Spatially resolved protein map of intact human cytomegalovirus virions.

Herpesviruses assemble large enveloped particles that are difficult to characterize structurally due to their size, fragility and complex multilayered proteome with partially amorphous nature. Here we used crosslinking mass spectrometry and quantitative proteomics to derive a spatially resolved interactome map of intact human cytomegalovirus virions. This enabled the de novo allocation of 32 viral proteins into four spatially resolved virion layers, each organized by a dominant viral scaffold protein. The viral protein UL32 engages with all layers in an N-to-C-terminal radial orientation, bridging nucleocapsid to viral envelope. We observed the layer-specific incorporation of 82 host proteins, of which 39 are selectively recruited. We uncovered how UL32, by recruitment of PP-1 phosphatase, antagonizes binding to 14-3-3 proteins. This mechanism assures effective viral biogenesis, suggesting a perturbing role of UL32-14-3-3 interaction. Finally, we integrated these data into a coarse-grained model to provide global insights into the native configuration of virus and host protein interactions inside herpesvirions.

Cross-regulation of viral kinases with cyclin A secures shutoff of host DNA synthesis.

Herpesviruses encode conserved protein kinases (CHPKs) to stimulate phosphorylation-sensitive processes during infection. How CHPKs bind to cellular factors and how this impacts their regulatory functions is poorly understood. Here, we use quantitative proteomics to determine cellular interaction partners of human herpesvirus (HHV) CHPKs. We find that CHPKs can target key regulators of transcription and replication. The interaction with Cyclin A and associated factors is identified as a signature of ß-herpesvirus kinases. Cyclin A is recruited via RXL motifs that overlap with nuclear localization signals (NLS) in the non-catalytic N termini. This architecture is conserved in HHV6, HHV7 and rodent cytomegaloviruses. Cyclin A binding competes with NLS function, enabling dynamic changes in CHPK localization and substrate phosphorylation. The cytomegalovirus kinase M97 sequesters Cyclin A in the cytosol, which is essential for viral inhibition of cellular replication. Our data highlight a fine-tuned and physiologically important interplay between a cellular cyclin and viral kinases.

The dynamic proteome of influenza A virus infection identifies M segment splicing as a host range determinant.

Pandemic influenza A virus (IAV) outbreaks occur when strains from animal reservoirs acquire the ability to infect and spread among humans. The molecular basis of this species barrier is incompletely understood. Here we combine metabolic pulse labeling and quantitative proteomics to monitor protein synthesis upon infection of human cells with a human- and a bird-adapted IAV strain and observe striking differences in viral protein synthesis. Most importantly, the matrix protein M1 is inefficiently produced by the bird-adapted strain. We show that impaired production of M1 from bird-adapted strains is caused by increased splicing of the M segment RNA to alternative isoforms. Strain-specific M segment splicing is controlled by the 3' splice site and functionally important for permissive infection. In silico and biochemical evidence shows that avian-adapted M segments have evolved different conserved RNA structure features than human-adapted sequences. Thus, we identify M segment RNA splicing as a viral host range determinant.

A viral kinase counteracts in vivo restriction of murine cytomegalovirus by SAMHD1.

The deoxynucleotide triphosphate (dNTP) hydrolase SAMHD1 inhibits retroviruses in non-dividing myeloid cells. Although antiviral activity towards DNA viruses has also been demonstrated, the role of SAMHD1 during cytomegalovirus (CMV) infection remains unclear. To determine the impact of SAMHD1 on the replication of CMV, we used murine CMV (MCMV) to infect a previously established SAMHD1 knockout mouse model and found that SAMHD1 inhibits the replication of MCMV in vivo. By comparing the replication of MCMV in vitro in myeloid cells and fibroblasts from SAMHD1-knockout and control mice, we found that the viral kinase M97 counteracts SAMHD1 after infection by phosphorylating the regulatory residue threonine 603. The phosphorylation of SAMHD1 in infected cells correlated with a reduced level of dNTP hydrolase activity and the loss of viral restriction. Together, we demonstrate that SAMHD1 acts as a restriction factor in vivo and we identify the M97-mediated phosphorylation of SAMHD1 as a previously undescribed viral countermeasure.

Synthetic lethal mutations in the cyclin A interface of human cytomegalovirus.

Generally, the antagonism between host restriction factors and viral countermeasures decides on cellular permissiveness or resistance to virus infection. Human cytomegalovirus (HCMV) has evolved an additional level of self-imposed restriction by the viral tegument protein pp150. Depending on a cyclin A-binding motif, pp150 prevents the onset of viral gene expression in the S/G2 cell cycle phase of otherwise fully permissive cells. Here we address the physiological relevance of this restriction during productive HCMV infection by employing a cyclin A-binding deficient pp150 mutant virus. One consequence of unrestricted viral gene expression in S/G2 was the induction of a G2/M arrest. G2-arrested but not mitotic cells supported viral replication. Cyclin A destabilization by the viral gene product pUL21a was required to maintain the virus-permissive G2-arrest. An HCMV double-point mutant where both pp150 and pUL21a are disabled in cyclin A interaction forced mitotic entry of the majority of infected cells, with a severe negative impact on cell viability and virus growth. Thus, pp150 and pUL21a functionally cooperate, together building a cell cycle synchronization strategy of cyclin A targeting and avoidance that is essential for productive HCMV infection.

PUL21a-Cyclin A2 interaction is required to protect human cytomegalovirus-infected cells from the deleterious consequences of mitotic entry.

Entry into mitosis is accompanied by dramatic changes in cellular architecture, metabolism and gene expression. Many viruses have evolved cell cycle arrest strategies to prevent mitotic entry, presumably to ensure sustained, uninterrupted viral replication. Here we show for human cytomegalovirus (HCMV) what happens if the viral cell cycle arrest mechanism is disabled and cells engaged in viral replication enter into unscheduled mitosis. We made use of an HCMV mutant that, due to a defective Cyclin A2 binding motif in its UL21a gene product (pUL21a), has lost its ability to down-regulate Cyclin A2 and, therefore, to arrest cells at the G1/S transition. Cyclin A2 up-regulation in infected cells not only triggered the onset of cellular DNA synthesis, but also promoted the accumulation and nuclear translocation of Cyclin B1-CDK1, premature chromatin condensation and mitotic entry. The infected cells were able to enter metaphase as shown by nuclear lamina disassembly and, often irregular, metaphase spindle formation. However, anaphase onset was blocked by the still intact anaphase promoting complex/cyclosome (APC/C) inhibitory function of pUL21a. Remarkably, the essential viral IE2, but not the related chromosome-associated IE1 protein, disappeared upon mitotic entry, suggesting an inherent instability of IE2 under mitotic conditions. Viral DNA synthesis was impaired in mitosis, as demonstrated by the abnormal morphology and strongly reduced BrdU incorporation rates of viral replication compartments. The prolonged metaphase arrest in infected cells coincided with precocious sister chromatid separation and progressive fragmentation of the chromosomal material. We conclude that the Cyclin A2-binding function of pUL21a contributes to the maintenance of a cell cycle state conducive for the completion of the HCMV replication cycle. Unscheduled mitotic entry during the course of the HCMV replication has fatal consequences, leading to abortive infection and cell death.

Chronic kidney disease in adolescent and adult patients with phenylketonuria.

OBJECTIVES: A lifelong phenylalanine-restricted diet with supplementation of a phenylalanine-free amino acid formula is recommended in patients with phenylketonuria (PKU). The effect of a long-term PKU diet on renal function and blood pressure has not been investigated yet. DESIGN: We analyzed renal function in 67 patients with PKU, aged 15-43 years, by measuring glomerular filtration rate (GFR) and effective renal plasma flow by isotope clearance ((51)Cr-EDTA, (123)J-Hippuran), estimated GFR, blood retention parameters, urinary protein and electrolyte excretion. Renal ultrasound and 24 h ambulatory blood pressure monitoring were performed additionally. Patients were divided into three groups according to their: 1) current diet (CD), i.e., daily protein intake: ICD <0.8 g/kg, IICD 0.8-1.04 g/kg, IIICD >1.04 g/kg; 2) life-long diet time (LDT), i.e., cumulative years of life in which daily protein intake exceeded dietary recommendations: ILDT <15 years, IILDT 15-19 years, IIILDT >19 years. RESULTS: GFR was decreased in 19 % of the patients. With increasing protein intake, GFR decreased significantly (ICD 111 ml/min; IICD 105 ml/min; IIICD 99 ml/min. ILDT 112 ml/min; IILDT 103 ml/min; IIILDT 99 ml/min). Proteinuria was detected in 31 %, microalbuminuria in 7 %, and hypercalciuria in 23 % of the patients. 23 % of the patients had arterial hypertension, and 41 % revealed a nocturnal non-dipping status. CONCLUSIONS: In patients with PKU on a lifelong diet we could detect impaired renal function in 19 %, proteinuria in 31 %, and arterial hypertension in 23 %. Thus, chronic kidney disease may develop in PKU patients, and routine renal function tests should be performed during long-term follow-up.

Long-term treatment with tetrahydrobiopterin in phenylketonuria: treatment strategies and prediction of long-term responders.

Tetrahydrobiopterin (BH4) responsive phenylketonuria has been described more than 10 years ago. However, criteria for the identification of long-term BH4 responsive patients are not yet established. 116 patients with phenylketonuria, aged 4-18 years, were screened for potential long-term BH4 responsiveness by at least two of the following criteria: positive neonatal BH4 loading test, putative BH4 responsive genotype, and/or milder phenotype. Patients had to be on permanent dietary treatment. 23 patients fulfilled these criteria and were tested for long-term BH4 responsiveness: 18/23 were long-term BH4 responsive, 5/23 were not. On long-term BH4 treatment over a period of 48 ± 27 months in a dose of 14.9 ± 3.3mg/kg/day phenylalanine tolerance was increased from 452 ± 201 mg/day to 1593 ± 647 mg/day, corresponding to a mean increase of 1141 ± 528 mg/day. Dietary phenylalanine intake was increased stepwise according to a clear defined protocol. In 8/18 patients, diet was completely liberalized; 10/18 patients still received phenylalanine-free amino acid formula with 0.63 ± 0.23 g/kg/day. The most predictive value for long-term BH4 responsiveness was the combination of pretreatment phenylalanine of < 1200 µmol/L, pretreatment phenylalanine/tyrosine ratio of <15, phenylalanine/tyrosine ratio of <15 on treatment, phenylalanine tolerance of >20mg/kg/day at age 3 years, positive neonatal BH4 loading, and at least one putative BH4 responsive mutation (p = 0.00024). Our data show that long-term BH4 responsiveness may be predicted already during neonatal period by determining maximum pretreatment phenylalanine and phenylalanine/tyrosine concentrations, neonatal BH4 loading and PAH genotype. A clear defined protocol is necessary to install long-term BH4 treatment.

The latency-associated UL138 gene product of human cytomegalovirus sensitizes cells to tumor necrosis factor alpha (TNF-alpha) signaling by upregulating TNF-alpha receptor 1 cell surface expression.

Many viruses antagonize tumor necrosis factor alpha (TNF-α) signaling in order to counteract its antiviral properties. One way viruses achieve this goal is to reduce TNF-α receptor 1 (TNFR1) on the surface of infected cells. Such a mechanism is also employed by human cytomegalovirus (HCMV), as recently reported by others and us. On the other hand, TNF-α has also been shown to foster reactivation of HCMV from latency. By characterizing a new variant of HCMV AD169, we show here that TNFR1 downregulation by HCMV only becomes apparent upon infection of cells with HCMV strains lacking the so-called ULb' region. This region contains genes involved in regulating viral immune escape, cell tropism, or latency and is typically lost from laboratory strains but present in low-passage strains and clinical isolates. We further show that although ULb'-positive viruses also contain the TNFR1-antagonizing function, this activity is masked by a dominant TNFR1 upregulation mediated by the ULb' gene product UL138. Isolated expression of UL138 in the absence of viral infection upregulates TNFR1 surface expression and can rescue both TNFR1 reexpression and TNF-α responsiveness of cells infected with an HCMV mutant lacking the UL138-containing transcription unit. Given that the UL138 gene product is one of the few genes recognized to be expressed during HCMV latency and the known positive effects of TNF-α on viral reactivation, we suggest that via upregulating TNFR1 surface expression UL138 may sensitize latently infected cells to TNF-α-mediated reactivation of HCMV.

Features and outcome of galactokinase deficiency in children diagnosed by newborn screening.

Galactokinase deficiency (GALK-D), an autosomal recessive disorder in the Leloir pathway, results in accumulation of galactose, galactitol, and galactonate and leads to early onset of juvenile bilateral cataract. Highest incidence of GALK-D is found in Romani populations. The migration wave due to the Yugoslavian civil war has changed the spectrum of inborn errors of metabolism within Europe. Hence, newborn screening (NBS) in the Berlin region, performed from 1991 until 2010 in 683,675 neonates, revealed an increased incidence of GALK-D of 1:40,000, comparable to that of galactose-1-phosphate-uridyltransferase deficiency. A total of 44% of GALK-D patients were of Romani origin. All patients of Bosnian or Serbian origin were homozygous for the Romani founder mutation p.P28T. Detection of GALK-D by NBS and early start of galactose-restricted diet resulted in regression or prevention of cataracts. Slight cataracts without visual impairment occurred in 50% of the patients, 56% of whom were noncompliant. Further clinical symptoms, e.g., hypoglycemia, mental retardation, microcephaly, and failure to thrive, were associated with noncompliance. With treatment, galactose in blood decreased from 8,892 ± 5,243 to 36.5 ± 49.3 µmol/l, galactose in urine from 31,820 ± 32,103 to 30.0 ± 36.1 µmol/mmol creatinine, galactitol in RBC from 1,584 ± 584 to 12.3 ± 9.4 µmol/l, and galactitol in urine from 11,724 ± 4,496 to 236 ± 116 µmol/mmol creatinine. This is the first presentation of outcome and clinical features in GALK-D patients diagnosed by NBS. As our data suggest, GALK-D should be considered for inclusion in NBS in populations expected to have substantial numbers of GALK-D carriers, e.g., Yugoslavian immigrants.

Long-term treatment with tetrahydrobiopterin increases phenylalanine tolerance in children with severe phenotype of phenylketonuria.

Hyperphenylalaninemia caused by phenylalanine hydroxylase (PAH) deficiency requires lifelong rigorous diet starting in early infancy to prevent severe neurodevelopmental handicap. In a considerable number of children with mild hyperphenylalaninemia, long-term tetrahydrobiopterin (BH4) treatment significantly improves phenylalanine (phe) tolerance, but it has never been investigated in classic phenylketonuria (PKU). We performed a BH4-loading test in 40 consecutive infants with phe serum concentrations exceeding 240 microM, who had been detected by newborn screening programs. Eighteen out of 40 infants were found to be BH4 responsive. Five of them, responding to the neonatal BH4-loading test, showed a phe tolerance of less than 20 mg/kg/day and a phe pretreatment level of >1000 microM. They were treated with BH4 (20 mg/kg/day) over a period of 24 months. All five children had a sustained response to BH4, allowing substantial easing of dietary restrictions. Before BH4 treatment daily phe tolerance was 18-19 mg/kg, increasing to 30-80 mg/kg on BH4 treatment and decreasing again to 12-17 mg/kg after termination of BH4 treatment. Mutation analysis revealed compound heterozygosity for a putative null and a variant PAH mutation in four patients and homozygosity for a variant PAH mutation in one patient. We conclude that BH4 sensitivity is not restricted to mild hyperphenylalaninemia and that long-term BH4 treatment may also improve phenylalanine tolerance in a considerable number of children with a more severe PKU phenotype.

[The influence of normobaric hyperoxia on hepatic oxygenation--experience with an animal model]. / Der Einfluss von normobarer Hyperoxie auf die lebervenöse Oxygenierung--Erfahrungen mit einem Tiermodell.

We investigated the effect of a ventilation with an FiO2 of 1.0 on arterial and hepatic venous oxygenation in 23 Göttingen minipigs. Under balanced anaesthesia (isoflurane/fentanyl), a fibreoptic catheter was placed into a hepatic vein. The correct position of the tip of the catheter was controlled manually after laparotomy. After measurement of baseline values (arterial and hepatic blood gases, ShvO2), in 13 minipigs normoventilation with an FiO2 of 1.0 was performed for 15 minutes. Thereafter, ventilation was continued with an FiO2 of 0.4. In the control group (n = 10), the animals were oxygenated with an FiO2 of 0.4 permanently. The changes due to hyperoxia were measured in hepatic venous oxygen saturation (ShvbgaO2: from 81.2 +/- 1.43% to 87.5 +/- 1.77%, ShvoximO2: from 82.6 +/- 1.14% to 90.5 +/- 0.90%), arterial (from 217.5 +/- 5.0 mmHg to 467.2 +/- 22.0 mmHg) and hepatic venous (from 51.8 +/- 2.0 mmHg) oxygen partial pressure. We found a correlation between hepatic venous oxygen partial pressure und ShvbgaO2 in the blood (r = 0.84, p < 0.001) and between ShvO2 (ShvbgaO2/ShvoximO2), which was either measured directly in the blood or by a fibreoptic catheter (r = 0.6, p < 0.001). Whereas the increase in ShvO2 during hyperoxia may be a result of increased arterial supply, the decrease in ShvO2 after the end of hyperoxia below baseline values needs further investigations. The continuous fibreoptic measurement of ShvoximO2, also under hyperoxic conditions is a valuable parameter for the monitoring of hepatic venous oxygenation.

Homozygous and double heterozygous Factor V Leiden and Factor II G20210A genotypes predispose infants to thromboembolism but are not associated with an increase of foetal loss.

Prospective and controlled data about the individual risk profile in asymptomatic children with homozygous or double heterozygous risk genotypes for Factor V Leiden (FVL) and factor II (FII) G20210A are currently unavailable. The systematic and prospective observational study presented here was designed to determine the impact of the homozygous and double heterozygous FVL and FII G20210A genotypes on the prenatal and postnatal risk profiles of affected children. Risk infants and heterozygous controls were identified by screening of 85,304 neonates. Follow-up included the comparison of prenatal and postnatal development, ultrasonography of brain and kidneys, and a panel of independent determinants of thrombophilia. The numbers of identified or expected FVL homozygotes and double heterozygotes did not differ significantly (FVL: 116 versus 91, p=0.08; FVL/FII: 94 versus 76, p=0.17), indicating the absence of a prenatal disadvantage. A prenatal advantage was suggested in FII homozygotes, whose identified number far exceeded the expected (19 versus 4, p=0.002). Clinical and/or imaging abnormalities indicated spontaneous thromboembolic events in 4 of 129 risk infants (3%) but in none of the 178 controls (p=0.02). Physical and neurological development was normal in both groups during the first 2 years of life. The risk genotypes appear to confer a significant predisposition for spontaneous thromboembolic events in infancy without impeding development within the first two years of life. Foetal risk genotypes do not cause an increased foetal loss rate. Moreover, homozygous FII G20210A appears to be associated with a prenatal advantage.

An unexpectedly high frequency of hypergalactosemia in an immigrant Bosnian population revealed by newborn screening.

In galactokinase (GALK) deficiency, galactose cannot be phosphorylated into galactose-1-phosphate, which leads to cataract formation. Neonatal screening for hypergalactosemia in Berlin has been performed by thin-layer chromatography since 1978, which detects classical galactosemia and GALK deficiency. Until 1991, GALK deficiency has not been identified in a total of approximately 260,000 samples. In contrast, from 1992 to 1999, nine patients were detected in a total of approximately 240,000 screened newborns. One Turkish patient was homozygous for two novel S142I/G148C GALK mutations in close proximity to the putative ATP-binding site of the enzyme. The other eight children were born to five families belonging to the Bosnian refugee population consisting of approximately 30,000 individuals who have arrived in Berlin since 1991. In two of these families, GALK deficiency was subsequently diagnosed in siblings who had cataract surgery at 4 and 5 y of age, respectively. In all these 10 Bosnian patients, a homozygous P28T mutation located near the active center of the enzyme was identified. We propose that neonatal screening of populations with a significant proportion of Bosnians and possibly other southeastern Europeans, e.g. Romani, should be particularly directed toward GALK deficiency, an inborn error of metabolism that is readily amenable to effective treatment.