An integrated ontology resource to explore and study host-virus relationships.

Our growing knowledge of viruses reveals how these pathogens manage to evade innate host defenses. A global scheme emerges in which many viruses usurp key cellular defense mechanisms and often inhibit the same components of antiviral signaling. To accurately describe these processes, we have generated a comprehensive dictionary for eukaryotic host-virus interactions. This controlled vocabulary has been detailed in 57 ViralZone resource web pages which contain a global description of all molecular processes. In order to annotate viral gene products with this vocabulary, an ontology has been built in a hierarchy of UniProt Knowledgebase (UniProtKB) keyword terms and corresponding Gene Ontology (GO) terms have been developed in parallel. The results are 65 UniProtKB keywords related to 57 GO terms, which have been used in 14,390 manual annotations; 908,723 automatic annotations and propagated to an estimation of 922,941 GO annotations. ViralZone pages, UniProtKB keywords and GO terms provide complementary tools to users, and the three resources have been linked to each other through host-virus vocabulary

High efficiency of alphaviral gene transfer in combination with 5-fluorouracil in a mouse mammary tumor model

Copyright: Copyright 2014 Elsevier B.V., All rights reserved.Background: The combination of virotherapy and chemotherapy may enable efficient tumor regression that would be unachievable using either therapy alone. In this study, we investigated the efficiency of transgene delivery and the cytotoxic effects of alphaviral vector in combination with 5-fluorouracil (5-FU) in a mouse mammary tumor model (4 T1).Methods: Replication-deficient Semliki Forest virus (SFV) vectors carrying genes encoding fluorescent proteins were used to infect 4 T1 cell cultures treated with different doses of 5-FU. The efficiency of infection was monitored via fluorescence microscopy and quantified by fluorometry. The cytotoxicity of the combined treatment with 5-FU and alphaviral vector was measured using an MTT-based cell viability assay. In vivo experiments were performed in a subcutaneous 4 T1 mouse mammary tumor model with different 5-FU doses and an SFV vector encoding firefly luciferase.Results: Infection of 4 T1 cells with SFV prior to 5-FU treatment did not produce a synergistic anti-proliferative effect. An alternative treatment strategy, in which 5-FU was used prior to virus infection, strongly inhibited SFV expression. Nevertheless, in vivo experiments showed a significant enhancement in SFV-driven transgene (luciferase) expression upon intratumoral and intraperitoneal vector administration in 4 T1 tumor-bearing mice pretreated with 5-FU: here, we observed a positive correlation between 5-FU dose and the level of luciferase expression.Conclusions: Although 5-FU inhibited SFV-mediated transgene expression in 4 T1 cells in vitro, application of the drug in a mouse model revealed a significant enhancement of intratumoral transgene synthesis compared with 5-FU untreated mice. These results may have implications for efficient transgene delivery and the development of potent cancer treatment strategies using alphaviral vectors and 5-FU.publishersversionPeer reviewe

New World and Old World Alphaviruses Have Evolved to Exploit Different Components of Stress Granules, FXR and G3BP Proteins, for Assembly of Viral Replication Complexes

<div><p>The positive-strand RNA viruses initiate their amplification in the cell from a single genome delivered by virion. This single RNA molecule needs to become involved in replication process before it is recognized and degraded by cellular machinery. In this study, we show that distantly related New World and Old World alphaviruses have independently evolved to utilize different cellular stress granule-related proteins for assembly of complexes, which recruit viral genomic RNA and facilitate formation of viral replication complexes (vRCs). Venezuelan equine encephalitis virus (VEEV) utilizes all members of the Fragile X syndrome (FXR) family, while chikungunya and Sindbis viruses exploit both members of the G3BP family. Despite being in different families, these proteins share common characteristics, which determine their role in alphavirus replication, namely, the abilities for RNA-binding and for self-assembly into large structures. Both FXR and G3BP proteins interact with virus-specific, repeating amino acid sequences located in the C-termini of hypervariable, intrinsically disordered domains (HVDs) of viral nonstructural protein nsP3. We demonstrate that these host factors orchestrate assembly of vRCs and play key roles in RNA and virus replication. Only knockout of all of the homologs results in either pronounced or complete inhibition of replication of different alphaviruses. The use of multiple homologous proteins with redundant functions mediates highly efficient recruitment of viral RNA into the replication process. This independently evolved acquisition of different families of cellular proteins by the disordered protein fragment to support alphavirus replication suggests that other RNA viruses may utilize a similar mechanism of host factor recruitment for vRC assembly. The use of different host factors by alphavirus species may be one of the important determinants of their pathogenesis.</p></div

G3BP-nsP3 complexes bind viral G RNA in CHIKV-infected cells.

<p>(A) Colocalization of CHIKV nsP3-Cherry (pseudocolored in green) fusion proteins with CHIKV G RNA. Images are presented as MIP of 6 x-y sections (1 μm) through the middle plane of nucleus. Bars: 10 and 3 μm. (B) Colocalization of CHIKV nsP3 with G3BP2, stained with specific Abs at the plasma membrane at 2 h PI. Bars: 10 μm and 3 μm. (C) At 2 h PI, G3BP2 and dsRNA form stripes at the plasma membrane in CHIKV-infected cells. Staining was performed with specific Abs. (D) CHIKV G RNA and G3BP1-GFP strongly colocalize at the plasma membrane in the infected cells. The dsRNA-containing vRCs are formed in close proximity to G3BP-GFP/G RNA complexes and often overlap. Bars: 2 μm. Images in B-D are presented as MIP of x-y sections (1 μm) at the plasma membrane. Yellow arrowheads indicate pre-vRCs, which appear as dsRNA-free G3BP2-positive spots (red) in (C) or G RNA/G3BP1-GFP spots (yellow) in (D). Turquoise arrowheads indicate fully formed, dsRNA-positive vRCs, which contain low levels of G3BP1-GFP. Pink arrowheads indicate pre-vRCs, which contain a dsRNA signal of low intensity and volume and, likely, represent vRCs in the process of synthesis of dsRNA intermediate.</p

Repeating aa sequences in VEEV and CHIKV HVDs determine virus replication.

<p>(A) The schematic presentation of the recombinant VEEV genome and modifications introduced into nsP3 HVD sequence. Replacement of repeating sequences in VEEV HVD by those derived from CHIKV HVD makes VEEV/chikvR/GFP replication dependent on G3BP. VEEV/mutHVD/GFP titers were determined at 7 h PI, and VEEV/chikvR/GFP titers were determined at 8 h PI (MOI 0.01). (B) The schematic presentation of the recombinant CHIKV genome and modifications introduced into nsP3 HVD sequence. SINV-specific sequence in CHIKV HVD, but not VEEV repeating element, can support CHIKV replication. Virus titers were determined at 24 h PI (MOI of 0.05). Data in (A) and (B) are presented as mean±SD of 3 biological repeats. NV indicates that the designed mutants were not viable. (C) Colocalization of wt and mutant VEEV nsP3 proteins with FXR1 and G3BP2. Images are presented as MIP of x-y 1-μm-thick sections at the plasma membrane. Pearson's colocalization coefficients are shown in overlay panel (mean±SD, n>6). Bars: 2 μm</p

Model of FXR- and G3BP-mediated alphavirus replication complex formation.

<p>Binding of FXRs or G3BPs to the PM-bound P123 and P123+nsP4 complexes promotes their interaction with newly synthesized G RNAs and formation of pre-vRCs. In the absence of FXRs or G3BPs, G RNAs are acquired by P123+nsP4 complexes less efficiently and are likely degraded.</p