NF90 Binds the Dengue Virus RNA 3′ Terminus and is a Positive Regulator of Dengue Virus Replication

Background Viral RNA translation and replication are regulated by sequence and structural elements in the 5′ and 3′ untranslated regions (UTR) and by host cell and/or viral proteins that bind them. Dengue virus has a single-stranded RNA genome with positive polarity, a 5′ m7GpppG cap, and a conserved 3′-terminal stem loop (SL) that is linked to proposed functions in viral RNA transcription and translation. Mechanisms explaining the contributions of host proteins to viral RNA translation and replication are poorly defined, yet understanding host protein-viral RNA interactions may identify new targets for therapeutic intervention. This study was directed at identifying functionally significant host proteins that bind the conserved dengue virus RNA 3′ terminus. Methodology/Principal Findings Proteins eluted from a dengue 3′ SL RNA affinity column at increasing ionic strength included two with double-strand RNA binding motifs (NF90/DRBP76 and DEAH box polypeptide 9/RNA helicase A (RHA)), in addition to NF45, which forms a heterodimer with NF90. Although detectable NF90 and RHA proteins localized to the nucleus of uninfected cells, immunofluorescence revealed cytoplasmic NF90 in dengue virus-infected cells, leading us to hypothesize that NF90 has a functional role(s) in dengue infections. Cells depleted of NF90 were used to quantify viral RNA transcript levels and production of infectious dengue virus. NF90 depletion was accompanied by a 50%-70% decrease in dengue RNA levels and in production of infectious viral progeny. Conclusions/Significance The results indicate that NF90 interacts with the 3′ SL structure of the dengue RNA and is a positive regulator of dengue virus replication. NF90 depletion diminished the production of infectious dengue virus by more than 50%, which may have important significance for identifying therapeutic targets to limit a virus that threatens more than a billion people worldwide.Ruth L. Kirschstein National Research Service Award (NIH-NRSA GM64985)UNCF-Merck Postdoctoral FellowshipNational Institute of Allergy and Infectious Diseases (U.S.)Ellison Medical Foundatio

Dengue-2 Structural Proteins Associate with Human Proteins to Produce a Coagulation and Innate Immune Response Biased Interactome

<p>Abstract</p> <p>Background</p> <p>Dengue virus infection is a public health threat to hundreds of millions of individuals in the tropical regions of the globe. Although Dengue infection usually manifests itself in its mildest, though often debilitating clinical form, dengue fever, life-threatening complications commonly arise in the form of hemorrhagic shock and encephalitis. The etiological basis for the virus-induced pathology in general, and the different clinical manifestations in particular, are not well understood. We reasoned that a detailed knowledge of the global biological processes affected by virus entry into a cell might help shed new light on this long-standing problem.</p> <p>Methods</p> <p>A bacterial two-hybrid screen using DENV2 structural proteins as bait was performed, and the results were used to feed a manually curated, global dengue-human protein interaction network. Gene ontology and pathway enrichment, along with network topology and microarray meta-analysis, were used to generate hypothesis regarding dengue disease biology.</p> <p>Results</p> <p>Combining bioinformatic tools with two-hybrid technology, we screened human cDNA libraries to catalogue proteins physically interacting with the DENV2 virus structural proteins, Env, cap and PrM. We identified 31 interacting human proteins representing distinct biological processes that are closely related to the major clinical diagnostic feature of dengue infection: haemostatic imbalance. In addition, we found dengue-binding human proteins involved with additional key aspects, previously described as fundamental for virus entry into cells and the innate immune response to infection. Construction of a DENV2-human global protein interaction network revealed interesting biological properties suggested by simple network topology analysis.</p> <p>Conclusions</p> <p>Our experimental strategy revealed that dengue structural proteins interact with human protein targets involved in the maintenance of blood coagulation and innate anti-viral response processes, and predicts that the interaction of dengue proteins with a proposed human protein interaction network produces a modified biological outcome that may be behind the hallmark pathologies of dengue infection.</p

Citotoxicidad y actividad antiviral de extractos de chiles (Capsicum spp)

Abstract: In this work cytotoxicity and antiviral activity against herpes simplex virus type 1 (HSV-1) were evaluated for the pepper extracts of jalapeño, serrano, guajillo, ancho and sweet pepper (Capsicum annuum L. var. annuum.) and habanero (Capsicum chinense Jacq.), as well as for pure phenylpropanoids and flavonoids previously identified in the peppers. The concentration causing 50% toxicity in Vero cells was also determined (CC50). The antiviral potential of pepper extracts and pure compounds was expressed as the concentration required to achieve 50 % protection against cytopathic effects in Vero cells (IC50) infected with HSV-1. The selective index was calculated as the ratio of the CC50 to IC50. The sweet pepper extract showed the lowest cytotoxicity (CC50 = 9.82 ± 0.06 mg/mL) and the highest antiviral activity (IC50 = 0.56 ± 0.02 mg/mL), with the highest selective index of 17.5. High antiviral capacities were also observed for ancho and guajillo. The habanero pepper extract presented the highest cytotoxicity and the lowest selective index. Phenolic compounds also showed antiviral activity and are probably responsible for the antiviral properties of pepper extracts.Resumen: En este trabajo se evaluaron la citotoxicidad y la actividad antiviral en contra del virus herpes simplex tipo 1 (VHS-1) de extractos de chile jalapeño, serrano, guajillo, ancho, pimiento (Capsicum annuum L. var. annuum.) y habanero (Capsicum chinense Jacq.), así como de los fenilpropanoides, ácidos fenólicos y flavonoides puros que fueron identificados por HPLC-MS en los extractos de chile. La concentración que causa el 50% de toxicidad en células Vero fue determinada (CC50) así como el potencial antiviral de los extractos de chiles y de los compuestos puros, expresado como la concentración inhibitoria del 50% (CI50) en contra de los efectos citopáticos en células Vero infectadas con el virus. El índice de selectividad fue calculado como la relación de CC50 entre CI50. El extracto de pimiento mostró las más baja citotoxicidad (CC50 = 9.82 ± 0.06 mg/mL) y la más alta actividad antiviral (IC50 = 0.56 ± 0.02 mg/mL), con el mayor índice de selectividad de 17.5. Los extractos de chile ancho y guajillo mostraron también una alta capacidad antiviral. El extracto de chile habanero presentó la mayor citotoxicidad y el menor índice de selectividad. Los compuestos fenólicos presentes en los extractos también mostraron actividad antiviral, lo que sugiere que pueden ser estos compuestos los responsables de la actividad antiviral de los chiles

Comparative genomic analysis of pre-epidemic and epidemic Zika virus strains for virological factors potentially associated with the rapidly expanding epidemic

Less than 20 sporadic cases of human Zika virus (ZIKV) infection were reported in Africa and Asia before 2007, but large outbreaks involving up to 73% of the populations on the Pacific islands have started since 2007, and spread to the Americas in 2014. Moreover, the clinical manifestation of ZIKV infection has apparently changed, as evident by increasing reports of neurological complications, such as Guillain–Barré syndrome in adults and congenital anomalies in neonates. We comprehensively compared the genome sequences of pre-epidemic and epidemic ZIKV strains with complete genome or complete polyprotein sequences available in GenBank. Besides the reported phylogenetic clustering of the epidemic strains with the Asian lineage, we found that the topology of phylogenetic tree of all coding regions is the same except that of the non-structural 2B (NS2B) coding region. This finding was confirmed by bootscan analysis and multiple sequence alignment, which suggested the presence of a fragment of genetic recombination at NS2B with that of Spondweni virus. Moreover, the representative epidemic strain possesses one large bulge of nine bases instead of an external loop on the first stem-loop structure at the 3′-untranslated region just distal to the stop codon of the NS5 in the 1947 pre-epidemic prototype strain. Fifteen amino acid substitutions are found in the epidemic strains when compared with the pre-epidemic strains. As mutations in other flaviviruses can be associated with changes in virulence, replication efficiency, antigenic epitopes and host tropism, further studies would be important to ascertain the biological significance of these genomic changes