Nuclease Activity of the Junín Virus Nucleoprotein C-Terminal Domain.

The mammarenavirus Junín (JUNV) is the causative agent of Argentine hemorrhagic fever, a severe disease of public health concern. The most abundant viral protein is the nucleoprotein (NP), a multifunctional, two-domain protein with the primary role as structural component of the viral nucleocapsids, used as template for viral polymerase RNA synthesis activities. Here, we report that the C-terminal domain (CTD) of the attenuated Candid#1 strain of the JUNV NP can be purified as a stable soluble form with a secondary structure in line with known NP structures from other mammarenaviruses. We show that the JUNV NP CTD interacts with the viral matrix protein Z in vitro, and that the full-length NP and Z interact with each other in cellulo, suggesting that the NP CTD is responsible for this interaction. This domain comprises an arrangement of four acidic residues and a histidine residue conserved in the active site of exoribonucleases belonging to the DEDDh family. We show that the JUNV NP CTD displays metal-ion-dependent nuclease activity against DNA and single- and double-stranded RNA, and that this activity is impaired by the mutation of a catalytic residue within the DEDDh motif. These results further support this activity, not previously observed in the JUNV NP, which could impact the mechanism of the cellular immune response modulation of this important pathogen.

cis-Acting Element at the 5' Noncoding Region of Tacaribe Virus S RNA Modulates Genome Replication.

Tacaribe virus (TCRV) is the prototype of New World mammarenaviruses, a group that includes several members that cause hemorrhagic fevers in humans. The TCRV genome comprises two RNA segments, named S (small) and L (large). Both genomic segments contain noncoding regions (NCRs) at their 5' and 3' ends. While the 5'- and 3'-terminal 19-nucleotide sequences are known to be essential for promoter function, the role of their neighboring internal noncoding region (iNCR) sequences remains poorly understood. To analyze the relevance of the 5' and 3' iNCRs in TCRV S RNA synthesis, mutant S-like minigenomes and miniantigenomes were generated. Using a minireplicon assay, Northern blotting, and reverse transcription-quantitative PCR, we demonstrated that the genomic 5' iNCR is specifically engaged in minigenome replication yet is not directly involved in minigenome transcription, and we showed that the S genome 3' iNCR is barely engaged in this process. Analysis of partial deletions and point mutations, as well as total or partial substitution of the 5' iNCR sequence, led us to conclude that the integrity of the whole genomic 5' iNCR is essential and that a local predicted secondary structure or RNA-RNA interactions between the 5' and 3' iNCRs are not strictly required for viral S RNA synthesis. Furthermore, we employed a TCRV reverse genetic approach to ask whether manipulation of the S genomic 5' iNCR sequence may be suitable for viral attenuation. We found that mutagenesis of the 5' promoter-proximal subregion slightly impacted recombinant TCRV virulence in vivo. IMPORTANCE The Mammarenavirus genus of the Arenaviridae family includes several members that cause severe hemorrhagic fevers associated with high morbidity and mortality rates, for which no FDA-approved vaccines and limited therapeutic resources are available. We provide evidence demonstrating the specific involvement of the TCRV S 5' noncoding sequence adjacent to the viral promoter in replication. In addition, we examined the relevance of this region in the context of an in vivo infection. Our findings provide insight into the mechanism through which this 5' viral RNA noncoding region assists the L polymerase for efficient viral S RNA synthesis. Also, these findings expand our understanding of the effect of genetic manipulation of New World mammarenavirus sequences aimed at the rational design of attenuated recombinant virus vaccine platforms.

The Virus-Host Interplay in Junín Mammarenavirus Infection.

Junín virus (JUNV) belongs to the Arenaviridae family and is the causative agent of Argentine hemorrhagic fever (AHF), a severe human disease endemic to agricultural areas in Argentina. At this moment, there are no effective antiviral therapeutics to battle pathogenic arenaviruses. Cumulative reports from recent years have widely provided information on cellular factors playing key roles during JUNV infection. In this review, we summarize research on host molecular determinants that intervene in the different stages of the viral life cycle: viral entry, replication, assembly and budding. Alongside, we describe JUNV tight interplay with the innate immune system. We also review the development of different reverse genetics systems and their use as tools to study JUNV biology and its close teamwork with the host. Elucidating relevant interactions of the virus with the host cell machinery is highly necessary to better understand the mechanistic basis beyond virus multiplication, disease pathogenesis and viral subversion of the immune response. Altogether, this knowledge becomes essential for identifying potential targets for the rational design of novel antiviral treatments to combat JUNV as well as other pathogenic arenaviruses.

Deconstructing virus condensation.

Viruses have evolved precise mechanisms for using the cellular physiological pathways for their perpetuation. These virus-driven biochemical events must be separated in space and time from those of the host cell. In recent years, granular structures, known for over a century for rabies virus, were shown to host viral gene function and were named using terms such as viroplasms, replication sites, inclusion bodies, or viral factories (VFs). More recently, these VFs were shown to be liquid-like, sharing properties with membrane-less organelles driven by liquid-liquid phase separation (LLPS) in a process widely referred to as biomolecular condensation. Some of the best described examples of these structures come from negative stranded RNA viruses, where micrometer size VFs are formed toward the end of the infectious cycle. We here discuss some basic principles of LLPS in connection with several examples of VFs and propose a view, which integrates viral replication mechanisms with the biochemistry underlying liquid-like organelles. In this view, viral protein and RNA components gradually accumulate up to a critical point during infection where phase separation is triggered. This yields an increase in transcription that leads in turn to increased translation and a consequent growth of initially formed condensates. According to chemical principles behind phase separation, an increase in the concentration of components increases the size of the condensate. A positive feedback cycle would thus generate in which crucial components, in particular nucleoproteins and viral polymerases, reach their highest levels required for genome replication. Progress in understanding viral biomolecular condensation leads to exploration of novel therapeutics. Furthermore, it provides insights into the fundamentals of phase separation in the regulation of cellular gene function given that virus replication and transcription, in particular those requiring host polymerases, are governed by the same biochemical principles.

Development of a Reverse Genetic System to Generate Recombinant Chimeric Tacaribe Virus that Expresses Junín Virus Glycoproteins.

Mammarenaviruses are enveloped and segmented negative-stranded RNA viruses that comprise several pathogenic members associated with severe human hemorrhagic fevers. Tacaribe virus (TCRV) is the prototype for the New World group of mammarenaviruses and is not only naturally attenuated but also phylogenetically and antigenically related to all South American pathogenic mammarenaviruses, particularly the Junín virus (JUNV), which is the etiological agent of Argentinian hemorrhagic fever (AHF). Moreover, since TCRV protects guinea pigs and non-human primates from lethal challenges with pathogenic strains of JUNV, it has already been considered as a potential live-attenuated virus vaccine candidate against AHF. Here, we report the development of a reverse genetic system that relies on T7 polymerase-driven intracellular expression of the complementary copy (antigenome) of both viral S and L RNA segments. Using this approach, we successfully recovered recombinant TCRV (rTCRV) that displayed growth properties resembling those of authentic TCRV. We also generated a chimeric recombinant TCRV expressing the JUNV glycoproteins, which propagated similarly to wild-type rTCRV. Moreover, a controlled modification within the S RNA 5' non-coding terminal sequence diminished rTCRV propagation in a cell-type dependent manner, giving rise to new perspectives where the incorporation of additional attenuation markers could contribute to develop safe rTCRV-based vaccines against pathogenic mammarenaviruses.

Estructura poblacional y hábitat de un árbol tropical con frutos comestibles, Annona purpurea (Annonaceae), en el occidente de México / Population structure and habitat of a tropical tree with edible fruits, Annona purpurea (Annonaceae), in Western Mexico

Introducción: Annona purpurea es un árbol Mesoamericano, distribuido por la vertiente pacífica y atlántica de México hasta Sudamérica. Diferentes partes de la planta son de utilidad y los frutos son comestibles. Objetivo: Describir la estructura poblacional, el hábitat y los factores ambientales que influyen en la distribución y abundancia de A. purpurea en el occidente de México. Métodos: De abril a noviembre 2015, se establecieron 24 unidades de muestreo de 500 m2 cada una, con presencia de A. purpurea. En cada sitio se midieron e identificaron todas las especies leñosas con diámetro a la altura de pecho (dap) ≥ 2.5 cm y se registró información ambiental, geográfica y climática. Se obtuvo información sobre la repoblación de la especie. Se estimó la estructura de A. purpurea y se examinaron sus relaciones con las variables ambientales. El índice de asociación de Whittaker se determinó utilizando la matriz de índice de valor de importancia (IVI). Con la matriz de variables ambientales y la de IVI, se realizó un Análisis de Correspondencia Canónica para determinar la influencia de las variables ambientales sobre la especie y visualizar la distribución de las especies en el espacio multidimensional. Resultados: A. purpurea presentó 1 108 tallos en 1.2 ha, con el 85 % de ellos concentrados en las tres primeras categorías diamétricas. Su densidad presentó una relación positiva con la presencia de tocones, mientras que el área basal y el IVI se relacionaron de manera similar con la incidencia de incendios. Hubo poca o nula regeneración de A. purpurea debajo de su dosel. Los mayores IVI en la comunidad se registraron para A. purpurea, Tabebuia rosea, Quercus magnoliifolia y Enterolobium cyclocarpum y la prueba de perfiles de semejanza separó a A. purpurea y Guazuma ulmifolia como un grupo diferente. Las variables ambientales con mayor influencia en la distribución y abundancia de A. purpurea fueron la precipitación anual, incidencia de incendios, elevación, temperatura media anual, pedregosidad y pastoreo. Conclusión: A. purpurea presenta los mayores atributos estructurales en la comunidad, sus poblaciones se ven favorecidas en sitios con mayor precipitación y temperatura, con poca pedregosidad, donde hay incidencia de perturbaciones por fuego y ganado que generan claros grandes y su mayor asociación la presenta con G. ulmifolia.

Introduction: Annona purpurea is a Mesoamerican tree, distributed from the Pacific and Atlantic slopes of Mexico to South America. Different parts of the plant are of utility and the fruits are edible. Objective: To describe the population structure, habitat and environmental factors that influence the distribution and abundance of A. purpurea in Western Mexico. Methods: From April to November 2015, 24 sampling units were established, each of which were 500 m2 in area and had a presence of A. purpurea. At each site, all woody species with a diameter at breast height (dap) ≥ 2.5 cm were measured and identified, and environmental, geographic and climatic information recorded. Information regarding the regeneration of the species was obtained. The structure of A. purpurea was estimated and its relationships with environmental variables examined. The Whittaker association index was determined using the importance value index matrix (IVI). With the matrix of environmental variables and that of IVI, a Canonical Correspondence Analysis was performed to determine the influence of environmental variables on the species and to visualize the distribution of the species in multidimensional space. Results: A. purpurea presented 1 108 stems in 1.2 ha, with 85 % of these concentrated in the first three diametric categories. Its density presented a positive relationship with the presence of stumps, while the basal area and IVI were similarly related to the incidence of fire. There was little or no regeneration of A. purpurea under its canopy. The highest IVI in the community were found for A. purpurea, Tabebuia rosea, Quercus magnoliifolia and Enterolobium cyclocarpum, and the similarity profiles test separated A. purpurea and Guazuma ulmifolia as a distinct group. The environmental variables with the greatest influence on the distribution and abundance of A. purpurea were annual precipitation, fire incidence, elevation, mean annual temperature, stoniness and livestock. Conclusion: A. purpurea presents the greatest structural attributes in the community, its populations are favored in places with greater precipitation and temperature, with little stoniness and with incidence of disturbances by fire and livestock that generate large gaps and its greatest association is with G. ulmifolia.

Cuerpos extraños radiopacos con doble contorno en la vía digestiva: ¿son siempre pilas de botón? / Are strange radiopaque bodies with double contours in the digestive tract always button cell batteries?

Resumen La ingesta accidental de cuerpos extraños constituye una emergencia en los servicios de atención pediátrica y el grupo más frecuentemente afectado se encuentra en los pacientes entre los 6 meses y los 3 años. Asimismo, la ingesta de pilas de botón es un factor de riesgo importante para el desarrollo de complicaciones tempranas severas como la perforación esofágica y mediastinitis, y a largo plazo, la aparición de estenosis. Por este motivo, su identificación temprana y la extracción constituyen las estrategias más importantes de manejo. Se ha descrito como característico el hallazgo del doble contorno en la radiografía frontal cérvico-toracoabdominal en estos pacientes. Sin embargo, se ha reportado el mismo signo radiológico en cuerpos extraños diferentes. En el presente artículo se presentan dos casos con estas características.

Abstract Accidental ingestion of foreign bodies constitutes an emergency for pediatric care services especially since the group most frequently affected consists of children between 6 months and 3 years of age. Ingestion of button cell batteries has high risks for severe early complications such as esophageal perforations and mediastinitis, as well as the long term risk of stenosis. Early identification and extraction are the most important management strategies. A double contour on a frontal cervical, thoracic, or abdominal x-ray is characteristic, but this same radiological sign has been reported for other foreign bodies. We present two cases with these characteristics.

Virus⁻Host Interactions Involved in Lassa Virus Entry and Genome Replication.

Lassa virus (LASV) is the causative agent of Lassa fever, a human hemorrhagic disease associated with high mortality and morbidity rates, particularly prevalent in West Africa. Over the past few years, a significant amount of novel information has been provided on cellular factors that are determinant elements playing a role in arenavirus multiplication. In this review, we focus on host proteins that intersect with the initial steps of the LASV replication cycle: virus entry and genome replication. A better understanding of relevant virus⁻host interactions essential for sustaining these critical steps may help to identify possible targets for the rational design of novel therapeutic approaches against LASV and other arenaviruses that cause severe human disease.

Role of the ERK1/2 Signaling Pathway in the Replication of Junín and Tacaribe Viruses.

We have previously shown that the infection of cell cultures with the arenaviruses Junín (JUNV), Tacaribe (TCRV), and Pichindé promotes the phosphorylation of mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinases 1 and 2 (ERK1/2) and that this activation is required for the achievement of a productive infection. Here we examined the contribution of ERK1/2 in early steps of JUNV and TCRV multiplication. JUNV adsorption, internalization, and uncoating were not affected by treatment of cultured cells with U0126, an inhibitor of the ERK1/2 signaling pathway. In contrast, U0126 caused a marked reduction in viral protein expression and RNA synthesis, while JUNV RNA synthesis was significantly augmented in the presence of an activator of the ERK1/2 pathway. Moreover, U0126 impaired the expression of a reporter gene in a TCRV-based replicon system, confirming the ability of the compound to hinder arenavirus macromolecular synthesis. By using a cell-based assay, we determined that the inhibitor did not affect the translation of a synthetic TCRV-like mRNA. No changes in the phosphorylation pattern of the translation factor eIF2α were found in U0126-treated cells. Our results indicate that U0126 impairs viral RNA synthesis, thereby leading to a subsequent reduction in viral protein expression. Thus, we conclude that ERK1/2 signaling activation is required for an efficient arenavirus RNA synthesis.

Regulation of Tacaribe Mammarenavirus Translation: Positive 5' and Negative 3' Elements and Role of Key Cellular Factors.

Mammarenaviruses are enveloped viruses with a bisegmented negative-stranded RNA genome that encodes the nucleocapsid protein (NP), the envelope glycoprotein precursor (GPC), the RNA polymerase (L), and a RING matrix protein (Z). Viral proteins are synthesized from subgenomic mRNAs bearing a capped 5' untranslated region (UTR) and lacking 3' poly(A) tail. We analyzed the translation strategy of Tacaribe virus (TCRV), a prototype of the New World mammarenaviruses. A virus-like transcript that carries a reporter gene in place of the NP open reading frame and transcripts bearing modified 5' and/or 3' UTR were evaluated in a cell-based translation assay. We found that the presence of the cap structure at the 5' end dramatically increases translation efficiency and that the viral 5' UTR comprises stimulatory signals while the 3' UTR,specifically the presence of a terminal C+G-rich sequence and/or a stem-loop structure, down-modulates translation. Additionally, translation was profoundly reduced in eukaryotic initiation factor (eIF) 4G-inactivated cells, whereas depletion of intracellular levels of eIF4E had less impact on virus-like mRNA translation than on a cell-like transcript. Translation efficiency was independent of NP expression or TCRV infection. Our results indicate that TCRV mRNAs are translated using a cap-dependent mechanism, whose efficiency relies on the interplay between stimulatory signals in the 5' UTR and a negative modulatory element in the 3' UTR. The low dependence on eIF4E suggests that viral mRNAs may engage yet-unknown noncanonical host factors for a cap-dependent initiation mechanism.IMPORTANCE Several members of the Arenaviridae family cause serious hemorrhagic fevers in humans. In the present report, we describe the mechanism by which Tacaribe virus, a prototypic nonpathogenic New World mammarenavirus, regulates viral mRNA translation. Our results highlight the impact of untranslated sequences and key host translation factors on this process. We propose a model that explains how viral mRNAs outcompete cellular mRNAs for the translation machinery. A better understanding of the mechanism of translation regulation of this virus can provide the bases for the rational design of new antiviral tools directed to pathogenic arenaviruses.

CERVANTESIACEAE: NUEVO REGISTRO DE UNA FAMILIA NATURALIZADA PARA LA FLORA DE MÉXICO / Cervantesiaceae: New Record of a Naturalized Family for the Flora of Mexico

Se registra por primera vez para México la especie naturalizada Acanthosyris glabrata (familia Cervantesiaceae), un árbol hemiparásito sólo conocido del noroeste de Sudamérica. Se proporciona información sobre su hábitat y se presenta una ilustración de la especie. Se propone una hipótesis de comercio maritimo entre Ecuador y México para explicar la presencia del taxón en el occidente de México, en un área que dista 3500 km de su distribución original.

We register for the first time in Mexico the naturalized species Acanthosyrisglabrata (family Cervantesiaceae), a hemiparasitic tree only known from northwestern South America. We provide information about its habitat and present an illustration of the species. We propose a hypothesis of maritime commerce between Ecuador and Mexico to account for the presence of the taxon in western Mexico, in an area 3500 km away from its original known distribution.

Cálculo biométrico en pacientes operados de catarata / Biometrical calculation in patients operated for cataract

Introducción: para obtener resultados satisfactorios en el implante de lentes intraoculares, se requiere de un estudio previo a la operación de catarata y del cálculo correcto del lente. Objetivo: determinar los resultados refractivos de esta cirugía mediante el cálculo biométrico. Métodos: se realizó un estudio descriptivo y prospectivo de 149 ojos de pacientes operados de catarata, en el Centro Oftalmológico del estado venezolano de Apure, desde enero hasta abril del 2014. Resultados: predominaron el sexo femenino (61,7 %) y el grupo etario de 60-69 años (41,6 %). Los valores de la queratometría oscilaron entre las 43-44 dioptrías (67,0 %) y la longitud axial fue de 22-24 mm (70,5 %). El mayor porcentaje de los ojos operados correspondió a los corregidos con el lente intraocular implantado (79,2 %). Conclusiones: los resultados refractivos de los pacientes intervenidos quirúrgicamente demostraron la importancia del cálculo del lente intraocular previo a la extracción de catarata.

Introduction: to obtain satisfactory results in the implant of intraocular lens, a previous study to the cataract surgery and to the correct calculation of the lens is required. Objective: to determine the refractive results of this surgery by means of the biometrical calculation. Methods: a descriptive and prospective study of 149 cataract operated patients was carried out in the Ophthalmology Center in the Venezuelan state of Apure, from January to April, 2014. Results: feminine sex (61.7%) and the age group 60-69 years (41.6%) prevailed. The keratometry values fluctuated between 43-44 diopters (67.0%) and the axial longitude was of 22-24 mm (70.5%). The highest percentage in the operated eyes corresponded to those corrected with the implanted intraocular lens (79.2%). Conclusions: the refractive results of the surgically treated patients demonstrated the importance of the calculation obtained from the intraocular lens previous to the cataract extraction.

Caracterización de pacientes operados de catarata por la técnica de Blumenthal / Characterization of patients operated on for cataract through Blumenthal technique

Introducción: la catarata es la afección ocular más frecuente en todo el mundo, y representa 47,8 % de todas las causas de ceguera. Así, el progresivo envejecimiento de la población ha aumentado su prevalencia, y actualmente el único tratamiento eficaz y reparador es el quirúrgico. Objetivo: caracterizar a los pacientes operados de catarata por la técnica de Blumenthal. Métodos: se realizó un estudio descriptivo longitudinal y retrospectivo en los 149 afectados de catarata, que fueron intervenidos quirúrgicamente durante el primer semestre del 2013 en el Centro Oftalmológico del Estado Apure, en Venezuela. Resultados: en la serie predominaron el grupo etario de 60-69 años (41,6 %) y el sexo masculino (61,7 %); igualmente, se observó una primacía de la catarata senil (67,1 %), así como de la retinopatía diabética (26,1 %) y la miopía (22,9 %) como antecedentes patológicos oculares, y de la hipertensión arterial (44,2 %) y la diabetes mellitus como enfermedades sistémicas. La ruptura de la cápsula posterior (51,8 %) y su opacidad (45,6 %) constituyeron las complicaciones preponderantes, mientras que la agudeza visual más frecuente antes de la intervención fue de 0,1-0,3 (55,0 %) y posterior al procedimiento resultó de 0,8-1,0 y de 0,4-0,7 (54,0 y 30,0 %, respectivamente). Conclusiones: se apreció una concordancia entre lo obtenido en esta investigación y lo notificado en la bibliografía nacional e internacional en cuanto a las variables clinicoepidemiológicas descritas.

Introduction: cataract is the most frequent ocular disorder in the whole world, and it represents 47.8% of all the causes of blindness. Thus, the progressive aging of the population has increased its prevalence, and at present the only effective and repairing treatment is the surgical one. Objective: to characterize the operated patients through the Blumenthal technique. Methods: a longitudinal and retrospective descriptive study was carried out in the 149 patients affected with cataract who were surgically treated during the first semester of 2013 in the Ophthalmological Center of Apure State, Venezuela. Results: the group 60-69 years (41.6%) and the male sex (61.7%) prevailed in the series; likewise, a primacy of the senile cataract was observed (67.1%), as well as of diabetic retinopathy (26.1%) and myopia (22.9%) as ocular pathological history, and of hypertension (44.2%) and diabetes mellitus as systemic diseases. The rupture of the posterior capsule (51.8%) and its opacity (45.6%) constituted the predominant complications, while the most frequent visual acuity before the surgery was of 0.1-0.3 (55.0%) and after the procedure it was 0.8-1.0 and 0.4-0.7 (54.0 and 30.0%, respectively). Conclusions: a concordance was observed between what was obtained in this investigation and what was notified in the national and international as for the clinical and epidemiological variables described.

Differential contributions of tacaribe arenavirus nucleoprotein N-terminal and C-terminal residues to nucleocapsid functional activity.

UNLABELLED: The arenavirus nucleoprotein (NP) is the main protein component of viral nucleocapsids and is strictly required for viral genome replication mediated by the L polymerase. Homo-oligomerization of NP is presumed to play an important role in nucleocapsid assembly, albeit the underlying mechanism and the relevance of NP-NP interaction in nucleocapsid activity are still poorly understood. Here, we evaluate the contribution of the New World Tacaribe virus (TCRV) NP self-interaction to nucleocapsid functional activity. We show that alanine substitution of N-terminal residues predicted to be available for NP-NP interaction strongly affected NP self-association, as determined by coimmunoprecipitation assays, produced a drastic inhibition of transcription and replication of a TCRV minigenome RNA, and impaired NP binding to RNA. Mutagenesis and functional analysis also revealed that, while dispensable for NP self-interaction, key amino acids at the C-terminal domain were essential for RNA synthesis. Furthermore, mutations at these C-terminal residues rendered NP unable to bind RNA both in vivo and in vitro but had no effect on the interaction with the L polymerase. In addition, while all oligomerization-defective variants tested exhibited unaltered capacities to sustain NP-L interaction, NP deletion mutants were fully incompetent to bind L, suggesting that, whereas NP self-association is dispensable, the integrity of both the N-terminal and C-terminal domains is required for binding the L polymerase. Overall, our results suggest that NP self-interaction mediated by the N-terminal domain may play a critical role in TCRV nucleocapsid assembly and activity and that the C-terminal domain of NP is implicated in RNA binding. IMPORTANCE: The mechanism of arenavirus functional nucleocapsid assembly is still poorly understood. No detailed information is available on the nucleocapsid structure, and the regions of full-length NP involved in binding to viral RNA remain to be determined. In this report, novel findings are provided on critical interactions between the viral ribonucleoprotein components. We identify several amino acid residues in both the N-terminal and C-terminal domains of TCRV NP that differentially contribute to NP-NP and NP-RNA interactions and analyze their relevance for binding of NP to the L polymerase and for nucleocapsid activity. Our results provide insight into the contribution of NP self-interaction to RNP assembly and activity and reveal the involvement of the NP C-terminal domain in RNA binding.

Targeting of arenavirus RNA synthesis by a carboxamide-derivatized aromatic disulfide with virucidal activity.

Several arenaviruses can cause severe hemorrhagic fever (HF) in humans, representing a public health threat in endemic areas of Africa and South America. The present study characterizes the potent virucidal activity of the carboxamide-derivatized aromatic disulfide NSC4492, an antiretroviral zinc finger-reactive compound, against Junín virus (JUNV), the causative agent of Argentine HF. The compound was able to inactivate JUNV in a time and temperature-dependent manner, producing more than 99 % reduction in virus titer upon incubation with virions at 37 °C for 90 min. The ability of NSC4492-treated JUNV to go through different steps of the multiplication cycle was then evaluated. Inactivated virions were able to bind and enter into the host cell with similar efficiency as control infectious particles. In contrast, treatment with NSC4492 impaired the capacity of JUNV to drive viral RNA synthesis, as measured by quantitative RT-PCR, and blocked viral protein expression, as determined by indirect immunofluorescence. These results suggest that the disulfide NSC4492 targets on the arenavirus replication complex leading to impairment in viral RNA synthesis. Additionally, analysis of VLP produced in NSC4492-treated cells expressing JUNV matrix Z protein revealed that the compound may interact with Z resulting in an altered aggregation behavior of this protein, but without affecting its intrinsic self-budding properties. The potential perspectives of NSC4492 as an inactivating vaccinal compound for pathogenic arenaviruses are discussed.

Uncovering viral protein-protein interactions and their role in arenavirus life cycle.

The Arenaviridae family includes widely distributed pathogens that cause severe hemorrhagic fever in humans. Replication and packaging of their single-stranded RNA genome involve RNA recognition by viral proteins and a number of key protein-protein interactions. Viral RNA synthesis is directed by the virus-encoded RNA dependent-RNA polymerase (L protein) and requires viral RNA encapsidation by the Nucleoprotein. In addition to the role that the interaction between L and the Nucleoprotein may have in the replication process, polymerase activity appears to be modulated by the association between L and the small multifunctional Z protein. Z is also a structural component of the virions that plays an essential role in viral morphogenesis. Indeed, interaction of the Z protein with the Nucleoprotein is critical for genome packaging. Furthermore, current evidence suggests that binding between Z and the viral envelope glycoprotein complex is required for virion infectivity, and that Z homo-oligomerization is an essential step for particle assembly and budding. Efforts to understand the molecular basis of arenavirus life cycle have revealed important details on these viral protein-protein interactions that will be reviewed in this article.

Molecular determinants of arenavirus Z protein homo-oligomerization and L polymerase binding.

The arenavirus Z is a zinc-binding RING protein that has been implicated in multiple functions during the viral life cycle. These roles of Z involve interactions with viral and cellular proteins that remain incompletely understood. In this regard, Z inhibits viral RNA transcription and replication through direct interaction with the viral L polymerase. Here, we defined the L-binding domain of Tacaribe virus (TCRV) Z protein and the structural requirements mediating Z homo-oligomerization. By using site-directed mutagenesis, coimmunoprecipitation, and functional assays, we showed that residues R37, N39, W44, L50, and Y57, located around the zinc coordination site I, play a critical role in the Z-L interaction. We also found that Z protein from either TCRV or the pathogenic Junin virus (JUNV) self-associates into oligomeric forms in mammalian cells. Importantly, mutation of the myristoylation site, the strictly conserved residue G at position 2, severely impaired the ability of both TCRV Z and JUNV Z to self-interact as well as their capacity to accumulate at the plasma membrane, strongly suggesting that Z homo-oligomerization is associated with its myristoylation and cell membrane targeting. In contrast, disruption of the RING structure or substitution of W44 or N39, which are critical for L protein recognition, did not affect Z self-binding. Overall, the data presented here indicate that homo-oligomerization is not a requirement for Z-L interaction or Z-mediated polymerase activity inhibition.

[Approaches to determine priorities and to analyze problems of health with a look from the equity: experience in the local level in Venezuela]. / Priorización y análisis de problemas de salud con una mirada desde la equidad: experiencia en el nivel local en Venezuela.

This article analyzes the application of the explanatory moment of the Strategic Situational Planning (SSP) and the Analysis of the Situation of Health (ASIS), as approaches that together, allow to prioritize with a look from the equity problems of health in the local level feasible of intervention. By using the case study developed in the parish Zuata of Aragua State, Venezuela, it can be observed the application of both approaches The main actors of the above mentioned parish prioritized the low coverage of drinkable water, like a health problem. On having analyzed the problem, the following causes were selected to prepare the proposed action: scarce community participation, weakness of governmental plans, absence of political town-planning, inadequate administration of the public resources and lack of conscience in the rational use of water. At the end one concludes that the joint PES-ASIS allows to generate inputs that concretized for the actors in an action plan, they can contribute in the reduction of inequities. Also, the active participation of the actors allows to demonstrate the real problems of the population and to construct a plan of demands.

Priorización y análisis de problemas de salud con una mirada desde la equidad: experiencia en el nivel local en Venezuela / Approaches to determine priorities and to analyze problems of health with a look from the equity: experience in the local level in Venezuela

En este artículo se analiza la aplicación del momento explicativo de la Planificación Estratégica Situacional (PES) y el Análisis de la Situación de Salud (ASIS), como enfoques que conjugados permiten priorizar con una mirada desde la equidad problemas de salud en el nivel local factibles de intervención. A través del estudio de un caso desarrollado en la parroquia Zuata del Estado Aragua, Venezuela, se presenta la aplicación de ambos enfoques. Los actores claves de dicha parroquia priorizaron la baja cobertura de agua potable, como problema de salud. Al analizar el problema se seleccionaron las siguientes causas claves para elaborar la propuesta de acción: escasa participación comunitaria, debilidad de planes gubernamentales, ausencia de políticas urbanísticas, inadecuada administración de los recursos públicos y poca conciencia en el uso racional del agua. Al final se concluye que la articulación PES-ASIS contribuye a generar insumos que concretizados por los actores en un plan de acción, pueden contribuir en la reducción de inequidades. Asimismo, la participación activa de los actores permite evidenciar los problemas reales de la población y construir un plan de demandas.

This article analyzes the application of the explanatory moment of the Strategic Situational Planning (SSP) and the Analysis of the Situation of Health (ASIS), as approaches that together, allow to prioritize with a look from the equity problems of health in the local level feasible of intervention. By using the case study developed in the parish Zuata of Aragua State, Venezuela, it can be observed the application of both approaches The main actors of the above mentioned parish prioritized the low coverage of drinkable water, like a health problem. On having analyzed the problem, the following causes were selected to prepare the proposed action: scarce community participation, weakness of governmental plans, absence of political town-planning, inadequate administration of the public resources and lack of conscience in the rational use of water. At the end one concludes that the joint PES-ASIS allows to generate inputs that concretized for the actors in an action plan, they can contribute in the reduction of inequities. Also, the active participation of the actors allows to demonstrate the real problems of the population and to construct a plan of demands.