Identification of Unusual E6 and E7 Proteins within Avian Papillomaviruses: Cellular Localization,Biophysical Characterization,and Phylogenetic Analysis

Papillomaviruses (PVs) are a large family of small DNA viruses infecting mammals, reptiles, and birds. PV infection induces cell proliferation that may lead to the formation of orogenital or skin tumors. PV-induced cell proliferation has been related mainly to the expression of two small oncoproteins, E6 and E7. In mammalian PVs, E6 contains two 70-residue zinc-binding repeats, whereas E7 consists of a natively unfolded N-terminal region followed by a zinc-binding domain which folds as an obligate homodimer. Here, we show that both the novel francolin bird PV Francolinus leucoscepus PV type 1 (FlPV-1) and the chaffinch bird PV Fringilla coelebs PV contain unusual E6 and E7 proteins. The avian E7 proteins contain an extended unfolded N terminus and a zinc-binding domain of reduced size, whereas the avian E6 proteins consist of a single zinc-binding domain. A comparable single-domain E6 protein may have existed in a common ancestor of mammalian and avian PVs. Mammalian E6 C-terminal domains are phylogenetically related to those of single-domain avian E6, whereas mammalian E6 N-terminal domains seem to have emerged by duplication and subsequently diverged from the original ancestral domain. In avian and mammalian cells, both FlPV-1 E6 and FlPV-1 E7 were evenly expressed in the cytoplasm and the nucleus. Finally, samples of full-length FlPV-1 E6 and the FlPV-1 E7 C-terminal zinc-binding domain were prepared for biophysical analysis. Both constructs were highly soluble and well folded, according to nuclear magnetic resonance spectroscopy measurements.Papillomaviruses (PVs) are nonenveloped, epitheliotropic, double-stranded DNA viruses that cause a variety of diseases in a multitude of hosts. Based on available whole-genome sequences and subgenomic amplicons, more than 200 human and over 55 nonhuman mammalian PV types have been described (7, 34, 35, 37, 38). To date, two avian PV types have been characterized (37, 38).The genomic organizations of the PVs are remarkably similar. The genome is ca. 8 kb in length and comprises an upstream regulatory region (URR), the early genes (E1, E2, E4, E6, and E7), and the late genes that encode the capsid proteins (L1 and L2). Although most PVs code for these seven open reading frames (ORFs), only the URR, the replicative proteins E1 and E2 (and possibly the E4 gene), and the capsid proteins L1 and L2 are strictly conserved in all PVs (11).Upon infection of the stratified squamous epithelia, PV gene expression is linked to the differentiation state of the infected epithelium cells. The expression of early PV proteins, in particular E6 and E7, primes the proliferation of the infected epithelium. This proliferation, which is absolutely required for viral replication, may become malignant depending on the PV strain considered. Several “high-risk” mucosal human PV (HPV) strains (predominantly HPV type 16 [HPV-16], HPV-18, and HPV-45) have been shown to be responsible for cervical cancer (19).The ability of PVs to induce proliferation of the infected cells has been attributed mainly to two small “oncoproteins,” E6 and E7. In genital high-risk HPVs, these proteins play a prominent role in cell immortalization and transformation (31). In most mammalian PVs, E6 is a small protein of about 150 amino acids, with two conserved N- and C-terminal zinc-binding domains, E6N and E6C, respectively (12). The solution structure of the HPV-16 E6C domain was recently determined (23). The sequence alignments pointed to a structural similarity between the E6C and E6N domains, suggesting that a single-domain protein possessing the same fold might have once existed. Earlier phylogenetic studies had suggested that gene duplication may have given rise to the current double-domain E6 proteins (5). Interestingly, although the E6 ORF has been found in most mammalian PVs (with the exception of bovine papillomavirus type 3 [BPV-3], BPV-4, BPV-6, HPV-101, and HPV-103 [3, 7]), it was not detected in the two avian PVs previously sequenced (37, 38).In this study, we present the full sequence of the genome of a novel PV from a francolin bird (Francolinus leucoscepus PV type 1 [FlPV-1]) and compare it to the two other avian PV genomes known to date (Psittacus erithacus PV [PePV] and Fringilla coelebs PV [FPV]). In light of recent structural data, we compare the unusual avian E6 and E7 ORFs to their mammalian orthologs. We describe the expression and purification of recombinant avian PV E6 and E7 proteins, their biophysical characterization, and cellular localization. Finally, we use phylogenetic techniques to investigate the evolutionary history of the E6 protein family.     

Impact of a Flood Event on the Planktonic Food Web of the Schelde Estuary (Belgium) in Spring 1998

To evaluate the effect of short-term changes in discharge on plankton dynamics, a station in the upper reaches of the Schelde estuary was monitored during 1 month in spring 1998 with a sampling frequency of 1–2 days. During this monitoring period, a flood event occurred during which discharge increased about 5-fold. This flood event had a strong effect on the composition of the planktonic community. Rotifers were strongly negatively affected by the flood event while densities of heterotrophic nanoflagellates and scuticociliates increased. Chlorophyll a concentration and abundance of bacteria, oligotrich ciliates and crustacean zooplankton did not respond clearly to the flood event. Although the flood event lasted only a few days it took more than 2 weeks for the planktonic community to return to its original composition.     

Involvement of the platelet-activating factor receptor in host defense against Streptococcus pneumoniae during postinfluenza pneumonia

Although influenza infection alone may lead to pneumonia, secondary bacterial infections are a much more common cause of pneumonia. Streptococcus pneumoniae is the most frequently isolated causative pathogen during postinfluenza pneumonia. Considering that S. pneumoniae utilizes the platelet-activating factor receptor (PAFR) to invade the respiratory epithelium and that the PAFR is upregulated during viral infection, we here used PAFR gene-deficient (PAFR-/-) mice to determine the role of this receptor during postinfluenza pneumococcal pneumonia. Viral clearance was similar in wild-type and PAFR-/- mice, and influenza virus was completely removed from the lungs at the time mice were inoculated with S. pneumoniae (day 14 after influenza infection). PAFR-/- mice displayed a significantly reduced bacterial outgrowth in their lungs, a diminished dissemination of the infection, and a prolonged survival. Pulmonary levels of IL-10 and KC were significantly lower in PAFR-/- mice, whereas IL-6 and TNF-alpha were only trendwise lower. These data indicate that the pneumococcus uses the PAFR leading to severe pneumonia in a host previously exposed to influenza A.     

Mathematical disabilities in young primary school children with velo-cardio-facial syndrome

The aim of the present study was to examine the previously reported mathematical disabilities (MD) of children with Velo-Cardio-Facial Syndrome (VCFS) in children of a younger age range. Fourteen children with VCFS (aged 6-10 years) participated in this study. These children were individually matched on sex, IQ, age and parental educational level to a control group of peers, selected from the same classes. A broad range of mathematical abilities were assessed, comprising number reading and writing, number comparison, counting, single-digit arithmetic, multidigit arithmetic and word problem solving. Consistent with previous reports, children with VCFS were significantly slower in counting numerosities and they tended to perform more poorly on number comparison. These results indicate that difficulties in low-level number processing in children with VCFS occur already at a very young age. Furthermore, children with VCFS demonstrated preserved retrieval of arithmetic facts, but, in contrast to older children with VCFS, no procedural difficulties in mathematics were observed. Finally, word problem solving appeared to be an important area of weakness, starting already at this young age.     

Granulomatous skin lesions complicating Varicella infection in a patient with Rothmund-Thomson syndrome and immune deficiency: case report

Acro-cardio-facial syndrome (ACFS) is a rare genetic disorder characterized by split-hand/split-foot malformation (SHFM), facial anomalies, cleft lip/palate, congenital heart defect (CHD), genital anomalies, and mental retardation. Up to now, 9 patients have been described, and most of the reported cases were not surviving the first days or months of age. The spectrum of defects occurring in ACFS is wide, and both interindividual variability and clinical differences among sibs have been reported. The diagnosis is based on clinical criteria, since the genetic mechanism underlying ACFS is still unknown. The differential diagnosis includes other disorders with ectrodactyly, and clefting conditions associated with genital anomalies and heart defects. An autosomal recessive pattern of inheritance has been suggested, based on parental consanguinity and disease's recurrence in sibs in some families. The more appropriate recurrence risk of transmitting the disease for the parents of an affected child seems to be up to one in four. Management of affected patients includes treatment of cardiac, respiratory, and feeding problems by neonatal pediatricians and other specialists. Prognosis of ACFS is poor.     

Biogeographic historical legacies in the net primary productivity of Northern Hemisphere forests

It has been suggested that biogeographic historical legacies in plant diversity may influence ecosystem functioning. This is expected because of known diversity effects on ecosystem functions, and impacts of historical events such as past climatic changes on plant diversity. However, empirical evidence for a link between biogeographic history and present‐day ecosystem functioning is still limited. Here, we explored the relationships between Late‐Quaternary climate instability, species‐pool size, local species and functional diversity, and the net primary productivity (NPP) of Northern Hemisphere forests using structural equation modelling. Our study confirms that past climate instability has negative effects on plant functional diversity and through that on NPP, after controlling for present‐day climate, soil conditions, stand biomass and age. We conclude that global models of terrestrial plant productivity need to consider the biogeographical context to improve predictions of plant productivity and feedbacks with the climate system.     

Mutations in the TGFβ binding-protein-like domain 5 of FBN1 are responsible for acromicric and geleophysic dysplasias

Geleophysic (GD) and acromicric dysplasia (AD) belong to the acromelic dysplasia group and are both characterized by severe short stature, short extremities, and stiff joints. Although AD has an unknown molecular basis, we have previously identified ADAMTSL2 mutations in a subset of GD patients. After exome sequencing in GD and AD cases, we selected fibrillin 1 (FBN1) as a candidate gene, even though mutations in this gene have been described in Marfan syndrome, which is characterized by tall stature and arachnodactyly. We identified 16 heterozygous FBN1 mutations that are all located in exons 41 and 42 and encode TGFβ-binding protein-like domain 5 (TB5) of FBN1 in 29 GD and AD cases. Microfibrillar network disorganization and enhanced TGFβ signaling were consistent features in GD and AD fibroblasts. Importantly, a direct interaction between ADAMTSL2 and FBN1 was demonstrated, suggesting a disruption of this interaction as the underlying mechanism of GD and AD phenotypes. Although enhanced TGFβ signaling caused by FBN1 mutations can trigger either Marfan syndrome or GD and AD, our findings support the fact that TB5 mutations in FBN1 are responsible for short stature phenotypes.