RESULTS:
1 - 4 of 4 for "Armando Arias"
Ubiquitin-like modifier-activating enzyme 1 interacts with Zika virus NS5 and promotes viral replication in the infected cell
Translation errors impaired folding or environmental stressors (e.g. infection) can all lead to an increase in the presence of misfolded proteins. These activate cellular responses to their removal including intracellular protein degradation activities. Protein ubiquitylation is involved in two major degradation pathways the ubiquitin-proteasome system and selective autophagy. In humans the ubiquitin-like modifier-activating enzyme 1 (UBA1) is the primary E1 enzyme in the ubiquitin conjugation cascade. Viruses have evolved to exploit protein degradation pathways to complete their infection cycles. Zika virus (ZIKV) is an emerging orthoflavivirus causing serious neurologic disorders in neonates (congenital microcephaly) and adults (Guillain–Barré syndrome). Non-structural protein 5 (NS5) the largest and most conserved protein in the orthoflaviviruses catalyses the synthesis and capping of new viral genomes. In addition to viral RNA replication in the cytoplasm ZIKV NS5 is translocated into the nucleus to interfere with host antiviral responses. Here we demonstrate that ZIKV NS5 co-immunoprecipitates with cellular UBA1. Immunofluorescence assays suggest that this interaction takes place primarily in the nucleus of an infected cell although colocalization of both proteins is also detected in the cytosol. RNA interference-mediated depletion of UBA1 leads to reduced virus titres in the infected cells while transient overexpression of UBA1 favours faster replication kinetics with higher virus titres and protein levels detected. Moreover UBA1-targeting drugs cause significant drops in virus infectivity. These results support a proviral role for UBA1 during ZIKV infection and encourage the potential use of inhibitors against this enzyme or its NS5-interacting epitopes as potential therapeutic targets.
Molecular dissection of a viral quasispecies under mutagenic treatment: positive correlation between fitness loss and mutational load
Low fidelity replication and the absence of error-repair activities in RNA viruses result in complex and adaptable ensembles of related genomes in the viral population termed quasispecies with important implications for natural infections. Theoretical predictions suggested that elevated replication error rates in RNA viruses might be near to a maximum compatible with viral viability. This fact encouraged the use of mutagenic nucleosides as a new antiviral strategy to induce viral extinction through increased replication error rates. Despite extensive evidence of lethal mutagenesis of RNA viruses by different mutagenic compounds a detailed picture of the infectivity of individual genomes and its relationship with the mutations accumulated is lacking. Here we report a molecular analysis of a foot-and-mouth disease virus population previously subjected to heavy mutagenesis to determine whether a correlation between increased mutagenesis and decreased fitness existed. Plaque-purified viruses isolated from a ribavirin-treated quasispecies presented decreases of up to 200-fold in infectivity relative to clones in the reference population associated with an overall eightfold increase in the mutation frequency. This observation suggests that individual infectious genomes of a quasispecies subjected to increased mutagenesis lose infectivity by their continuous mutagenic ‘poisoning’. These results support the lethal defection model of virus extinction and the practical use of chemical mutagens as antiviral treatment. Even when extinction is not achieved mutagenesis can decrease the infectivity of surviving virus and facilitate their clearance by host immune responses or complementing antiviral approaches.
Development of a reverse-genetics system for murine norovirus 3: long-term persistence occurs in the caecum and colon
Human noroviruses (HuNoV) are a major cause of viral gastroenteritis worldwide yet due to the inability to propagate HuNoV in cell culture murine norovirus (MNV) is typically used as a surrogate to study norovirus biology. MNV-3 represents an attractive strain to study norovirus infections in vivo because it establishes persistence in wild-type mice yet causes symptoms resembling gastroenteritis in immune-compromised STAT1−/− mice. The lack of reverse-genetics approaches to recover genetically defined MNV-3 has limited further studies on the identification of viral sequences that contribute to persistence. Here we report the establishment of a combined DNA-based reverse-genetics and mouse-model system to study persistent MNV-3 infections in wild-type (C57BL/6) mice. Viral RNA and infectious virus were detected in faeces for at least 56 days after inoculation. Strikingly the highest concentrations of viral RNA during persistence were detected in the caecum and colon suggesting that viral persistence is maintained in these tissues. Possible adaptive changes arising during persistence in vivo appeared to accumulate in the minor capsid protein (VP2) and the viral polymerase (NS7) in contrast with adaptive mutations selected during cell-culture passages in RAW264.7 cells that appeared in the major capsid protein (VP1) and non-structural protein NS4. This system provides an attractive model that can be readily used to identify viral sequences that contribute to persistence in an immunocompetent host and to more acute infection in an immunocompromised host providing new insights into the biology of norovirus infections.
Molecular intermediates of fitness gain of an RNA virus: characterization of a mutant spectrum by biological and molecular cloning
The mutant spectrum of a virus quasispecies in the process of fitness gain of a debilitated foot-and-mouth disease virus (FMDV) clone has been analysed. The mutant spectrum was characterized by nucleotide sequencing of three virus genomic regions (internal ribosome entry site; region between the two AUG initiation codons; VP1-coding region) from 70 biological clones (virus from individual plaques formed on BHK-21 cell monolayers) and 70 molecular clones (RT–PCR products cloned in E. coli). The biological and molecular clones provided statistically indistinguishable definitions of the mutant spectrum with regard to the distribution of mutations among the three genomic regions analysed and with regard to the types of mutations mutational hot-spots and mutation frequencies. Therefore the molecular cloning procedure employed provides a simple protocol for the characterization of mutant spectra of viruses that do not grow in cell culture. The number of mutations found repeated among the clones analysed was higher than expected from the mean mutation frequencies. Some components of the mutant spectrum reflected genomes that were dominant in the prior evolutionary history of the virus (previous passages) confirming the presence of memory genomes in virus quasispecies. Other components of the mutant spectrum were genomes that became dominant at a later stage of evolution suggesting a predictive value of mutant spectrum analysis with regard to the outcome of virus evolution. The results underline the observation that greater insight into evolutionary processes of viruses may be gained from detailed clonal analyses of the mutant swarms at the sequence level.