-depleted areas at the periphery of the nuclear envelope, which were detectable in transiently transfected and HCMV-infected cells. Using recombinant HCMV, which produces green fluorescent protein-labeled viral capsids, the direct transition of viral capsids through these areas could be visualized. This process was sensitive to an inhibitor of pUL97/PKC activity. The pUL97-mediated phosphorylation of lamin A/C at Ser22 generated a novel binding motif for the peptidyl-prolyl cis/trans-isomerase Pin1. In HCMV-infected fibroblasts, the physiological localization of Pin1 was altered, leading to recruitment of Pin1 to viral replication centers and to the nuclear lamina. The local increase in Pin1 peptidyl-prolyl cis/trans-isomerase activity may promote conformational modulation of lamins. Thus, we postulate a novel phosphorylation-triggered mechanism for the reorganization of the nuclear lamina in HCMV-infected cells.

Cytomegaloviral proteins that associate with the nuclear lamina: components of a postulated nuclear egress complex.

The nuclear egress of cytomegaloviral capsids traversing the nuclear envelope is dependent on a locally restricted destabilization of the rigid nuclear lamina. It has been suggested that the multi-component nuclear egress complex (NEC) that is formed is comprised of both viral and cellular proteins which act to recruit lamin-phosphorylating protein kinases. Recently, we reported that the lamina-associated human cytomegalovirus-encoded proteins pUL50 and pUL53, conserved among herpesviruses, interact with each other and recruit protein kinase C (PKC) to the nuclear envelope in transfected cells. The multiple interactions of the transmembrane protein pUL50 with pUL53, PKC and cellular PKC-binding protein p32, appear crucial to the formation of the NEC. In this study, we mapped individual interaction sequence elements of pUL50 by coimmunoprecipitation analysis of deletion mutants and yeast two-hybrid studies. Amino acids 1-250 were shown to be responsible for interaction with pUL53, 100-280 for PKC and 100-358 for p32. Interestingly, p32 specifically interacted with multiple NEC components, including the kinases PKC and pUL97, thus possibly acting as an adaptor for protein recruitment to the lamin B receptor. Notably, p32 was the only protein that interacted with the lamin B receptor. Immunofluorescence studies visualized the colocalization of NEC components at the nuclear rim in coexpression studies. The data imply that a tight interaction between at least six viral and cellular proteins leads to the formation of a postulated multi-protein complex required for nuclear egress.

Regulatory roles of protein kinases in cytomegalovirus replication.

Viral replication is a complex process relying on a network of interacting viral and cellular proteins, in which particularly protein kinases play an important regulatory role. The specific phosphorylation of substrate proteins induces activation, inactivation, or other functional modification and thus determines virus-host cell interregulation. During herpesviral infections, both viral and cellular protein kinases are expressed and provide activities crucial for the efficiency of virus replication. The protein kinase pUL97 encoded by human cytomegalovirus (HCMV) is a multifunctional regulatory enzyme which exerts strong regulatory effects on early and late steps of the viral replication cycle. A number of interacting proteins and substrates of pUL97 have been described, including retinoblastoma (Rb) protein, nuclear lamins and viral pUL69. Recently, it was demonstrated that pUL97 has structural and functional resemblance to cyclin-dependent protein kinases (CDKs) and thus represents a CDK ortholog. pUL97 can phosphorylate and inactivate Rb, resulting in a stimulation of cell cycle progression. In addition, the association of pUL97 activity with nucleocytoplasmic export of viral capsids has been demonstrated by several investigators. We could show that pUL97 is able to phosphorylate nuclear lamins and to contribute to the HCMV-induced reorganization of the nuclear lamina. On the basis of very recent findings, it is becoming increasingly clear that pUL97 is a component of a multiprotein nuclear egress complex (NEC). The NEC contains a small number of egress proteins involved in the recruitment of protein kinases, such as pUL97 and cellular protein kinase C (PKC), to specific sites of the nuclear lamina. Current information about the composition, function, and regulatory complexity of the NEC leads to a mechanistic concept which may set the key features of HCMV nuclear egress in a new light.

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.

Cytomegaloviral protein kinase pUL97 interacts with the nuclear mRNA export factor pUL69 to modulate its intranuclear localization and activity

Human cytomegalovirus encodes a number of phosphorylation-regulated proteins, including the autophosphorylating protein kinase pUL97 and the nuclear mRNA export factor pUL69. Recently, it was reported that the kinase inhibitor roscovitine induces an intranuclear aggregation of pUL69 in infected fibroblasts. Here, we demonstrate that pUL97-specific kinase inhibitors induce a similar pUL69 aggregation. Furthermore, a direct pUL69-pUL97 interaction was demonstrated by coimmunoprecipitation analyses. Deletion mapping identified the domains required for interaction in both proteins (1-140/478-532 in pUL69 and 231-336 in pUL97). Further analysis of the immunoprecipitates by in vitro kinase assays demonstrated the phosphorylation of pUL69 by pUL97. However, catalytically inactive mutants of pUL97 and interaction-negative fragments of pUL69 were phosphorylation-negative. Moreover, an analysis of the pUL69-mediated nuclear RNA export indicated a correlation of the export efficiency with the presence of active pUL97 kinase. These data suggest a specific pUL69-pUL97 interaction and pUL97-mediated phosphorylation which influences the regulatory activities of pUL69.

Sensitivity of human herpesvirus 6 and other human herpesviruses to the broad-spectrum antiinfective drug artesunate

BACKGROUND Antiviral therapy for HHV-6 infection with conventional anti-herpesviral drugs is problematic so novel drugs are required. Artesunate is a well-tolerated drug approved for malaria therapy which possesses antiviral activity. OBJECTIVE The artesunate sensitivity of HHV-6 was analyzed and compared to that of several other human herpesviruses. STUDY DESIGN Cultured human cells were productively infected with strains of HHV-6 or other human herpesviruses to measure artesunate inhibition of viral protein synthesis (Western blot analysis) or viral genome replication (qPCR), and to determine IC(50) values by immunofluorescence or plaque reduction assays. RESULTS Sensitivity of HHV-6 to artesunate was demonstrated with an IC(50) of 3.80+/-1.06microM. This is in a range similar to IC(50) values for HCMV and EBV. Artesunate treatment of HHV-6-infected cells significantly reduced viral early and late protein synthesis that occurred in the absence of drug-induced apoptosis or necrotic cytotoxicity. HHV-6A genome replication was markedly reduced by artesunate. CONCLUSIONS Artesunate possesses anti-HHV-6 activity in vitro and may be useful for treatment of HHV-6 infections.

The unique antiviral activity of artesunate is broadly effective against human cytomegaloviruses including therapy-resistant mutants.

Current therapy options to treat infections with human cytomegalovirus face severe limitations leading to a continued search for novel drug candidates. Here, we describe novel characteristics of the strong antiviral potency of the drug artesunate. In vitro virus replication systems were applied to analyze a number of laboratory and clinically relevant strains of human cytomegalovirus. An inhibitory block at a very early stage of infection was demonstrated. Time-of-addition experiments indicated that the antiviral efficacy could be optimized when artesunate was applied as fractional doses consecutively added post-infection. Artesunate showed a clearly higher anti-cytomegaloviral activity than its parental drug artemisinin (approximately 10-fold) or other artesunate-related compounds. Mean IC(50) values of artesunate for a variety of standard therapy-resistant virus mutants were within a 2-fold range compared to wild-type virus. Furthermore, a synergistic effect was identified when artesunate was combined with the mechanistically distinct antiviral compound maribavir. These findings point to unique antiviral properties of artesunate which may offer an advantage over standard antiviral therapy particularly in cases of drug resistance.

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

Background: The conserved cytomegalovirus proteins pUL50 and pUL53 heterodimerize and form a core nuclear egress complex. Results: The crystal structure of pUL50-pUL53 was solved at 2.44 Å resolution, revealing an N-terminal hook-like extension of pUL53. Conclusion: Data unravel the core NEC architecture, providing a scaffold for viral-cellular NEC protein interactions. Significance: The identified NEC structure will stimulate the development of novel antiviral strategies. 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 Å2 to the total interface area (1880 Å2). 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.

Specific Residues of a Conserved Domain in the N Terminus of the Human Cytomegalovirus pUL50 Protein Determine Its Intranuclear Interaction with pUL53

Background: Interaction between the cytomegalovirus proteins pUL50 and pUL53 is essential for formation of a nuclear egress complex. Results: Mutations within a globular domain interfere with the function of pUL50. Conclusion: Residues Glu-56 and Tyr-57 of pUL50 are essential for binding to pUL53. Significance: Identification of the mode of important viral protein interactions promotes the development of novel antiviral strategies. Herpesviral capsids are assembled in the host cell nucleus and are subsequently translocated to the cytoplasm. During this process it has been demonstrated that the human cytomegalovirus proteins pUL50 and pUL53 interact and form, together with other viral and cellular proteins, the nuclear egress complex at the nuclear envelope. In this study we provide evidence that specific residues of a conserved N-terminal region of pUL50 determine its intranuclear interaction with pUL53. In silico evaluation and biophysical analyses suggested that the conserved region forms a regular secondary structure adopting a globular fold. Importantly, site-directed replacement of individual amino acids by alanine indicated a strong functional influence of specific residues inside this globular domain. In particular, mutation of the widely conserved residues Glu-56 or Tyr-57 led to a loss of interaction with pUL53. Consistent with the loss of binding properties, mutants E56A and Y57A showed a defective function in the recruitment of pUL53 to the nuclear envelope in expression plasmid-transfected and human cytomegalovirus-infected cells. In addition, in silico analysis suggested that residues 3–20 form an amphipathic α-helix that appears to be conserved among Herpesviridae. Point mutants revealed a structural role of this N-terminal α-helix for pUL50 stability rather than a direct role in the binding of pUL53. In contrast, the central part of the globular domain including Glu-56 and Tyr-57 is directly responsible for the functional interaction with pUL53 and thus determines formation of the basic nuclear egress complex.

A Novel CDK7 Inhibitor of the Pyrazolotriazine Class Exerts Broad-Spectrum Antiviral Activity at Nanomolar Concentrations

ABSTRACT Protein kinases represent central and multifunctional regulators of a balanced virus-host interaction. Cyclin-dependent protein kinase 7 (CDK7) plays crucial regulatory roles in cell cycle and transcription, both connected with the replication of many viruses. Previously, we developed a CDK7 inhibitor, LDC4297, that inhibits CDK7 in vitro in the nano-picomolar range. Novel data from a kinome-wide evaluation (>330 kinases profiled in vitro) demonstrate a kinase selectivity. Importantly, we provide first evidence for the antiviral potential of the CDK7 inhibitor LDC4297, i.e., in exerting a block of the replication of human cytomegalovirus (HCMV) in primary human fibroblasts at nanomolar concentrations (50% effective concentration, 24.5 ± 1.3 nM). As a unique feature compared to approved antiherpesviral drugs, inhibition occurred already at the immediate-early level of HCMV gene expression. The mode of antiviral action was considered multifaceted since CDK7-regulated cellular factors that are supportive of HCMV replication were substantially affected by the inhibitors. An effect of LDC4297 was identified in the interference with HCMV-driven inactivation of retinoblastoma protein (Rb), a regulatory step generally considered a hallmark of herpesviral replication. In line with this finding, a broad inhibitory activity of the drug could be demonstrated against a selection of human and animal herpesviruses and adenoviruses, whereas other viruses only showed intermediate drug sensitivity. Summarized, the CDK7 inhibitor LDC4297 is a promising candidate for further antiviral drug development, possibly offering new options for a comprehensive approach to antiviral therapy.