Inclusion of Veterinary Services in national emergency management plans.

With human population growth, rapid urbanisation, increasing globalisation, and climate change, the interdependency of human health and animal health is mounting. Therefore, the importance of national emergency management plans (NEMPs) for the mitigation of, and preparedness for, all hazards, including disease epidemics, both zoonotic and zootic, is ever increasing. The authors decided to take a One Health approach by assessing the inclusion of Veterinary Services and animal health in NEMPs, based on geographical region, the date of the NEMP, national income status, and the proportion of the agricultural sector in national gross domestic product (GDP). To carry out the assessment, the authors analysed the publicly available NEMPs of 86 Members of the World Organisation for Animal Health. Of the 86 NEMPs reviewed, only a third expressly mentioned Veterinary Services, almost 60% mentioned zoonotic and/or zootic diseases, and about two-thirds mentioned animals to some extent. The highest correlating factor to the inclusion of animal health in NEMPs was the level of the agricultural sector's contributions to the national GDP. Fisheries and aquaculture were not a major consideration in any of the reviewed NEMPs, especially not in relation to diseases. Based on region, Latin America and the Caribbean exhibited the lowest inclusion rate of animal health in NEMPs. The results demonstrate that the omission of animal health is still a problem. A multi-disciplinary approach that includes veterinary medicine as well as human medicine is vital in the construction and/or revision of NEMPs. Future studies should consider whether or not there is a connection between countries' veterinary capacities and the inclusion of Veterinary Services in their NEMPs and whether or not they have the infrastructure and human resources to put into operation the roles of Veterinary Services as identified in their NEMPs.

La croissance démographique humaine, l'urbanisation accélérée, la mondialisation accrue et le changement climatique sont autant de facteurs qui intensifient l'interdépendance de la santé humaine et de la santé animale. De ce fait, les plans nationaux de gestion des urgences jouent un rôle de plus en plus important pour atténuer les dangers, quels qu'ils soient, et pour se préparer à leur survenue, y compris les dangers liés aux épidémies zoonotiques ou zootiques. Les auteurs ont entrepris d'évaluer le niveau d'intégration des Services vétérinaires et de la santé animale dans les plans nationaux de gestion des urgences dans une perspective Une seule santé, en se basant sur les critères suivants : la région géographique, la date du plan national de gestion des urgences, le niveau de revenu du pays et la part du secteur agricole dans le produit intérieur brut (PIB). Pour les besoins de cette évaluation, les auteurs ont analysé les plans nationaux de gestion des urgences publiés par 86 Membres de l'Organisation mondiale de la santé animale. Parmi ces 86 plans nationaux, un tiers seulement mentionnait expressément les Services vétérinaires, près de 60 % mentionnaient les maladies zoonotiques ou les épizooties et environ deux tiers prenaient en compte les animaux pour une raison ou pour une autre. Le facteur présentant la corrélation la plus élevée avec la prise en compte de la santé animale dans les plans nationaux de gestion des urgences était le niveau de contribution du secteur agricole dans le PIB national. Aucun des plans nationaux de gestion des urgences examinés ne prenait en compte la pêche et l'aquaculture en tant qu'aspect important, en particulier en lien avec des maladies. À l'échelle régionale, c'est en Amérique latine et aux Caraïbes que l'intégration de la santé animale dans les plans nationaux de gestion des urgences était la plus faible. Ces résultats montrent que le problème de l'omission de la santé animale est toujours d'actualité. Il est d'une importance capitale qu'une approche pluridisciplinaire intégrant la médecine vétérinaire et la médecine humaine soit adoptée lors de la conception et/ou de la révision des plans nationaux de gestion des urgences. Il conviendrait que de nouvelles études déterminent à l'avenir s'il existe ou non un lien entre les capacités vétérinaires des pays et la prise en compte des Services vétérinaires dans les plans nationaux de gestion des urgences, et si les pays disposent ou non des infrastructures et des ressources humaines permettant à leurs Services vétérinaires de mener à bien les interventions prévues dans les plans nationaux de gestion des urgences.

El crecimiento demográfico, la rápida urbanización, la creciente mundialización y el cambio climático son otros tantos factores que traen consigo una dependencia recíproca cada vez más acusada entre la salud humana y la sanidad animal. De ahí la creciente importancia que van adquiriendo los planes nacionales de gestión de emergencias destinados a prepararse para todo tipo de peligros, incluidas las enfermedades epidémicas, tanto zoonóticas como epizoóticas, y, llegado el caso, a mitigar sus consecuencias. Los autores, partiendo de las premisas de Una sola salud, decidieron evaluar la integración de los Servicios Veterinarios y la sanidad animal en los planes nacionales de gestión de emergencias, utilizando como criterios de evaluación la región geográfica, la fecha del plan nacional en cuestión, el nivel de renta del país y el porcentaje del producto interno bruto (PIB) que representa el sector agrícola. Para llevar a cabo la evaluación los autores analizaron los planes nacionales de gestión de emergencias que están a disposición pública de 86 Miembros de la Organización Mundial de Sanidad Animal. De esos 86 planes nacionales examinados, solo en un tercio se mencionaban explícitamente los Servicios Veterinarios, en casi un 60% se aludía a enfermedades zoonóticas y/o epizoóticas y en cerca de dos tercios se hablaba en alguna medida de los animales. El factor que mayor correlación presentaba con la integración de la sanidad animal en los planes nacionales de gestión de emergencias era la aportación del sector agrícola al PIB. En ninguno de los planes examinados ocupaban un lugar relevante ni la pesca ni la acuicultura, especialmente en relación con las enfermedades. Por regiones, América Latina y el Caribe presentaba el menor porcentaje de integración de la sanidad animal en los planes nacionales de gestión de emergencias. Los resultados demuestran que la omisión de la sanidad animal sigue suponiendo un problema. A la hora de elaborar o revisar los planes nacionales de gestión de emergencias es crucial hacerlo desde planteamientos multidisciplinares que incluyan tanto la medicina veterinaria como la humana. En estudios ulteriores convendría determinar si existe una correlación entre la capacidad veterinaria de los países y la integración de los Servicios Veterinarios en su plan nacional de gestión de emergencias y si los países disponen de la infraestructura y el personal requeridos para que los Servicios Veterinarios cumplan las funciones que se les asignan en el plan nacional de gestión de emergencias.

Psychosocial Stress During First Pregnancy Predicts Infant Health Outcomes in the First Postnatal Year.

OBJECTIVES: To evaluate the impact of psychosocial stress during pregnancy on infant health outcomes in the first postnatal year. METHODS: A sample of 3000 women completed a stress inventory (the Psychosocial Hassles Scale) during their third trimester before first childbirth. Infant health outcomes were measured via maternal report at 1, 6 and 12 months postpartum. Poisson regression was used to model the effect of maternal stress during pregnancy on infant health outcomes in the first year, controlling for age, race/ethnicity, education, insurance coverage, marital status, and cigarette smoking during pregnancy. RESULTS: Women who were younger, minority, unmarried, publicly insured and without a college degree were more likely to report high levels of prenatal stress. High prenatal stress was a significant predictor of maternal reporting of gastrointestinal illness (p < 0.0001), respiratory illness (p = 0.025), and total illness in the first year (p < 0.0001). High prenatal stress was also a significant predictor of urgent care visits (p < 0.0001) and emergency department visits (p = 0.001). It was not a significant predictor of hospitalizations (p = 0.36). CONCLUSIONS: Maternal prenatal stress is associated with increased maternal reporting of infant illness, as well as increased frequency of both urgent care visits and emergency department visits.

The impact of COVID-19 on health systems, mental health and the potential for nursing.

This paper offers a perspective on nursing and lived experience responses to the COVID-19 pandemic. It charts health systems and mental health impacts with a particular focus on children and adolescents, older people and people availing of mental health services. Issues of moral distress and the nursing reaction are considered alongside psychological and social concerns which continue to rapidly evolve. The perspective of a person attending adult community mental health services and the experience of engaging with a mental health service remotely is provided. Matters of note for acute inpatient mental health nursing are highlighted and informed by the lived experience of a mental health nurse. The need for integrated health systems responses across nursing disciplines and the wider interdisciplinary team is elucidated.

Intercellular delivery of functional p53 by the herpesvirus protein VP22.

The herpes simplex virus type 1 (HSV-1) virion protein VP22 exhibits the remarkable property of intercellular trafficking whereby the protein spreads from the cell in which it is synthesized to many surrounding cells. In addition to having implications for protein trafficking mechanisms, this function of VP22 might be exploited to overcome a major hurdle in gene therapy, i.e., efficient delivery of genes and gene products. We show that chimeric polypeptides, consisting of VP22 linked to the entire p53 protein, retain their ability to spread between cells and accumulate in recipient cell nuclei. Furthermore the p53-VP22 chimeric protein efficiently induces apoptosis in p53 negative human osteosarcoma cells resulting in a widespread cytotoxic effect. The intercellular delivery of functional p53-VP22 fusion protein is likely to prove beneficial in therapeutic strategies based on restoration of p53 function. These results, demonstrating intracellular transport of large functional proteins, indicate that VP22 delivery may have applications in gene therapy.

Piloting the older adult financial exploitation measure in adult safeguarding services.

Financial abuse is arguably the most complex form of elder abuse as it may occur remote to the older person and it is impacted by issues such as cultural values, perpetrator intent and family expectations. Financial abuse may not be recognised by either the older person or the perpetrator, thus, its prevention, early identification and amelioration are important. The (Irish) National Centre for the Protection of Older People undertook a study to determine the appropriateness of the Older Adult Financial Exploitation Measure for use by the national safeguarding older person services. Findings from a small pilot study involving 16 safeguarding staff's use of the Older Adult Financial Exploitation Measure with 52 community dwelling older people referred to their service demonstrate a higher suspicion of financial abuse as well as identifying multiple instances of possible financial exploitation in a single individual. Thus, the Older Adult Financial Exploitation Measure is considered appropriate to assist safeguarding personnel's assessment of older people related to a suspicion of financial abuse.

Ischemia/reperfusion-induced changes in membrane fluidity characteristics of brain capillary endothelial cells and its prevention by liposomal-incorporated superoxide dismutase.

The effect of global cerebral ischemia and reperfusion on cerebral capillary endothelial cell membrane fluidity was examined using electron paramagnetic resonance techniques following 8 minutes of global ischemia and 15 minutes of blood reperfusion. The luminal surface of the cerebral vasculature was perfused with a series of doxyl stearic acid reporters (5-, 12-, 16-doxyl stearic acid) which differ in the site of attachment of the nitroxide free radical on the fatty acid chain. Each doxyl stearic acid reports on membrane fluidity characteristics from different depths within the membrane. Ischemia/reperfusion produced a membrane ordering that was markedly dependent on intramembrane location, and was consistent with changes previously associated with lipid peroxidation. The effect of ischemia/reperfusion on membrane fluidity was maximal in the membrane environment reported by 12-doxyl stearic acid (12-DS). The utilization of a liposomal system was shown to enhance superoxide dismutase delivery to cerebral tissues as well as attenuating the change in membrane order seen following reperfusion-induced lipid peroxidation.

Functional Domains within the Nucleus of a Cell Infected with HSV-1.

HSV-1 is a nuclear replicating DNA virus capable of establishing both lytic and latent infections in mammalian cells. Expression of the more than 80 HSV genes (the majority of which do not contain introns) requires complex coordination of viral and cellular factors both temporally, at appropriate points during the infectious cycle, and spatially as the virus transcription, replication and DNA packaging factories develop in the cell nucleus. Whilst the HSV genome encodes sufficient proteins to sustain viral DNA replication, it is reliant upon its host cell for RNA polymerase II and RNA processing machinery, in addition to an increasing number of cellular cofactors, for gene expression. As HSV establishes a lytic infection, cellular gene expression and splicing are inhibited as cellular chromatin is displaced and a dramatic reorganisation of the host cell nucleus occurs. The formation of large protein-rich factories synthesising viral RNA and replicating and packaging the viral genomes is the most striking alteration. In addition to the synthetic factories, large clumps of cellular and viral intron-containing RNAs accumulate in the nucleus as a result of the inhibition of splicing, at locations which colocalise with splicing factors, but are separate from transcription sites. An essential HSV protein IE63, discussed here, has been identified with a role in the organisation of the nucleus at many levels including replication and transcription site formation, splicing factor organisation and the transport of RNA. This review is a summary of our present understanding of the organisation of the HSV infected cell nucleus, relating viral genomes, RNA, DNA and proteins in the context of the nucleus. However this is a rapidly evolving field and new factors (both viral and cellular) involved in the regulation of these functional domains are constantly being identified. Copyright 1997 by John Wiley & Sons, Ltd.

Selective visualization of choroidal neovascular membranes.

PURPOSE: Laser-targeted angiography has unique advantages over conventional angiography of the fundus. Its efficacy in visualizing choroidal neovascular membranes was tested in a rat model and compared to that of fluorescein angiography. METHOD: Laser-targeted angiography was performed in rats with choroidal neovascularization (CNV) by injecting heat-sensitive carboxyfluorescein liposomes intravenously, locally releasing a bolus of dye in the choroid with a weak laser pulse, and recording advancement of the bolus on a video camera. Conventional fluorescein angiography also was performed. RESULTS: Laser-targeted angiography revealed CNV as an abnormal pattern of brightly fluorescent vessels. The flow pattern of the bolus and histology, performed in some cases, confirmed the choroidal nature of the vessels. The angiographic visualization was not dependent on dye leakage through the vessels or staining of their walls. Laser-targeted angiography also provided visualization of new vessels that could not be diagnosed by fluorescein angiography. It demonstrated that blood flow was typically more sluggish in CNV than in normal choriocapillaris. Fluorescein angiography failed to demonstrate flow dynamics in all cases of CNV. CONCLUSIONS: This study, in an animal model of CNV, shows that laser-targeted angiography demonstrates CNV and its flow dynamics in a manner not provided by conventional fluorescein angiography. It holds clinical promise as a method to delineate CNV considered difficult or impossible to detect by fluorescein angiography. The method also may permit selective photocoagulation of feeding vessels in the choroid, thereby limiting damage to the overlying retina.

Propranolol-induced alterations in rat erythrocyte membrane fluidity and apparent phase-transition temperatures. A depth-dependent process.

Propranolol-induced alterations of membrane structure were studied in rat erythrocytes using electron spin resonance techniques. Propranolol produced a concentration-dependent change in membrane fluidity in hydrophobic membrane regions, while producing virtually no change in hydrophilic membrane regions. The changes were associated with depth-dependent alterations in "apparent" phase-transition profiles and transition temperatures. The effects of propranolol on these membrane characteristics were similar to those produced by cholesterol. Propranolol fluidized erythrocyte membranes in a depth-specific fashion, by virtue of its association with the rigid phospholipid acyl chains and cholesterol sterol rings in the hydrophilic regions of the membrane, which produced distant perturbations within the hydrophilic regions of the membrane.

Methylphenidate and seizure frequency in brain injured patients with seizure disorders.

BACKGROUND: Psychostimulant drugs such as methylphenidate are increasingly being used in patients with traumatic brain injury or other brain injuries for a variety of cognitive and behavioral problems. However, there is some reluctance among clinicians and family members to use methylphenidate in brain injured patients because of warnings of seizure occurrence, which are prominently reported by the product labeling included in the Physicians' Desk Reference. METHOD: We retrospectively studied the use of methylphenidate in 30 consecutive patients with active seizure disorders. We compared the seizure frequency in the 3 months before and after methylphenidate with the seizure frequency during methylphenidate treatment, using the Wilcoxon signed-rank test. RESULTS: Overall, the findings demonstrated a trend toward a lesser incidence of seizures in patients during methylphenidate treatment. Only 4 patients had greater seizure frequency during methylphenidate treatment, and 3 of these 4 were receiving tricyclic antidepressants. CONCLUSION: Methylphenidate can be safely used in brain injured patients, even those at high risk for seizures, as it was associated with a trend toward reduction (rather than increase) in seizure frequency in this population.

Athermal alterations in the structure of the canalicular membrane and ATPase activity induced by thermal levels of microwave radiation.

Sprague-Dawley rats (200-250 g) were exposed 30 min/day for 4 days to thermogenic levels (rectal temperature increase of 2.2 degrees C) of microwave radiation [2.45 GHz, 80 mW/cm2, continuous-wave mode (CW)] or to a radiant heat source resulting in an equivalent increase in body temperature of 2.2 degrees C. On the fifth day after the 4 days of exposure to microwave radiation, the animals were sacrificed and their livers removed. The canalicular membranes were isolated and evaluated for adenosinetriphosphatase (ATPase) activity, total fatty acid composition and membrane fluidity characteristics. Mg(++)-ATPase activity (Vmax) decreased by 48.5% in the group exposed to microwave radiation, with no significant change in the group exposed to radiant heat. The decrease in Mg(++)-ATPase was partially compensated by a concomitant increase in Na+/K(+)-ATPase activity (170% increase in Vmax over control) in animals exposed to microwave radiation, while no change occurred in the group exposed to radiant heat. This alteration in ATPase activity in the group exposed to microwave radiation is associated with a large decrease in the ratio of saturated to unsaturated fatty acids. Conversely, the group exposed to radiant heat had an increase in the ratio of saturated to unsaturated fatty acids. The most dramatic changes were found in the levels of arachidonic acid. Finally, the electron paramagnetic resonance (EPR) spin label technique used to measure the fluidity of the canalicular membranes of the animals in the three groups (sham, microwave radiation and radiant heat) indicated that the results were different in the three groups, reflecting the changes found in their fatty acid composition. The physiological response to "equivalent" thermal loads in rats is expressed differently for different types of energy sources. Possible mechanisms producing these divergent thermogenic responses are discussed.

In Vitro Systems to Analyze HSV Transcript Processing.

During lytic virus replication, herpes simplex virus (HSV) exhibits a closely regulated pattern of viral gene expression and of DNA replication, resulting in virion production (1). Broadly, HSV genes can be divided into immediate early, early, and late categories based on the kinetics of their expression. The five immediate early genes are expressed in the absence of prior viral protein synthesis although their expression is stimulated by a viral tegument protein. Two immediate early proteins are essential for virus replication in vitro and act at the transcriptional (IE 175) and posttranscriptional (IE63) levels to regulate early and late gene expression. Throughout infection, mRNA is synthesized using cellular RNA poly-merase II, which is modified by the action of an immediate early protein (2).

Health maintenance organizations and the treatment of substance abuse.

The deterioration of substance abusers to the point where they need to be hospitalized generally happens slowly, over a period of years if not decades. During this time, most of their medical care takes place in an ambulatory setting. Primary care physicians are thus in an ideal position to identify and help patients in relatively early stages of the problem. The HMO, with its ability to practice patient-oriented case management and its access to all aspects of the medical care delivery system without additional financial burden to the patient, is particularly suited to provide effective and efficient care. The next challenge for HMOs is to reach patients at much earlier stages of their disease.

Herpes simplex virus type 1 immediate early protein IE63 shuttles between nuclear compartments and the cytoplasm.

Herpes simplex virus type 1 (HSV-1) immediate early protein IE63, an essential nuclear protein, is pleiotropic in function and, at the post-transcriptional level, inhibits RNA splicing, interacts with cellular splicing small nuclear ribonucleoprotein particles (snRNPs), binds RNA and prevents the nucleocytoplasmic transport of intron-containing mRNAs. Here it is reported that IE63 is a nucleocytoplasmic shuttle protein able to travel from snRNP- and RNA-rich nuclear foci to the cytoplasm, where it accumulates during actinomycin D treatment. This newly identified property suggests that IE63 facilitates nuclear export of HSV-1 transcripts, in addition to retaining intron-containing transcripts in the nucleus. The mechanism by which IE63 controls RNA export has yet to be defined.

2-Iminothiolane: a reagent for the introduction of sulphydryl groups into oligosaccharides derived from asparagine-linked glycans.

A facile method for introducing reactive sulphydryl groups into oligosaccharides was developed. 1-Amino-oligosaccharides generated from asparagine-linked glycans by peptide-N4(N-acetyl-beta-D-glucosaminyl) asparagine amidase (PNGase F) digestion were monitored by high-performance anion-exchange chromatography with pulsed amperometric detection and derivatized under optimal conditions with 2-iminothiolane-HCl. The resulting mercapto-butyramido oligosaccharides, which were obtained in high yield, were alkylated with a fluorescent reagent and used to selectively assay for endoglycosidases that hydrolyse di-N-acetylchitobiose linkages.

Detection and quantification of peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidases.

A detailed study of the oligosaccharide specificity of the almond enzyme, peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase A, was undertaken by comparing the rate of release of intact oligosaccharide chains from defined glycopeptides of all significant classes. The oligosaccharide of a trisialo-triantennary pentaglycopeptide from fetuin was released at the highest rate. A procedure was developed for the isolation of this glycopeptide in high yield from 5 g fetuin. Sequence analysis established the structure as Leu-Ala-Asn(CHO)-Cys-Ser. The Cys(Cm) and the Cys(Ae) derivatives of the glycopeptide were reacted with 4-(dimethylamino)-azobenzene-4'-sulfonyl (dabsyl) chloride to yield a monosubstituted and a disubstituted glycopeptide respectively. This chromophore confers high sensitivity at 436 nm on a pentapeptide backbone having minimal bonds for protease cleavage. A procedure was developed wherein these dabsyl derivatives were used in a high-performance liquid chromatography assay. The dabsyl-pentapeptide was retarded significantly from the dabsyl-glycopeptide and provided a sensitive method (1-2 nmol) of detection of peptide-N4-(N-acetylglucosaminyl)asparagine amidase activity. Enzyme was detected in crude extracts of all eight seed sources surveyed. The enzyme from Pisum sativum was partially purified and its properties were compared with the corresponding enzyme from almonds.

Herpes simplex virus type 1 protein IE63 affects the nuclear export of virus intron-containing transcripts.

Using in situ hybridization labelling methods, we have determined that the herpes simplex virus type 1 immediate-early protein IE63 (ICP27) affects the cellular localization of virus transcripts. Intronless transcripts from the IE63, UL38, and UL44 genes are rapidly exported to and accumulate in the cytoplasm throughout infection, in either the presence or absence of IE63 expression. The intron-containing transcripts from the IE110 and UL15 genes, while initially cytoplasmic, are increasingly retained in the nucleus in distinct clumps as infection proceeds, and the clumps colocalize with the redistributed small nuclear ribonucleoprotein particles. Infections with the IE63 mutant virus 27-lacZ demonstrated that in the absence of IE63 expression, nuclear retention of intron-containing transcripts was lost. The nuclear retention of UL15 transcripts, which demonstrated both nuclear and cytoplasmic label, was not as pronounced as that of the IE110 transcripts, and we propose that this is due to the late expression of UL15. Infections with the mutant virus 110C1, in which both introns of IE110 have been precisely removed (R.D. Everett, J. Gen. Virol. 72:651-659, 1991), demonstrated IE110 transcripts in both the nucleus and the cytoplasm; thus, exon definition sequences which regulate viral RNA transport are present in the IE110 transcript. By in situ hybridization a stable population of polyadenylated RNAs was found to accumulate in the nucleus in spots, most of which were separate from the small nuclear ribonucleoprotein particle clumps. The IE63 protein has an involvement, either direct or indirect, in the regulation of nucleocytoplasmic transport of viral transcripts, a function which contrasts with the recently proposed role of herpes simplex virus type 1 Us11 in promoting the nuclear export of partially spliced or unspliced transcripts (J.-J. Diaz, M. Duc Dodon, N. Schaerer-Uthurraly, D. Simonin, K. Kindbeiter, L. Gazzolo, and J.-J. Madjar, Nature [London] 379:273-277, 1996), the significance of which is discussed.

Regulation of herpes simplex virus poly (A) site usage and the action of immediate-early protein IE63 in the early-late switch.

The essential herpes simplex virus type 1 (HSV-1) immediate-early IE63 (ICP27) is pleiotropic in function, promoting the switch from the early to late phase of virus gene expression, and has effects on the posttranscriptional processes of mRNA splicing and 3' processing. We have investigated the role of IE63 in the regulation of viral mRNA 3' processing and of late gene expression. Our in vitro 3' processing studies demonstrated that HSV-1 infection induces an activity, which requires IE63 gene expression, responsible for an observed increase in 3' processing of selected HSV-1 poly(A) sites. Processing efficiencies at the poly(A) sites of two late genes, UL38 and UL44, shown to be inherently weak processing sites, were increased by the IE63-induced activity. In contrast, 3' processing at the poly(A) sites of selected immediate-early and early genes, stronger processing sites, was unaffected by IE63 expression. UV cross-linking experiments demonstrated that HSV infection caused enhanced binding of protein factors, including the 64-kDa component of cleavage stimulation factor (CstF), to poly(A) site RNAs from virus genes of all temporal classes and that this enhanced binding required expression of IE63. By immunofluorescence, the homogeneous pattern of the 64-kDa CstF protein distribution became slightly clumped with infection, whereas the splicing small nuclear ribonucleoprotein particles were recognized into a highly punctate distribution away from the sites of virus transcription. This effect could create an increase in the relative concentration of 3' processing factors available to pre-mRNAs. Western blot (immunoblot) analysis showed that IE63 was required for expression of several true late genes and for the efficient and timely expression of the UL29 and UL42 early genes, integral components on the viral DNA synthesis machinery. Our data are consistent with two effects of IE63 on late gene regulation: firstly, a stimulation of pre-mRNA 3' processing and, secondly, as a requirement for expression of functions necessary for viral DNA synthesis.

Immediate early protein IE63 of herpes simplex virus type 1 binds RNA directly.

The herpes simplex virus type 1 (HSV-1) immediate early protein IE63 acts post-transcriptionally to affect RNA 3'-processing and splicing. Functional domains such as the RGG box and zinc-finger motifs potentially provide the protein with RNA binding capacity. Here, IE63 protein expressed in E. coli, purified by affinity chromatography and used in RNA binding assays, demonstrated similar binding to RNA substrates containing poly(A) sites from different temporal classes of HSV-1 genes, RNA containing splice site recognition sequences and RNA containing no recognized processing motifs. Competition binding assays showed that IE63 binding could be competed out, suggesting that IE63 binds RNA weakly. HSV-1 infection results in an increase or stabilization in vitro of protein binding to poly(A) site-containing RNAs; IE63 is required for this effect. RNA binding assays combining purified IE63 with protein from mock-infected and HSV-1 infected nuclear extracts demonstrated no effect on protein-RNA binding patterns.