Sabrina Auerochs

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.

Protein kinase inhibitors of the quinazoline class exert anti-cytomegaloviral activity in vitro and in vivo

Cytomegalovirus infection is associated with severe disease in immunocompromised individuals. Current antiviral therapy faces several limitations. In a search of novel drug candidates, we describe here the anti-cytomegaloviral properties of two compounds of the chemical class of quinazolines, gefitinib (Iressa) and Ax7396 (RGB-315389). Both compounds showed strong inhibitory effects in vitro against human and animal cytomegaloviruses with IC(50)s in a low micromolar range. Cytotoxicity did not occur at these effective concentrations. The antiviral mode of action was based on the inhibition of protein kinase activity, mainly directed to a viral target kinase (UL97/M97) in addition to cellular target candidates. This was demonstrated by a high sensitivity of the respective protein kinases in vitro and by infection experiments with viral mutants carrying genomic alterations in the ORF UL97/M97 modulating viral drug sensitivity. In a guinea pig model, gefitinib showed inhibition of cytomegaloviral loads in blood and lung tissue. Importantly, the rate of mortality of infected animals was reduced by gefitinib treatment. In contrast to the in vitro data, Ax7396 showed no significant antiviral activity in a mouse model. Further in vivo analyses have to assess the potential use of gefitinib in the treatment of cytomegalovirus disease.

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.

Cyclin-dependent Kinases Phosphorylate the Cytomegalovirus RNA Export Protein pUL69 and Modulate Its Nuclear Localization and Activity.

Replication of human cytomegalovirus (HCMV) is subject to regulation by cellular protein kinases. Recently, we and others reported that inhibition of cyclin-dependent protein kinases (CDKs) or the viral CDK ortholog pUL97 can induce intranuclear speckled aggregation of the viral mRNA export factor, pUL69. Here we provide the first evidence for a direct regulatory role of CDKs on pUL69 functionality. Although replication of all HCMV strains was dependent on CDK activity, we found strain-specific differences in the amount of CDK inhibitor-induced pUL69 aggregate formation. In all cases analyzed, the inhibitor-induced pUL69 aggregates were clearly localized within viral replication centers but not subnuclear splicing, pore complex, or aggresome structures. The CDK9 and cyclin T1 proteins colocalized with these pUL69 aggregates, whereas other CDKs behaved differently. Phosphorylation analyses in vivo and in vitro demonstrated pUL69 was strongly phosphorylated in HCMV-infected fibroblasts and that CDKs represent a novel class of pUL69-phosphorylating kinases. Moreover, the analysis of CDK inhibitors in a pUL69-dependent nuclear mRNA export assay provided evidence for functional impairment of pUL69 under suppression of CDK activity. Thus, our data underline the crucial importance of CDKs for HCMV replication, and indicate a direct impact of CDK9-cyclin T1 on the nuclear localization and activity of the viral regulator pUL69.

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.

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.

Influenza A virus proteins PB1 and NS1 are subject to functionally important phosphorylation by protein kinase C

The virulence of influenza A viruses depends on the activity of the viral RNA polymerase complex and viral regulatory phosphoproteins. We identified that the protein kinase C (PKC) inhibitor Gö6976 had a post-entry anti-influenza viral effect, by using a polymerase activity-based reporter assay. This inhibitory effect was observed for influenza virus-infected cells as well as for cells transiently transfected with constructs for the RNA polymerase complex. Importantly, the in vitro analysis of viral protein phosphorylation identified PKCalpha as a kinase phosphorylating PB1 and NS1, but not PB2, PA or NP. Gö6976 was able to block PKC-specific phosphorylation in vitro. Thus, our data suggest that PKC contributes to the phosphorylation of influenza PB1 and NS1 proteins which appears to be functionally relevant for both viral RNA polymerase activity and efficient viral replication.

Modification of the major tegument protein pp65 of human cytomegalovirus inhibits virus growth and leads to the enhancement of a protein complex with pUL69 and pUL97 in infected cells

The tegument protein pp65 of human cytomegalovirus (HCMV) is abundant in lytically infected human foreskin fibroblasts (HFF), as well as in virions and subviral dense bodies (DB). Despite this, we showed previously that pp65 is dispensable for growth in HFF. In the process of refining a DB-based vaccine candidate, different HCMV mutants were generated, expressing a dominant HLA-A2-presented peptide of the IE1 protein fused to pp65. One of the mutant viruses (RV-VM1) surprisingly showed marked impairment in virus release from HFF. We hypothesized that analysis of the phenotypic alterations of RV-VM1 would provide insight into the functions of pp65, poorly defined thus far. RV-VM1 infection resulted in nuclear retention of the fusion protein and reorganization of nuclear inclusion bodies. Coimmunoprecipitation experiments suggested that wild-type (wt) pp65 and pp65-VM1 were substrates of the viral pUL97 kinase in vitro and formed a complex with the viral RNA-export protein pUL69 and with pUL97 in lysates of infected cells. No evidence for an impairment of pUL97 within this complex was found. However, RV-VM1 replication in infected cells was resistant to a pUL97 inhibitor, and pUL97 inhibitors mimicked the mutant in terms of pp65 being retained in the nucleus. The results suggest that the life cycle of RV-VM1 was impeded at the stages of early-late transcription, RNA export or capsid maturation. wt-pp65 may play a role at these stages of infection, and complex formation with pUL69 and pUL97 may be important for that function.